WO2025006818A1

WO2025006818A1 - Synthetic proteins based on foldamers as targeted therapeutics for neurodegenerative disorders

Info

Publication number: WO2025006818A1
Application number: PCT/US2024/035930
Authority: WO
Inventors: Sunil Kumar; Ryan DOHONEY; Magzoub MAZIN; Palanikumar LOGANATHAN
Original assignee: University of Denver
Current assignee: University of Denver
Priority date: 2023-06-28
Filing date: 2024-06-28
Publication date: 2025-01-02
Anticipated expiration: 2025-12-28

Abstract

A synthetic protein molecule includes a scaffolding framework and at least one ligand attached to the scaffolding framework. The scaffolding framework is selected for mimicking a conformation of a naturally occurring monomer protein. A plurality of the naturally occurring monomer protein so selected exhibit a tendency for aggregation thereof to form a plurality of oligomers. The at least one ligand is selected for an affinity for the plurality of oligomers so as to inhibit aggregation of the plurality of oligomers. An associated method for synthesizing a synthetic protein molecule is also disclosed. The synthetic protein molecule may be a foldamer.

Description

Synthetic Proteins Based on Foldamers as Targeted Therapeutics for

Neurodegenerative Disorders

REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of US Pat. App. No. 63/523,642, filed 2023- 06-28 and titled "Synthetic Proteins Based on Bio-Mimicry Approaches as Targeted Therapeutics for Neurodegenerative Disorders," which is incorporated hereby in its entirety by reference.

FI ELD OF TH E I NVENTION

[0002] The present invention relates to therapeutics for neurodegenerative disorders. In particular, but not by way of limitation, the present invention relates to compounds suitable for use as therapeutics for neurodegenerative disorders involving undesirable protein aggregation.

DESCRIPTION OF RELATED ART

[0001] Various pharmaceutical companies that are working in the field of therapeutics for neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease (PD), involving impairment and/or death of cells of the central nervous system. For example, PD is a progressive neurodegenerative disorder for which there is no successful prevention or intervention. There are more than 10 million people worldwide living with PD and the number is expected to double in 2030. Unfortunately, there is currently no identified therapy to cure or slow the progression of PD. Therefore, there is a pressing need to develop therapeutic interventions for PD.

[0002] The pathological hallmark for PD involves the self-assembly of functional Alpha- Synuclein (aS), which is a neuronal protein expressed at high levels in dopaminergic (DA) neurons in the brain, into non-functional amyloid structures. These amyloid structures are known to impair the regulation of synaptic vesicle trafficking, recycling, and neurotransmitter release. The process of aS aggregation impairs the function of DA neurons by formation of pathological aS oligomers and aS fibers (aggregates). For example, the pathological events in PD include aS aggregation and the spread of pathogenic aS fibers from neuron-to-neuron (via prion-like spread) and template the soluble aS into insoluble aS fibers. These processes of aS aggregation are also associated with other neurodegenerative disease, including Lewy body dementia, Parkinson disease dementia, and multiple system atrophy.

[0003] As another example, involving impairment and/or death of cells of the central nervous system. For example, Aberrant protein-protein interaction involving amyloid beta 1-42 (AP1-42) aggregation, is linked to the onset of Alzheimer's disease (AD). AD is a progressive neurodegenerative disorder that is the leading cause of dementia affecting millions across the world. Under pathological conditions, the misfolding of API.₄₂ into aggregates of insoluble plaques in the hippocampus and its connected structures contributes to the impaired neuron function and cognitive decline associated with AD. Alzheimer's Disease is the most common neurodegenerative disease that affects memory, thought processes, and behavior. The aggregation of Alzheimer's associated AP into insoluble neuronal plaques is thought to be the main pathogenic hallmark of the disease.

[0004] Protein-protein interactions (PPIs) are crucial for the regulation of numerous biological processes. Accordingly, Aberrant Protein-Protein Interactions (APPIs) can lead to the development of numerous pathological conditions, including cancer, infectious disease, neurodegenerative diseases, and diabetes. Thus, these APPIs make for attractive targets in the development of new therapeutics for APPI-related diseases.

[0005] The most prominent challenge in abrogating these APPIs lies in the surface topology of the protein targets. APPIs can span large surface areas at the site of interaction, involving numerous chemical and side-chain interactions. For this reason, the development and discovery of small-molecule therapeutics capable of prohibiting these interactions is of the utmost challenge.

[0006] Several mechanisms for the modulation of APPIs have been demonstrated in the literature. For instance, while peptide intervention is one strategy toward the modulation of APPIs, the impermeability of peptide-based drugs in combination with the lack of chemical and structural stability in the cellular milieu, and poor ability to cross the blood brain barrier (BBB) significantly limits their pharmaceutical applications.

[0007] The physiological and pathophysiological mechanisms of this impairment due to such conditions are illustrated in FIGS. 1A and IB, respectively. FIG. 1A shows a physiological model of neurons with an expanded view of a portion of adjacent neurotransmitter and receptor pair, showing the expression of aS in dopaminergic neurons, where it is localized on dopamine-filled synaptic vesicles and help in dopamine release. As shown in FIG. IB, illustrating a pathophysiological model of the blockage of the release of dopamine as a result of aS aggregation, leading to cell death.

[0008] One model of such protein aggregation is illustrated in FIG. 2. As shown in FIG. 2, a monomer may exhibit a tendency to cluster to form a small oligomer. The oligomerization may involve, for example, the adhesion of monomers in close proximity due to the chemical interactions between monomers that promote aggregation. A sufficient aggregation of monomers may turn into a plurality of large oligomers, which in turn may polymerize to form protofibrils. The protofibrils may cluster together to form amyloid fibers, which are known to be associated with neurodegenerative diseases.

[0009] Modulation of aggregation and the prion-like spread of aS is considered to be a promising potential therapeutic intervention for PD. However, the bottleneck towards achieving this goal is the identification of aS domains/ sequences that are essential for aggregation.

[0010] There are more than 10 million people worldwide living with PD and the number is expected to double in 2030 and there is no identified therapy to cure or slow the progression of PD. Therefore, there is a need for an improved mechanistic and therapeutic insights into aS aggregation and its role in mediating PD phenotypes, which will pave the way for effective treatments for PD.

SUMMARY OF THE INVENTION

[0011] The following presents a simplified summary relating to one or more aspects and/or embodiments disclosed herein. As such, the following summary should not be considered an extensive overview relating to all contemplated aspects and/or embodiments, nor should the following summary be regarded to identify key or critical elements relating to all contemplated aspects and/or embodiments or to delineate the scope associated with any particular aspect and/or embodiment. Accordingly, the following summary has the sole purpose to present certain concepts relating to one or more aspects and/or embodiments relating to the mechanisms disclosed herein in a simplified form to precede the detailed description presented below.

[0012] In an embodiment, a synthetic protein molecule built on a scaffolding framework mimicking the conformation of a naturally occurring monomer protein is disclosed. The naturally occurring monomer protein may, for example, be inclined to aggregate to form toxic oligomers. The synthetic protein molecule includes one or more side chains for enhancing the affinity of the synthetic protein molecule to an oligomer formed by the naturally occurring monomer protein such that the synthetic protein molecule inhibits aggregation of a plurality of oligomers. Further, in mimicking the conformation of the naturally occurring monomer protein, the synthetic protein molecule efficiently crosses a blood-brain barrier in a treatment subject, such as a patient.

[0013] In another embodiment, a method for producing a synthetic protein molecule is disclosed. The method includes selecting a naturally occurring monomer protein, the naturally occurring monomer protein having a tendency to aggregate with another naturally occurring monomer protein to form an oligomer. The method further includes selecting a scaffolding framework that mimics the conformation of the naturally occurring monomer protein, and selecting one or more side chains. The method further includes synthesizing the synthetic protein molecule, wherein selecting the one or more side chains includes selecting the one or more side chains for enhancing the affinity of the synthetic protein molecule so produced to the oligomer.

[0014] In a further embodiment, a synthetic protein molecule includes a scaffolding framework and at least one ligand attached to the scaffolding framework. The scaffolding framework is selected for mimicking a conformation of a naturally occurring monomer protein. A plurality of the naturally occurring monomer protein so selected exhibit a tendency for aggregation thereof to form a plurality of oligomers. The at least one ligand is selected for enhancing an affinity of the synthetic protein molecule to the plurality of oligomers so as to inhibit aggregation of the plurality of oligomers.

[0015] In embodiments, the synthetic protein molecule is a foldamer.

[0016] In embodiments, the scaffolding framework is selected for mimicking at least one of helicity , molecule composition, length, curvature, spirality, and compatibility with at least one sidechains to be selected as the at least one ligand.

[0017] In certain embodiments, the scaffolding framework is a foldamer-based scaffolding. In embodiments, the scaffolding framework includes an oligoquinoline scaffolding. In embodiments, the oligoquinoline scaffolding is a foldamer. In embodiments, the oligoquinoline scaffolding includes at least one of SK-129 and SK-131. In certain embodiments, the naturally occurring monomer protein is at least one of an alpha-Synuclein monomer and amyloid beta 1-

[0018] In embodiments, a method for producing a synthetic protein molecule is disclosed. In certain embodiments, the synthetic protein molecule is a foldamer. The method includes selecting a naturally occurring monomer protein, the naturally occurring monomer protein exhibiting a tendency for aggregation to form a plurality of oligomers, selecting a scaffolding framework mimicking a conformation of the naturally occurring monomer protein, and attaching at least one ligand to the scaffolding framework to synthesize the synthetic protein molecule. Attaching the at least one ligand to the scaffolding framework includes selecting the at least one ligand to enhance an affinity of the synthetic protein molecule to the plurality of oligomers so as to inhibit aggregation of the plurality of oligomers.

[0019] In certain embodiments, selecting the scaffolding framework includes choosing the scaffolding framework based on at least one of helicity, molecule composition, length, curvature, spirality, and compatibility with at least one side chain selected as the at least one ligand.

[0020] In certain embodiments, selecting the scaffolding framework includes selecting an oligoquinoline scaffolding. In embodiments, the oligoquinoline scaffolding is a foldamer. In embodiments, selecting the oligoquinoline scaffolding includes choosing at least one of SK-129 and SK-131. In embodiments, selecting the naturally occurring monomer protein includes choosing at least one of an alpha-Synuclein monomer and amyloid beta 1-42.

[0021] These and other features, and characteristics of the present technology, as well as the methods of operation and functions of the related elements of structure and the combination of parts and economies of manufacture, will become more apparent upon consideration of the following description and the appended claims with reference to the accompanying drawings, all of which form a part of this specification, wherein like reference numerals designate corresponding parts in the various figures. It is to be expressly understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limits of the invention. As used in the specification and in the claims, the singular form of 'a', 'an', and 'the' include plural referents unless the context clearly dictates otherwise.

[0022] For simplicity and clarity of illustration, the drawing figures illustrate the general manner of construction, and descriptions and details of well-known features and techniques may be omitted to avoid unnecessarily obscuring the embodiments detailed herein. Additionally, elements in the drawing figures are not necessarily drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements to help improve understanding of the described embodiments. The same reference numerals in different figures denote the same elements.

[0023] The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments. In the following detailed description, references are made to the accompanying drawings that form a part hereof, and in which are shown by way of illustrations or specific examples. These aspects may be combined, other aspects may be utilized, and structural changes may be made without departing from the present disclosure. Example aspects may be practiced as methods, systems, or apparatuses. The following detailed description is therefore not to be taken in a limiting sense, and the scope of the present disclosure is defined by the appended claims and their equivalents.

BRI EF DESCRI PTION OF DRAWINGS

[0024] FIGS. 1A illustrates a physiological model of neurons, including an inset showing an expanded view of a portion of an adjacent neurotransmitter and receptor pair.

[0025] FIG. IB illustrates a pathophysiological model of the blockage of the release of dopamine between an adjacent neurotransmitter and receptor pair as a result of aS aggregation.

[0026] FIG. 2 shows an exemplary model of protein aggregation.

[0027] FIG. 3 shows an exemplary illustration of an aS oligomer associated with PD, including an inset showing further detail of a portion of the aS oligomer as compared with a synthetic protein mimetic, in accordance with an embodiment.

[0028] FIG. 4 shows an isolated view of the chemical structure of a portion of a variant of SK- 129, in accordance with an embodiment.

[0029] FIG. 5A shows a top view of an oligoquinoline (OQ) scaffolding framework or structure, in accordance with the embodiment.

[0030] FIG. 5B shows a side view of the OQ scaffolding structure, illustrating the helical nature of the OQ scaffolding, in accordance with an embodiment.

[0031] FIG. 6, including insets, illustrates the physiological mechanisms involved in the transmission of dopamine between neurons in a healthy brain and in a brain affected by Parkinson's Disease and the rescue of the physiological role of neurons by introduction of the synthetic foldamer, in accordance with an embodiment.

[0032] FIG. 7 shows a detailed view of an OQ structure suitable for use as the basis of a scaffolding structure, in accordance with certain embodiments.

[0033] FIG. 8 shows exemplary options for side chains attachable to the scaffolding structure of FIG. 7, in accordance with certain embodiments.

[0034] FIG. 9 shows an illustration of possible binding sites of SK129, suitable for use with certain embodiments.

[0035] FIG. 10 shows a fluorescent analog of SK-129, suitable for use with certain embodiments.

[0036] FIG. 11 shows a process for using a synthetic protein molecule, in accordance with embodiments.

[0037] FIG. 12 shows a graph illustrating the results of Thioflavin T aggregation assays, in accordance with embodiments.

[0038] FIG. 13A shows a graph illustrating the in vivo pharmacokinetics of SK-129 in the plasma of treated mice, in accordance with embodiments.

[0039] FIG. 13B shows a graph illustrating the in vivo pharmacokinetics of SK-129 in the brain tissue of treated mice, in accordance with embodiments.

[0040] FIG. 14 illustrates a synthesis approach for monomer precursors with various side chains, in accordance with embodiments.

[0041] FIG. 15 illustrates a synthesis approach for SK-129 analogs, in accordance with embodiments.

[0042] FIG. 16 shows a bar graph illustrating the fluorescence intensity measurements for treated and untreated GMC101 C. elegans worms, in accordance with an embodiment.

[0043] FIG. 17 shows a bar graph illustrating the motility rate of N2 and GMC101 C. elegans worms with and without 50 pM SK-131.

[0044] FIG. 18 shows a graph illustrating the intracellular reactive oxygen species (ROS) levels, demonstrating the rescuing effect of SK-131 on aggregation-induced ROS elevation, in certain embodiments.

[0045] FIG. 19 shows a graph of the biodistribution of SK-131 in plasma through 72 hours post administration, in certain embodiments. [0046] FIG. 20 shows a graph of the biodistribution of SK-131 in the brain tissue through 72 hours post administration, in certain embodiments.

[0047] FIG. 21 shows the absorption of SK-131 in the human liver over time, in certain embodiments.

[0048] FIG. 22 shows the difference in the area beneath the curve in FIG. 21 for the measured absorption in the human liver versus a control sample, in certain embodiments.

[0049] For simplicity and clarity of illustration, the drawing figures illustrate the general manner of construction, and descriptions and details of well-known features and techniques may be omitted to avoid unnecessarily obscuring the embodiments detailed herein. Additionally, elements in the drawing figures are not necessarily drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements to help improve understanding of the described embodiments. The same reference numerals in different figures denote the same elements.

[0050] The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments. In the following detailed description, references are made to the accompanying drawings that form a part hereof, and in which are shown by way of illustrations or specific examples. These aspects may be combined, other aspects may be utilized, and structural changes may be made without departing from the present disclosure. Example aspects may be practiced as methods, systems, or apparatuses. The following detailed description is therefore not to be taken in a limiting sense, and the scope of the present disclosure is defined by the appended claims and their equivalents.

DETAI LED DESCRIPTION OF TH E I NVENTION

[0051] It would be desirable to identify and synthesize molecules that inhibit the formation of amyloid fiber structures by, for example, specifically binding to intermediate toxic oligomer structures of aS. Further, it would be desirable to produce such molecules that may be used in therapeutic approaches to deliver these therapeutic molecules through the blood-brain barrier.

[0052] It is recognized herein that synthetic molecules that mimic the molecular features of naturally occurring molecules may be able to exhibit, in embodiments, higher affinity to specific target molecules that are similar in shape to those synthetic molecules. As a specific example, the aS oligomers associated with PD are by nature helical, as illustrated in the left-hand side of FIG. 3. These aS oligomers are considered to be highly neurotoxic structures and considered to be the main causal agents for the PD. It is further recognized herein that, in general, a helical ligand with the appropriate side-chains may attach itself to the aS structure so as to inhibit the formation of amyloid fibers. Additionally, in mimicking the helical nature of naturally occurring aS oligomers, which are neurotoxic, such helical structures are uniquely suited for being deliverable through the blood-brain barrier to the cells of the central nervous system. An inset in FIG. 3 show further detail of a portion of the aS oligomer as compared with an oligopyridylamides scaffolding, which is a synthetic protein mimetic.

[0053] Certain specific molecules based on foldamer scaffolding, such as SK-129 based on an OQ scaffolding framework or structure have been previously studied for their potential effects on disrupting self-assembly of aS oligomers into non-functional amyloid structures (see, for example, Ahmed, et aL, "Foldamers reveal and validate therapeutic targets associated with toxic a-synuclein self-assembly," Nature Communications, vol. 13, article number 2272 (2022)). FIG.

4 shows an isolated view of the chemical structure of a portion of a variant of SK-129 shown on the right side of FIG. 3. Large arrow heads indicate locations on the OQ scaffolding structure at which selected side chains may be attached to tune the properties of the resulting ligand. In a model illustration, FIGS. 5A and 5B, showing a top view and a side view of the OQ scaffolding framework or structure, illustrating the helical nature of the OQ scaffolding.

[0054] FIG. 6 illustrates the physiological mechanisms involved in the transmission of dopamine between neurons in a healthy brain and in a brain affected by Parkinson's Disease, as well as the rescue of the physiological role of neurons by introduction of the synthetic foldamer, in accordance with an embodiment. As shown in the upper left inset, in a healthy brain, dopamine is readily transmitted between neuron endings to promote healthy dopaminergic neurons within the substantia nigra portion of the brain. However, in a brain affected by Parkinson's disease, as illustrated in the upper right inset of FIG. 6, the self-assembly of aS oligomers into non-functional amyloid structures prevents the transfer of dopamine, thus leading to loss of dopaminergic neurons and, consequently, progression of PD.

[0055] Referring to the bottom inset of FIG. 6, to inhibit the self-assembly of aS oligomers, a synthetic protein with a high affinity for aS oligomers may be introduced, thus resulting in the rescue of dopaminergic neurons. In other words, the introduction of the foldamers of the present disclosure inhibits aggregation of the synuclein protein, thus rescuing the physiological role of the neurons. Further, the synthetic protein may be selected to mimic the conformation of the naturally occurring aS monomer to facilitate delivery of the synthetic protein through the blood-brain barrier and to the desired location of the brain. For instance, the synthetic protein illustrated in the bottom inset of FIG. 6 is the first reported foldamer with the ability to cross the BBB with high efficiency.

[0056] Further details of an OQ structure are shown in FIG. 7 , with a unit of OQ enclosed in brackets. The OQ structure may be used as scaffolding for the synthesis of foldamers with specific surface functionalities. Some exemplary options for side chains attachable to the scaffolding structure of FIG. 7 are illustrated in FIG. 8. Synthesis of molecules with selected scaffolding and side chain structures may be performed using known protein synthesis protocols, such as described below.

[0057] It is recognized herein that, more broadly, synthetic molecules that mimic the structure of certain proteins provide advantages, for instance, in increasing the affinity of such synthetic molecules to the target molecules so mimicked. For example, molecules that mimic the helical structure of certain proteins may be particularly suitable for therapeutic uses in inhibiting the self-assembly of proteins into non-functional amyloid structures as well as for delivery to the target therapeutic locations through the blood-brain barrier. Other features of target molecules that may be mimicked include, and are not limited to, molecule composition, length, curvature or spirality, and size and composition of the sidechains. For instance, the chemical diversity of the side chains of the selected scaffolding may be synthetically tuned for the optimization of their interactions with protein targets of interest. The scaffolding/backbone selected as a basis of the synthesized molecule may be optimized, for example, for antagonist activity toward target molecules.

[0058] As a specific example, the recognition that the helical structure of the alpha-Synuclein oligomers make them exhibit high affinity to synthetic foldamers, such as those based on OQ scaffolding may be valuable in selecting features of synthetic molecules for treating conditions associated with aS aggregation. That is, the helical nature of both alpha-Synuclein oligomers and the foldamer scaffolding is such that, with the appropriate sidechains selected and attached to the scaffolding, the resulting foldamer may be used as targeted therapeutics for crossing the blood brain barrier, thus preventing unwanted aggregation of amyloid proteins and slowing the progression of neurodegenerative diseases such as PD.

[0059] The OQ scaffold approach can be applied to numerous disease models including Parkinson's disease, Alzheimer's disease, Lewy body dementia, multiple system atrophy, and diabetes, which are known to be caused by the aggregation of Synuclein proteins. These foldamer scaffolds are synthetically tunable meaning they can be engineered to specifically target a large variety of diseases that stem from aberrant protein-protein interactions. Further, the selection of the appropriate scaffolding structures and side chains enable targeting other proteins known to be associated with other diseases.

[0060] As an example, a selected foldamer (such as SK-129) based on the OQ scaffold may be specifically designed to interact with toxic aS oligomers to inhibit aS aggregation and the formation of aS fibers. For instance, whereas aS monomers occur naturally as native conformation in humans, it has been demonstrated in model studies that SK-129 binds with a ten-fold higher affinity to pathological high molecular weight aS oligomers than aS monomers. In this way, a foldamer such as SK-129 may inhibit intracellular aS aggregation to promote rescue of degeneration of midbrain dopaminergic neurons, motility recovery, and improved behavioral deficits. Further, by selecting synthetic proteins such as SK-129 and variants that mimic the helical structure of aS monomers, such proteins may be used to as therapeutics that efficiently cross the blood-brain barrier. Additionally, by swapping out the side chains to form analogs of the foldamer, the interaction between the synthesized molecules with target molecules may be further tuned for therapeutic purposes.

[0061] More specifically, OQ scaffolding may be synthesized in a stepwise process via amide coupling of functionalized quinoline monomers to elongate the backbone. The OQ backbone presents chemically diverse sidechains capable of specific chemical interactions with mutant aS. For instance, SK129 presents carboxylic acid and isopropyl functional groups that interact with high specificity for the N-terminal lysine and hydrophobic amino acid residues of aS. Since these OQs are constructed through a series of amide coupling reactions, the foldamer scaffold can be synthetically tuned to increase its interaction with mutant aS to optimize the antagonist activity of OQs against aS aggregation. Various side chain compositions may be selected to synthesize analogs of the synthesized proteins to tune the interaction. For example, the use sulfonate and phosphonate sidechains in place of the carboxylic acid moiety and/or hydrophobic analogs of the isopropyl group will change the behavior of the synthesized molecules with respect to the target aS oligomers. In an example, OQs may be synthesized from a pool of functionalized monomers to enable rapid generations of analogs of SK129 to optimize its antagonist activity against a specific phenomenon, such as aS aggregation. [0062] For instance, an OQ based library may be used to identify a potential candidate. In the present example, SK-129 may be selected as a potent antagonist of aS aggregation as it is known to specifically bind to aS oligomers (pathological conformation) with 10-fold higher affinity than aS monomers (native conformation) in cellular, neuronal, and C. elegans models and efficiently crosses the BBB without demonstrating any toxicity in a mouse model. Further, the OQ scaffolding structure is then synthetically tuned to further enhance their antagonist activity against aS aggregation and PD phenotypes.

[0063] Further, other scaffold frameworks or structures, such as oligopyridylamides, may be tailored to target other proteins. That is, the scaffold framework may be selected for compatibility with side chains or ligands that are attachable to the scaffold framework to enhance the affinity of the resulting molecule to the target protein, such as the selected toxic oligomer. In addition to the helical nature of the scaffold framework, the selection of the specific scaffolding may be predicated on helicity, molecule composition, length, curvature, spirality, and compatibility with side chains or ligands, including compatibility with ligands with helical nature. Whereas SK-129 may present greater tunability due to the greater number and variety of binding sites (see FIG. 9), NS-163 may still be used for therapeutic purposes in specific contexts. In other words, oligopyridylamides may be an appropriate scaffolding approach for certain types of synthetic protein treatments. The oligoquinoline (foldamers) are one example of ligands that target the synuclein oligomers, and oligopyridylamides is another example of synthetic protein mimetic ligands that target the synuclein oligomers. These and other scaffolds that target the synuclein oligomers to inhibit aggregation and rescue toxicity, using the component selection and protein synthesis approach described herein.

[0064] Similarly, an OQ-based molecule may be enhanced with a fluorescent probe. For example, SK-129 may also be used as a probe to identify high affinity ligands for aS oligomers and potent antagonists of aS aggregation from other existing libraries of ligands. For instance, a fluorescent analog of SK-129 (SK-129F as shown in FIG. 10) may be used as a probe that binds with high affinity to aS oligomers. In examples, a high-throughput fluorescence polarization displacement assay may be implemented to screen libraries of ligands against the SK-129-F- aS oligomers complex. The high affinity ligands for aS oligomers may be identified by detecting changes in the fluorescence polarization.

[0065] In embodiments, a method for using a synthetic protein molecule may include a process as illustrated in FIG. 11. As shown in FIG. 11, a process 1100 begins with a start step 1102 and proceeds to a step 1112 to select specific features of a target molecule (such as aS monomers) to be mimicked by the synthetic protein molecule. A scaffolding structure with characteristics similar to the selected specific features of the target molecule is selected in a step 1114. For instance, if the target molecule exhibits a helical structure, then a scaffolding structure also exhibiting a helical structure may be selected in step 1114.

[0066] Process 1100 then proceeds to a step 1116 to select specific side-chains to enhance the affinity of the synthesized molecule to the target molecule. The synthetic molecule, based on the selected scaffolding and side-chains, is produced in a step 1118. Optionally, the synthetic molecule so produced may be administered to a patient to react with the target molecule, and the process terminates in an end step 1130.

[0067] Example - Parkinson's Disease (PD)

[0068] As described above with respect to FIG. 1A, Alpha-Synuclein (aS) is a neuronal protein expressed at high levels in dopaminergic (DA) neurons in the brain and it is implicated in the regulation of synaptic vesicle trafficking, recycling, and neurotransmitter release. As illustrated in FIG. IB, the process of aS aggregation impairs the function of DA neurons, a key pathophysiological event in Parkinson's Disease (PD), which proceeds through the formation of pathological aS oligomers and aS fibers (aggregates). The pathological events in PD include aS aggregation and the spread of pathogenic aS fibers from neuron-to-neuron (via prion-like spread) and template the soluble aS into insoluble aS fibers.

[0069] It is recognized herein that modulation of the aggregation and the prion-like spread of aS is a promising potential therapeutic intervention for PD. In particular, the usefulness of OQ scaffold based foldamers, which can specifically interact with toxic aS oligomers, is recognized herein as a potent approach to inhibit aS aggregation and aS fibers templated aggregation, and the approach is validated in cellular, neuronal, and C. elegans PD models.

[0070] As discussed above, OQs are unique foldamers with the ability to present chemically diverse side chains, which mimic the side chains of the secondary structure of proteins. OQs have also been shown to inhibit the aggregation of various amyloid proteins. Additionally, the chemical diversity of the side chains of OQs can be synthetically tuned for the optimization of their interactions with protein targets.

[0071] The present disclosure demonstrates the ability of synthetically tuned OQs, which has been optimized for antagonist activity on aS aggregation in PD phenotypes, to rescue aS aggregation-mediated PD phenotypes, such as in PD mouse models. [0072] In an exemplary embodiment, efficacy of OQs in rescuing PD phenotypes is demonstrated in a PD mouse model. Further, SK-129 analogs are synthesized to demonstrate the potential to optimize their antagonist activity against aS aggregation mediated PD phenotypes.

[0073] In other words, it is demonstrated herein that specific targeting of toxic aS oligomers can lead to a potent rescue of PD phenotypes and slow the progression of PD. While SK-129 has been selected from a library of OQs for the demonstrations described herein, other oligomers may be selected and appropriately synthesized with ligands optimized for specific diseases, such as PD.

[0074] In embodiments, OQs are demonstrated herein to rescue PD phenotypes in an in vivo PD models. From an OQ library, SK-129 was selected as a potent antagonist (SK-129) of aS aggregation. It was found that SK-129 binds with a 10-fold higher affinity to pathological high molecular weight (HMW) aS oligomers than aS monomers (native conformation). In fact, it is demonstrated herein that SK-129 rescues PD phenotypes in various cellular, neuronal, and C. elegans PD models, including inhibition of intracellular aS aggregation, the rescue of degeneration of DA neurons, motility recovery, and improved behavioral deficits. Significantly, experimental preliminary data shows that SK-129 efficiently crosses the blood-brain barrier (BBB).

[0075] SK-129 was tested in a well-established aS fibers-treated M83 PD mouse model, referred to as [M83(F)]. The M83(F) model presents a significant acceleration of PD phenotypes and this model is known to resemble clinical symptoms of PD. The immunohistochemistry (IHC) images of various brain regions in the M83 model showed Lewy body (LB)-like inclusions (a pathological biomarker for PD), aS aggregates, significant weight loss, and less than 20% survival after 270 days as previously reported.

[0076] In an initial study, the M83(F) model was treated with SK-129 (IV, 20 mg/kg, 10 doses/every two days). SK-129 completely rescued PD phenotypes in the M83(F) model at this dose as no Lewy bodies, aS aggregates, nor weight loss was observed, and the survival rate was 100%. Further refinement of the treatment protocol, such as by a dose study (e.g., 2 mg, 5 mg, 10 mg/kg) may be implemented to identify the minimum dose of SK-129 required to completely rescue PD phenotypes in the M83(F) model. Additional assessment of PD phenotypes, including survival rate, LBs in various brain regions, dopaminergic (DA) neuron health in the substantia nigra (SN), motor function using the rotarod test, and weight loss in M83(F) mice dosed with SK- 129 compared to non-treated mice, may be implemented to refine the treatment protocol for optimal outcomes.

[0077] The initial study involved the screening of an OQ-based library against 100 pM aS aggregation using a Thioflavin T (ThT) aggregation assay, which quantifies the amount of aS fibers. The screening resulted in the identification of SK-129, which was verified to suppress aS aggregation even at a sub-stoichiometric ratio (see FIG. 12). As visible in FIG. 12, the aggregation of the synuclein protein and its inhibition is confirmed by the ThT aggregation assay. The inhibition of 100 pM aS aggregation by SK-129 (100 pM) was also confirmed by transmission electron microscopy (TEM), which demonstrated a sample having an abundance of aS fibers was reduced to having essentially no fibers after the application of SK-129. SK-129 also demonstrated good cell permeability, as confirmed by a parallel artificial membrane permeation assay (PAMPA) and confocal imaging.

[0078] Using fluorescence polarization (FP) titration between the fluorescent analog of SK-129 (SK-129F) and aS monomer and aS oligomers (HMW), it was determined that the Kd values for SK-129F against aS monomers, aS oligomers, and aS fibers were 800 ± 60 nM, 60 + 5 nM, and 280 ± 21 nM, respectively. I other words, SK-129 interacts with approximately 10-fold (aS oligomers) and approximately 5-fold (aS fibers) higher affinities (to pathological conformation) than aS monomers (native conformation). Thus, SK-129 likely inhibits aS aggregation without interfering with the native function of aS.

[0079] The neuroprotective effect of SK-129 on aS aggregation-mediated degeneration of DA neurons in a well-established C. elegans PD model (UA196) was also assessed. The UA196 worms are known to express both human aS and green fluorescent protein (GFP) in six DA neurons and aggregation of aS leads to progressive degeneration of DA neurons during aging.

[0080] The DA neurons in UA196 worms degenerated and decreased from 6 (day 3) to 1 neuron (day 15), represented by a gradual decline in GFP fluorescence in DA neurons in experiment. In contrast, there was no degeneration of DA neurons in UA 196 worms that were treated with 10 pM SK-129 on day 2. This result is significant as other reported ligands do not exhibit such neuroprotective effects even at 1 mM dose (see, for example, Garcia-Moreno JC, et al., "Tyrosol, a simple phenol from EVOO, targets multiple pathogenic mechanisms of neurodegeneration in a C. elegans model of Parkinson's disease," Neurobiol Aging, 2019; 82:60- 68). [0081] The degeneration of DA neurons has been directly linked with the slow motility rate of UA196, as characterized using the WMicroTracker® ARENA plate reader of Phylum Tech. There was a significant decline in the motility rate of UA196 worms over the course of the experiment, in comparison to control worms. The motility rate of UA196 worms treated with 10 pM SK-129 (day two) was significantly improved during the aging process, likely due to the rescue of DA neurons by SK-129.

[0082] Initial studies to confirm the ability of SK-129 to cross the BBB was conducted using a mouse model. The mice were housed under environmentally controlled standard conditions with a 12 hour light/dark cycle and free access to food and water. Six to eight-week-old control mice (C3H mice, male and female) were used to evaluate long-term blood levels of SK-129. A single dose of SK-129 (IV, 20 mg/kg) was administered and the mice were humanely euthanized by CO₂ inhalation at the end of each evaluation time point (six time points over 0 to 72 hours). Whole blood was collected via heart puncture. Treatment with this dose of SK-129 was used to compare the efficacy of SK-129 with other reported ligands at the same dose. Collected plasma was processed by centrifugation at 3000xg for 15 minutes at 4°C. Brain samples were also collected at multiple time points (over 0 to 72 hours) then homogenized in a saline buffer (lx Phosphate-buffered saline (PBS)). SK-129 was extracted from both plasma and brain homogenates and characterized using liquid chromatography with tandem mass spectrometry (LC-MS/MS).

[0083] FIGS. 13A and 13B show in vivo pharmacokinetics of SK-129, with a dose of 20 mg/kg using intravenous injection. Specifically, the concentration of SK-129 (dose = 20 mg/kg) in plasma (FIG. 13A) and in brain tissue (FIG. 13B) of mice (n=6 per point) are shown. The inset table in FIG. 13B summarizes the in vitro physicochemical properties of SK-129 as calculated with StarDrop small molecule discovery software available from Optibrium.

[0084] SK-129 demonstrated rapid absorption (T1/2 = 1.78 hours) with a maximum concentration reaching approximately 140 pg/mL (FIG. 13A). This value corresponds to approximately 10 times higher concentration of SK-129 in the blood plasma in comparison to other potential drugs for PD. In other words, SK-129 crosses the BBB with a maximum concentration of 10 pg/mL, which is greater than 10-fold higher concentration than other potential drugs for PD21 (FIG. 13B). Moreover, SK-129 was detectable in the brain tissue for more than 70 hours post-injection, suggesting a high efficacy of SK-129 due to longer time in the brain tissue. Additionally, the in vitro physicochemical properties of SK-129 (FIG. 13B inset) are comparable to ligands, which are known to efficiently cross the BBB.

[0085] Further, the M83 mouse model was used to study the effects of SK-129 on various PD phenotypes. The M83 transgenic mice [male and female, (B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J) from the Jackson Laboratory] express the mutant A53T aS. PD neuropathology can be accelerated in M83 mice with injected pre-formed aS fibers. Previous studies have shown that aS fibers decrease the survival time (median approximately 182 days) in comparison to the natural survival time (median approximately 359 days) of M83 mice. As most of the current therapeutic strategies for PD rely on post-diagnosis PD symptoms, aS fibers treated M83 PD mouse model (referred to as M83(F)) is considered to closely resemble a post-diagnosed PD mouse model.

[0086] In the study, 8-week-old M83 mice were anesthetized and stereotactically injected (SN, coordinates: AP -3-4, ML ± 1-4, DH -4-0) with 100 pM aS fibers (200 n L). Two weeks following aS fibers injection, the mice were randomly divided into two groups. One group was treated with vehicle (IV, 100 pL/mice, saline buffer+2% dimethyl sulfoxide (DMSO)) and the second group was treated with SK-129 (IV, 20 mg/kg, 100 pL/mice, saline buffer+2% DMSO) for 21 days (every second day). The body weight and survival of mice were monitored every 10 days until 270 days of age.

[0087] The median survival time for M83(F) was approximately 175 days and less than 20% of mice survived after 270 days. The untreated M83(F) generally displayed quadriparesis, arched back, significant weight loss, and were unable to stand up and support their body weight.

[0088] In contrast, the survival of M83(F) in the treated group was 100% in the presence of SK- 129 (20 mg/kg) even after 270 days. The SK-129-treated M83(F) appeared to be healthy (similar to the control mice) and did not display any signs of quadriparesis or arched back, and their weight was similar to the weight of mice in the control group.

[0089] Immunohistochemistry (IHC) was used to assess the effects of SK-129 on various brain regions of M83(F). Pathological deposits of aS-pS-129 and p62 proteins, which are key pathological hallmarks of PD, were detected in 270-day old untreated mice. The untreated M83(F) mice displayed deposits of both aS-pS-129 and p62 proteins throughout the motor cortex, striatum, hypothalamus, hippocampus, amygdala, and SN. However, in the presence of SK-129, no aggregates of aS-pS-129 and p62 proteins were observed in brain regions of the treated M83(F). The LB biomarkers were quantified by calculating the number of stained regions in a defined area and at least 10 regions were analyzed in each mouse. The collected data demonstrate that SK-129 is an effective ligand in rescuing PD phenotypes in the M83(F) model.

[0090] A Structure-Activity Relationship (SAR) study to enhance the efficacy of SK-129 against PD phenotypes

[0091] Carboxylic acid (COOH) and isopropyl side chains on SK-129 appear to be essential for its antagonist activity as they interact with the N-terminal lysine and hydrophobic amino acid side chains of aS, respectively. Further modification of the side chains of SK-129 may further optimize its antagonist activity against aS aggregation. As described above, FIGS. 7 and 8 show design and synthesis of proposed analogs of SK-129, including the generic chemical structure of SK-129 analogs (FIG. 7) with proposed side-chains (FIG. 8). While only selected ligands are shown in FIG. 8, additional side-chains are also contemplated and are considered a part of the present disclosure.

[0092] Synthesis of monomers

[0093] FIG. 14 illustrates the synthesis of monomer precursors with various side chains, according to certain embodiments. Top path shows Ligand 1 placed in Hz gas, catalyst of Carbon with Palladium (Pd/C), and Methanol, and kept at room temperature for 12 hours. Second path shows Ligand 1 placed in lithium hydroxide (LiOH) and tetrahydrofuran (THF)/water, kept at room temperature for 6 hours. The monomer precursors with distinct side chains may be synthesized using known standard protocols. The monomer precursors with sulphonic (FIG. 14, Ligand 1) or phosphonic acids (FIG. 14, Ligand 4) may be synthesized using known protocols with a yield of approximately 70 to 80%. The monomer precursors will be either reduced (FIG. 14, Ligand 2,5) or saponified (FIG. 14, Ligand 3,6) to introduce the primary amine or COOH groups, respectively. The introduction of these functional groups is important to oligomerize the monomers via amide coupling. The monomer precursors with various hydrophobic side chains (FIG. 14, Ligand 7) may also be synthesized using standard protocols with a yield of approximately 70 to 80%. The monomer precursors may be either reduced (FIG. 14, Ligand 8) or saponified (FIG. 14, Ligand 9) to introduce the primary amine or COOH groups, respectively.

[0094] FIG. 15 illustrates the synthesis of SK-129 analogs, in accordance with certain embodiments. Two monomer precursors with sulphonic/phosphonic acid side chains (FIG. 15, Ligand 2/5 or 3/6) may be alternatively linked with two hydrophobic side chains (FIG. 15, Ligand 8,9) using iterative steps of reduction and the amide coupling (FIG. 15, Ligand 11,12,13) using standard protocols with a yield of approximately 50 to 70%. The linking of these side chains may allow the formation of SK-129 analogs (FIG. 15, Ligand 13).

[0095] Further experimental details

[0096] Materials and Methods

[0097] Materials: Acetone, acetonitrile, bovine serum albumin ( BSA), citric acid, dimethyl sulfoxide (DMSO), Dulbecco's phosphate buffered saline (PBS), ethanol, methanol, sodium citrate, sodium chloride (NaCI), sodium hydroxide (NaOH), xylene was all purchased from Sigma Aldrich (St. Louis, MO, USA). Hydrogen peroxide was obtained from Fisher Scientific (Loughborough, Leicestershire, UK). Mounting medium with 4',6-diamidino-2-phenylindole (DAPI) - Aqueous, Fluoroshield (abl04139), mouse specific HRP/DAB (ABC) detection IHC kit (ab64259) were obtained from Abeam (Trumpington, Cambridge, UK). Phospho-Tau (AT8), Wheat Germ Agglutinin (Alexa Fluor-488 conjugate, W11261 and Alexa Fluor-594 conjugate, W11262) was obtained from Thermo-Fisher Scientific (Waltham, Massachusetts, USA). Anti- pS129-a-syn/81a (825702) was obtained from Biolegend (San Diego, California, USA). Anti-p62 (P0067) was obtained from Millipore Sigma (Burlington, MA, USA).

[0098] aS protein purification

[0099] The aS protein was expressed and purified from the periplasm according to previously described protocol. Briefly, the aS sequence cloned into pETll vector (Addgene, Watertown, MA) was chemically transformed into Escherichia coli (E. coli) BL21(DE3) cells. Transformed cells were grown at 37 °C and shook at a rate of 200 rounds per minute (rpm) until the O.D. (optical density) reached a value of 0.8. Protein expression was induced by adding isopropyl 0-D- thiogalactoside (IPTG) at a final concentration of 1 mM. The induced cells were kept shaking at 200 rpm at 37 °C for 5 hours. Cells were then collected by centrifugation (8217xg at 4 °C for 10 minutes) and resuspended in an osmotic shock buffer (30 mM Tris pH 7.2, 30% sucrose, 2 mM Ethylenediaminetetraacetic acid (EDTA)) and stirred for 15 minutes. Subsequently, cells were collected again from the osmotic shock buffer by centrifugation (7177xg for 10 minutes at

4 °C) and reconstituted in cold Milli-Q water and stirred for another 10 minutes. A solution of

5 mM MgCI₂ was added and stirred cells for an additional 5 minutes. Cells were removed by centrifugation at 5635xg for 10 minutes and the solution was boiled at 95 °C for 15 minutes for further purification.

[0100] The resulting protein precipitate was centrifuged (6000xg for 20 minutes) and loaded on Bio-Scale Macro-Prep High Q. ion-exchange column (Bio-Rad, Hercules, CA) (20 mM Tris pH 8.0, 25 mM NaCI, 1 mM EDTA). The protein was eluted with a high salt buffer (20 mM Tris pH 8.0, 1 M NaCI, 1 mM EDTA). The purified protein was buffer exchanged and concentrated in Milli-Q. water using Amicon ultra 3 K filters (Millipore Sigma, Burlington, MA). The concentration was determined using NanoDrop One (e280 = 5960 M-l cm-1), lyophilized, and stored at -80°C.

[0101] Tau protein Purification

[0102] 2N4R tau was expressed and purified according to published protocols using BL21 (DE3)- competent Escherichia coli (E. coli) cells containing pET-28-2N4R plasmids were grown in LB media + 20 pg/mL kanamycin, and again grown under agitation at 37°C until OD600 reached 0.8. Protein expression was induced with 0.5 mM isopropyl p-D-l-thiogalactopyranoside (IPTG), and the cells were allowed to continue growing at 37°C for 3.5 hours before being pelleted and resuspended into buffer (500 mM NaCI, 20 mM PIPES pH = 7.5, 1 mM EDTA, and 50 mM 0Me). Cells were frozen overnight at -80°C, then heated to 80°C for 10 minutes. Cells were then sonicated on ice for 1 minute. Separation of protein from cellular debris was done by centrifugation for 30 minutes at 15000 xg. 60% ammonium sulfate was added to the supernatant and incubated on a shaker at room temperature overnight. Protein was sedimented out by centrifugation for 20 minutes at 20000 xg. Protein pellets were washed with deionized water (DI H₂O) twice before being resuspended into 50 mL DI H₂O, sonicated for 2 minutes, and syringe filtered before loading on to anion exchange column. A linear NaCI gradient was used to purify (50-100 mM NaCI, 20 mM PIPES pH = 7.5, 2 mM EDTA, 2 mM DL- Dithiothreitol (DTT)). Elutions were assessed and by SDS-PAGE and pooled. Protein was lyophilized, resuspended in DI H₂O, and dialyzed into DI H₂O before lyophilization and storage at -80°C. 0N3R tau was purchased from rPeptide and resuspended to a stock of 100 pM in 1 x PBS for use at working concentration (25 pM).

[0103] aS purification (for FLIM study)

[0104] WT aS and a single-cysteine variant (cysteine added at position 122, hereinafter aS-Cys) were cloned into a pT7-7 vector and expressed in BL21 (DE3)-competent Escherichia coli competent cells. Cultures were grown at 37 °C, 180 rpm until O.D. reached a value of 0.7. Protein expression was induced by adding IPTG at 1 mM for 10 hours at 28 °C. Cells were then harvested by centrifugation at 17.000xg and resuspended in lysis buffer (10 mM Tris, pH 7.7, 500 mM NaCI, 1 mM EDTA, 100 pM leupeptine and 50 pM benzamidine) and sonicated on ice for at least 5 pulses (1 minute on/1 minute off). The lysate was centrifuged (20,000xg for 30 minutes at 4 °C) and the supernatant was boiled to 95 °C for 20 minutes. DNA was precipitated by dissolving streptomycin sulphate (10 mg per mL of supernatant) and removed by centrifugation (20,000xg for 30 minutes at 4 °C). aS was precipitated by dissolving ammonium sulphate (361 mg per mL of supernatant) and after a centrifugation step (20,000xg for 30 minutes at 4 °C), the pellet was dissolved and dialyzed overnight in anion exchange chromatography buffer (25 mM Tris pH 7.7). The protein solution was then loaded into an anion exchange column (HiPrep Q XL, Cytiva, MA, USA) and fractions containing aS were pooled, concentrated and loaded into a size exclusion chromatography column (HiLoad 26/600 Superdex, Cytiva, MA, USA) equilibrated in PBS pH 7.4. The monomeric fractions of aS were pooled, concentrated to ca. 200-300 pM, and the aliquots were flash frozen in liquid nitrogen and stored at -80 °C. 5 mM DTT were added in all purification steps for the cysteine-containing aS variants.

[0105] Tau purification (for FLIM study)

[0106] WT 2N4R tau isoform, a truncated variant (A275-311) deficient in amyloid aggregation, AggDef-tau, and a single-cysteine tau variant (the natural cysteine residues in positions 291 and 332 mutated to alanine, and a single cysteine added at position 260, hereinafter tau-Cys), were cloned into a pET29b vector and expressed in BL21 (DE3)-competent Escherichia coli competent cells. Cultures were grown at 37 °C, 180 rpm until O.D. reached a value of 0.7. Protein expression was induced by adding IPTG at 1 mM for 3 hours at 37 °C. Cells were harvested by centrifugation at 17.000xg and resuspended in lysis buffer (20 mM HEPES pH 6.8, 500 mM NaCI, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM EDTA, 0.2 mM MgCI₂, 5 mM DTT, 100 pM leupeptine and 50 pM benzamidine), sonicated on ice for at least 5 pulses (1 minute on/1 minute off) and boiled to 95 °C for 20 minutes. Cellular debris were removed by ultracentrifugation (127,000xg for 40 minutes at 4 °C). The resulting supernatant was dialyzed overnight in cation exchange chromatography buffer (20 mM HEPES pH 6.8, 50 mM NaCI, 0.1 mM PMSF, 1 mM EDTA, 0.2 mM MgCI2, 2 mM DTT) and then loaded into a cation exchange column (HiTrap SPFF, Cytiva, MA, USA). Tau variants were then eluted by an increasing NaCI concentration, and the fractions containing tau were pooled and concentrated. Aliquots were then immediately flash frozen in liquid nitrogen and stored at -80 °C in 10 mM HEPES pH 7.4, 500 mM NaCI.

[0107] Protein labeling

[0108] aS-Cys and tau-Cys were fluorescently labeled by maleimide reactions with either AlexaFluor488 (AF488) or Atto647N, respectively. 100 pM of each protein were equilibrated in labeling buffer (25 mM Tris pH 7.1, 150 mM NaCI, 2 mM TCEP) and a molar excess of dye of 2.5x was slowly added. The dye stock concentrations were higher than 10 mM to avoid the addition of >2.5% DMSO to the labeling reaction. After an overnight incubation at 4 °C under mild shaking conditions, 2.5 mM DTT was added to the labeling reaction and the excess of dye was removed by a PD 10 desalting column (Cytiva Life Sciences, Uppsala, Sweden). Degree of labeling (>0.90%) was confirmed by absorbance.

[0109] Thioflavin T (ThT) aggregation assay

[0110] To study the aggregation of aS +/- tau (2N4R and 0N3R), the ThT dye at a final concentration of 50 pM was added to 70 pM aS and 25 pM tau solution in the aggregation buffer (1 x PBS buffer) +/- 70 pM SK-129 in a Costar black 96-well plate (Corning Inc., Kennebunk, ME). SK-129 were dissolved in dimethyl sulfoxide (DMSO; final DMSO concentration = 0.5%, v/v). The final volume in each well was 100 pL. The aggregation was determined by the final ThT fluorescence intensity after 96 hours for the aggregation of aS and aS-129 (with or without ligands) as an average of three separate experiments using an Infinite M200PRO plate reader (Tecan, Mannedorf, Switzerland).

[0111] In this method, protein (synuclein) was aggregated in the absence and presence of various ligands at the indicated molar ratios 0.1-1 equivalent. The experiments were conducted three separate times and the reported ThT intensity was an average of three separate experiments. The ThT intensity was reported as relative intensity where the highest and lowest intensity were used from the protein sample and the control (ThT, DMSO, and buffer conditions only), respectively. The concentration of DMSO was kept constant (0.5%, v/v) in protein (aS), control, and molecule solutions.

[0112] Tissue culture and microscopy

[0113] The Lipofectamine solution (Lipofectamine+P3000 reagent, Thermo Fisher Scientific, Waltham, MA) was diluted to a ratio of 1:20 (v/v) in the OptiMEM (Fisher Scientific, Pittsburgh, PA) media. Simultaneously, the aS +/- tau fiber solution (Stock solution cone. = 70/25 pM) was diluted in OptiMEM media to 5 pM with respect to the aS. The aS fiber solution was sonicated for 10 minutes at room temperature, followed by the addition of the Lipofectomine solution (in the OptiMEM media) at 1:1 ratio. Subsequently, this solution was incubated for another 10 minutes and then added to the Human Embryonic Kidney (HEK) cells media with a dilution factor of 10 (10 pL of the combined aS fiber solution + Lipofectomine solution, in 90 pL HEK cells media). [0114] The HEK cells expressing aSA53T-YFP or aS-YFP (400,000 cells/mL) were plated in a p- slide eight-well plate (Ibidi, Grafelfing, Germany) (250 pL/well) and incubated at 37 °C and 5%

CO2 (g) and allowed to adhere to the plate for 24 hours in complete media (Dulbecco's Modified Eagle Medium (DMEM), 10% FBS, 1% pen/strep). After 24 hours, the media was aspirated and 250 pL of OptiMEM containing aS +/- tau fibrils, 5 pM (with respect to the aS from aS/tau 70/25 pM stock) recombinant protein in the presence of Lipofectamine 3000, was added. The plate was incubated for 48 hours after the addition of fibers. The cells were treated with a Hoechst 33342 dye solution for 1 hour by adding 3 pL to each well (from 1 mg/ml solution in lx PBS buffer). The Hoechst 33342 solution was carefully mixed with media by gently rocking the plate. . The HEK cells were washed with the lx PBS buffer (twice) to remove excess traces of dyes and used for the live-cell confocal imaging. The confocal imaging was performed on an Olympus Fluoview FV3000 confocal/2-photon microscope, using a 40* 1.3 numerical aperture (NA) objective with Differential Interference Contrast (DIC) capability. The confocal images of the HEK cells were processed using the OlympusViewer in ImageJ processing software. Puncta were quantified by counting puncta and nuclei across 5 images and 70 cells.

[0115] Similar conditions were used to monitor the effect of aS + tau + 10 pM SK-129 on the aggregation of A53T-YFP aS in HEK cells. A solution of 70/25 pM aS/tau was aggregated for seven days in lx PBS buffer in the absence and presence of SK-129 at an equimolar ratio. The fibers of aS in the absence and presence of SK-129 in the presence of Lipofectamine 3000 were used in HEK cells.

[0116] MTT toxicity assay

[0117] The HEK293T cells that stably express aSA53T-YFP were grown in DMEM with 10% FBS and 1% pen/strep and cultured in an incubator at 37 °C and 5% CO2(g). A total of 60,000 cells per well in 100 pL media were plated in a sterile, clear 96-well plate and incubated for 24 hours to adhere to the plate. After 24 hours, the media was aspirated and 100 pL of OptiMEM (Fisher Scientific, Pittsburgh, PA) containing aS+/-tau fibrils, 5 pM of the recombinant protein, was added in the absence and presence of SK-129 and NS132 at an equimolar ratio in the presence of Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA). Next, the plate was incubated for 24 hours, followed by the addition of 100 pL of lx PBS buffer containing MTT dye (in lx PBS buffer, 5 mg/mL) to each well. The plates were wrapped in aluminum foil and incubated for 3 hours. After 3 hours, all liquid was aspirated carefully without disturbing the formazan crystals. To each well, 100 pL of DMSO was added to dissolve the crystals. The absorbance was read on a 96-well plate reader at 570 nm. The cytotoxicity of the HEK cells was taken 48 hours post transfection.

[0118] The seeds were generated by aggregating recombinant aS+tau (70/25 ptM) in the absence and presence of SK-129 at an equimolar ratio under the ThT aggregation kinetic conditions for four days.

[0119] C. elegans Experiments

[0120] Media and buffers for various worm assays

[0121] The worms were maintained at standard conditions (20 °C at all times) on nematode growth media (NGM) agar in 60 mm plates (CytoOne, Ocala, FL) using E. coli OP50 strain as the food source following the previous protocols. To ensure that worm's colonies don't starve, they were always transferred on new plates with E. coli OP50 strain as the food source. All worm strains were replaced with new worm colony after every six months to avoid any genetic mutation, which might affect the disease phenotypes. NGM agar plates, M9 buffer (3 g KH2PO4, 6 g NajHPC , 5 g NaCI, 1 mL 1 M MgSO«, milli-Q H2O to IL), Chemotaxis (CTX) media plates (2% Agar, 5 mM KHPO₄, 1 mM CaCI₂, and 1 mM MgSO₄), and CTX buffer (5 mM KH₂PO₄ 1 mM CaCI₂, and 1 mM MgSO₄) were prepared using previous protocols.

[0122] NGM agar plates are prepared from an autoclaved solution containing 15 g agar, 2.4 g NaCI, 2 g Tryptone, and 2.72 g KH₂PO₄ in 1 L of Milli Q water. After cooling to 60 °C in warm water, the following are added per liter: 0.8 ml of 1 M CaCI₂, 1 mL cholesterol (5 mg/ml in ethanol), and ImL 1 M MgSO₄, and (to prevent bacterial and fungal contamination) 1 mL streptomycin (100 mg/ml) and 1 mL nystatin (10 mg/ml). The 60 mm plates are then filled to 2/3 of their volume (10 mL) and left completely still to dry.

[0123] CTX media 6-well plates are prepared by adding 3 mL per well of the following: 2 g of agar in 100 mL of Milli Q water is autoclaved, before adding 1 M KH2PO4 (0.5 mL), IM CaCb (0.1 mL), and 1 MgSO₄ (0.1 mL) and the solution mixing by shaking.

[0124] Strains

[0125] The N2 (wild-type C. elegans Bristol strain)and Escherichia coli OP50 (E coli, a uracil requiring mutant) strains were obtained from Caenorhabditis Genomics Center (CGC, Minneapolis, MN). The UA196 strain was generously donated by the laboratory of Dr. Guy Caldwell (Department of Biological Science, University of Alabama, Tuscaloosa, AL, United States). The sex ratio for all strains is 99.5% female (or hermaphroditic), with the remaining 0.5% being facultative male. The age and number of worms used varies by experiment and are indicated in their respective methods sections.

[0126] UA196 strain [Pdat-l::a-syn+Pdat-l::GFP]. In UA196 strain, human aS and GFP are expressed in DA neurons under control of a dopamine transporter-specific promoter [Pdat-l::a- syn+Pdat-l::GFP], which results in age-dependent neurodegeneration of six DA neurons.

[0127] Culture methods for C. eleqans strains

[0128] The standard worm conditions were used for the culturing of various worm strains.

Briefly, the worms were bleached and synchronized using hypochlorite solution, followed by the incubation of the eggs (at 20 °C) on NGM plates (35 mm, CellTreat Scientific, Pepperell, MA), which were seeded with OP50 (350 pL, 0.5 OD600nm) as a food source, referred to as PLATE 1. The cultures of OP50 were prepared by incubating 50 mL of LB medium with OP50 18 hours at 37 °C and the final OD value was adjusted to 0.5 at 600 nm. The NGM plates were prepared by treating them with 350 pL OP50 and leaving the plates at 20 °C for 3 days. On day two, the worms were transferred (using M9 buffer) to NGM plates (35 mm, CellTreat Scientific, Pepperell, MA) containing 75 pM Fluorodeoxyuridine (FUDR; to prevent worm reproduction and ensure that equal ages of worms were used for the experiment)24,83 and OP50 as food source, referred to as PLATE 2. These NGM plates were prepared using autoclaved NGM media supplemented with 75 pM FUDR (2 mL total liquid). After 12 hours, the NGM plates were seeded with 350 pL of OP50 (with OD = 0.5 at 600 nm) at 20 °C. For treating various worm strains with ligands, the worms were transferred on day 2 to the NGM plates treated with ligands\.

[0129] Preparation of NGM plates with Ligands

[0130] The NGM plates (with NGM media) were used for the treatment of different worm strains with ligands. The NGM media containing 75 pM FUDR was autoclaved and poured in NGM plates (2 mL total liquid). After 12 hours, the NGM plates were seeded with 350 pL of OP50 (with OD = 0.5 at 600 nm) at 20 °C. After 12 hours, different doses of ligands (10-50 pM, stock cone. = 10 mM in DMSO, DMSO from 0.1-0.5%, v/v) were dissolved in M9 buffer (total volume = 300 pL) and were spotted atop the NGM plates at 20 °C, referred to as PLATE 3. The NGM plates treated with the ligands were placed in sterile laminar flow hood at 20 °C for 1 hour. The NGM plates treated with ligands were prepared and used within 24 hours. We used these plates to treat various disease strains for different assays.

[0131] Motility assay for C. elegans (N2and UA196) [0132] Briefly, all worm strains were bleached at the same time and the eggs were incubated on PLATE 1. On day two, the worms were divided into two batches. One half of the worms were transferred (using M9 buffer) on PLATE 2 (without ligand) and the second half of the worms were transferred to PLATE 3 (with ligand). The concentration of the ligands varied from 10-50 pM as described in the main manuscript. The worms were incubated on PLATE 2 or PLATE 3 up to day 4 at 20 °C in an incubator with constant humidity. On day four, various worm strains (with and without ligands from PLATE 2 or PLATE 3) were transferred to sterile 24 well plate (CellTreat Scientific, Pepperell, MA) containing liquid media (500 pL/well), referred to as PLATE 4A and containing liquid media with ligands (500 pL/well with 10-50 pM ligand in M9 buffer with 0.1%-0.5% DMSO, v/v), referred to as PLATE 4B. The liquid media for PLATE 4A/B was prepared with 67.28% (v/v) of M9 buffer, 75 pM FUDR, 0.1% of 1 M magnesium sulfate (v/v), 0.1% of 1 M calcium chloride (v/v), 2.5% of 1 M potassium phosphate solution (pH 6, v/v), and 30% (v/v) of OP50 (0.5 OD600nm). A total of 50 worms per well were manually transferred into PLATE 4A/B at 20 °C and a total of four wells (4 technical replicates) were used for each condition.

Subsequently, the worms were incubated for 6 hours at 20 °C with constant shaking (rpm = 100) to get acclimated with the solution conditions before starting the motility assay experiment.

[0133] To test the effect of each ligand on the disease worm strain (UA196), there were four conditions were used in PLATE 4A/B: (1) N2 worms, (2) N2 worms treated with ligand, (3) UA196 worms, and (4) UA196 worms treated with the ligand (10-50 pM). The assay was started on day four using the WMicroTracker ARENA plate reader (Phylumtech, Santa Fe, Argentina) at 20 °C for 1 hour per day over a 14-day period at intervals of 24 hours. The WMicroTracker ARENA plate reader uses a large array of infrared light microbeams in each well of the plate to detect the interference caused by the movement of the worms. The output by the ARENA plate reader is an average of the overall movement of all worms present in each well, which is denoted as the motility of the worms in each well. Each day, a total of 20 activity scores per well were collected in 1 hour. After collecting the data each day, the PLATE 4A/B was again placed on the shaker (100 rpm) at 20 °C in the incubator. The PLATE 4A/B was on the on the shaker (100 rpm) at 20 °C in the incubator the whole duration of the experiment except only when the reading was collected on the WMicroTracker ARENA plate reader for 1 hour. For each condition, four biological replicates were performed, and each biological replicate included four technical replicates (four wells). For each condition, the data were expressed as mean and the error bars report the standard error of the mean (s.e.m.) (n = 4 independent experiments and each n included a total of four technical replicates).

[0134] Motility assay for C. elegans (N 2 and UA196) in the presence of dopamine and SK-129

[0135] This motility assay was performed in a manner similar to the previous assay with slight adjustment in the preparation of the plates for the treatment of UA196 worms. For this assay, the only difference is the 24 well plates were prepared with 2 mM dopamine (referred to as PLATE 5) and both 2 mM dopamine and 50 pM SK-129 (referred to as PLATE 6). To prepare PLATE 5, the NGM media containing 75 pM FUDR was autoclaved and poured in NGM plates (2 mL total liquid). After 12 hours, the NGM plates were seeded with 350 pL of OP50 (with OD = 0.5 at 600 nm) at 20 °C. After 12 hours, 2 mM dopamine dissolved in M9 buffer (total volume = 300 pL) was spotted atop the NGM plates at 20 °C. To prepare PLATE 6, the NGM media containing 75 pM FUDR was autoclaved and poured in NGM plates (2 mL total liquid). After 12 hours, the NGM plates were seeded with 350 pL of OP50 (with OD = 0.5 at 600 nm) at 20 °C. After 12 hours, SK-129 (50 pM, stock cone. = 10 °mM in DMSO) and dopamine (2 mM in M9 buffer) were dissolved in M9 buffer (total volume = 300 pL) and were spotted atop the NGM plates at 20 °C. The NGM plates treated with dopamine and SK-129 and dopamine were placed in sterile laminar flow hood at 20 °C for 1 hour. The NGM plates treated with dopamine and SK- 129 and dopamine were prepared and used within 24 hours.

[0136] Similar to the previous section, the motility assay was performed for N2 and UA196 worms in the absence and presence of SK-129+dopamine in sterile 24 well plate (CellTreat Scientific, Pepperell, MA) containing liquid media (500 pL/well), liquid media + 2 mM dopamine, and liquid media + 2 mM dopamine + 50 pM SK-129. There were six conditions for this experiment: (1) N2 worms, (2) N2 worms treated with 2 mM dopamine, (3) UA196 worms, (4) UA196 worms treated with 2 mM dopamine, (5) UA196 worms treated with 50 pM SK-129, and (6) UA196 worms treated with 2 mM dopamine and 50 pM SK-129. For each condition, four biological replicates were performed, and each biological replicate included four technical replicates (four wells). For each condition, the data were expressed as mean and the error bars report the s.e.m. (n = 4 independent experiments and each n included a total of four technical replicates).

[0137] Confocal microscopy imaging of early stage treated C. elegans (UA196) with ligands

[0138] This experiment was performed based on previously described protocols with slight modifications. Briefly, the worm strain (UA196) were bleached at the same time and the eggs were incubated on PLATE 1. On day two, the worms were divided into two batches. One half of the worms were transferred (using M9 buffer) on PLATE 2 (without ligand) and the second half of the worms were transferred to PLATE 3 (with ligand). The concentration of the ligands varied from 10-50 pM as described in the main manuscript. The worms were incubated on PLATE 2 or PLATE 3 up to day 3 at 20 °C in an incubator with constant humidity. On day four, the ligand (10- 50 pM, stock cone. = 10 mM in DMSO, DMSO from 0.1-0.5%, v/v) was added again to PLATE 3. For confocal imaging, at least 10 worms per condition (from PLATE 2 or PLATE 3) were transferred to a cover slide containing an anesthetic (40 mM sodium azide) and mounted on a glass microscope slide containing 2% agarose pads using a reported protocol. The images of the worms were collected using an Olympus Fluoview FV3000 confocal/2-photon microscope (40 x Plan-Apo/1.3 NA objective with DIC capability) and processed using the OlympusViewer in ImageJ software. For UA196 strain, the number of healthy DA neurons (fluorescence due to GFP in DA neurons) were counted (10 worms per condition) using confocal imaging of the worms on day 3, day 5, day 10, and day 15. For each condition, six biological replicates were performed and at least 10 technical replicates were used for each biological replicate. The data were expressed as mean and the error bars report the s.e.m. (n = 6 independent experiments and each n included a minimum of ten technical replicates).

[0139] Chemotaxis Assay for C. elegans (N2 and UA196) in the presence of ligands

[0140] For chemotaxis assay, the Chemotaxis (CTX) media plates (2% Agar, 5 mM KH₂PO₄, 1 mM CaCI₂, 1 mM MgSO₄, 75pM FUDR), referred to as PLATE 7, and CTX buffer (5 mM KH₂PO₄, 1 mM CaCI₂, and 1 mM MgSO₄) were prepared using previous protocols. The PLATE 7 (without ligand) were treated with ligand (50 pM, stock cone. = 10 mM in DMSO) that was dissolved in CTX buffer (total volume = 300 pL) and was spotted atop the CTX media plates at 20 °C, referred to as PLATE 8.

[0141] For CTX assay, we prepared another plate referred to as PLATE 9, which was PLATE 7 divided into four equal quadrants and designated as A and C (diagonally opposite), B and D (diagonally opposite). A solution of E coli (50 pL of 0.5 OD at 600 nm, as an attractant) was placed at approximately 0.4 cm from the edge of quadrants B and D and the plate was allowed to dry for 1 hour at 20 °C. Subsequently, ethanol (10 pL, repellant) was added at approximately 0.4 cm from the edge of quadrants A and C at 20 °C. To ensure that the ethanol did not dry, the worms (N2 or UA196) were transferred to this plate within 10 minutes and the lid of the plate was closed. [0142] The experiment on the WMicroTracker ARENA plate reader (Phylumtech, Santa Fe, Argentina) started within 1 hour of the transfer of the worms at 20 °C. To carry out the experiment, the worms (N2 or UA196) were bleached at the same time and the eggs were incubated on PLATE 1.

[0143] On day two, the worms were divided into two batches. One half of the worms were transferred (using CTX buffer) on PLATE 7 (without ligand) and the second half of the worms were transferred to PLATE 8 (with 50 pM ligand) and kept at 20 °C in an incubator.

[0144] On day 3, a total of 50 worms were transferred to the center of the PLATE 9 using CTX buffer (approximately 20 pL) and the number of worms were counted under an Olympus microscope (SZ-6145, Waltham, MA). Subsequently, the lid of the plate was closed with parafilm (Bemis Company, Inc., Neenah, Wl). The worm activity was monitored using the WMicroTracker ARENA plate reader (Phylumtech, Santa Fe, Argentina) at 20 °C for 2 hours.

[0145] We used the same conditions for day 10 experiment except the ligand was added again (50 pM and 300 pL in CTX buffer, stock cone. = 10 mM in DMSO, DMSO = 0.5%, v/v) to PLATE 8. For day 10, 50 worms (N2 or UA196) from PLATE 7 (without ligand) or PLATE 8 (with 50 pM ligand) were transferred to PLATE 9 using CTX buffer (approximately 20 pL) and the number of worms were counted under an Olympus microscope (SZ-6145, Waltham, MA). Subsequently, the lid of the plate was closed with parafilm (Bemis Company, Inc., Neenah, Wl). The worm activity was monitored using the WMicroTracker ARENA plate reader (Phylumtech, Santa Fe, Argentina) at 20 °C for 2 hours. The report was generated using MapPlot option on the WMicroTracker ARENA plate reader.

[0146] The experiment was conducted for both N2 and UA196 worms (in the absence and presence of 50 pM SK-129) on day three and day 10 of the aging process. For each condition, three biological replicates were performed and at least two technical replicates were used for each biological replicate. The data were expressed as mean and the error bars report the s.e.m. (n = 3 independent experiments and each n included two technical replicates).

[0147] Measurement of the reactive oxygen species (ROS) level in UA196 worms in the presence of ligands (Quantification of ROS and confocal imaging)

[0148] The worm strains were bleached at the same time and the eggs were incubated on PLATE 1. On day two, the worms were divided into two batches. One half of the worms were transferred (using M9 buffer) on PLATE 2 (without ligand) and the second half of the worms were transferred to PLATE 3 (with ligand). The concentration of the ligands was 50 pM as described in the main manuscript. On day four, the ligand (10-50 pM in M9 buffer with 0.1%- 0.5% DMSO, v/v) was spotted again atop the PLATE 3 at 20 °C. The worms were incubated on PLATE 2 or PLATE 3 up today eight at 20 °C in an incubator with constant humidity. On day eight, the worms were transferred into 1.7 mL microcentrifuge tubes using M9 buffer (1 mL) and washed with M9 buffer (1 mL and three times) by centrifugation for 2 minutes at 700xg and 20 °C. Subsequently, a total of 100 worms/well were transferred to a Costar 96-well black plate (Corning, Kennebunk, ME) containing 100 pL solution, including M9 buffer (89 pL), worm solution (10 pL and 100 worms), and 2',7'-dichlorofluorescein diacetate (H2DCFDA) dye (1 pL, 50 pM, stock solution = 5 mM in cell culture grade DMSO, 99% pure). As a control, 99 pL of M9 buffer and 1 pL of 5 mM H2DCFDA reaction solution was placed in the wells. Subsequently, the Costar 96-well plate was gently shaken for 30 sec at 20 °C and the fluorescence intensity was measured (A_ez = 485 nm and X_em = 530 nm) at multiple time points (0 to 120 minutes) using the Infinite M200 Pro Plate Reader (Tecan, Mannedorf, Switzerland).

[0149] For confocal imaging, the same UA196 worms (in the absence and presence of ligands) used for the quantification of ROS, were transferred from the 96-well plate into PLATE 2, airdried into the sterile laminar flow hood. The worms (approximately 10 worms) were transferred to a cover slide containing an anesthetic (40 mM sodium azide) and mounted on a glass microscope slide containing 2% agarose pads for the confocal imaging as described in the previous sections. This experiment included three biological replicates and each biological replicate included three technical replicates. The data were expressed as mean and the error bars report the sd's (n = 3 independent experiments and each n included three technical replicates).

[0150] Confocal imaging of a post-disease onset PD model of UA196 worms in the absence and presence of ligands

[0151] The UA196 worms were bleached, and the eggs were incubated on PLATE 1. On day two, the UA196 worms were transferred (using M9 buffer) on PLATE 2 (without ligand). On day five, ten UA196 worms were used to determine the number of healthy DA worms using confocal imaging (as described earlier) prior to the treatment with ligands on day five. In tandem, approximately 50 worms were transferred to PLATE 3 (with ligand) using M9 buffer (10-20 piL) and the plate was incubated at 20 °C in the incubator. On days 10 and 15, 10 UA196 worms from PLATE 2 (without ligand) and PLATE 3 (with ligand) were used to determine the number of healthy DA worms using confocal imaging (as described earlier). For each condition, six biological replicates were performed and at least ten technical replicates were used for each biological replicate. The data were expressed as mean and the error bars report the s.e.m. (n = 6 independent experiments and each n included a minimum of ten technical replicates).

[0152] Measurement of intracellular ROS level in a post-disease onset PD model of UA196 worms in the absence and presence of ligands

[0153] The UA196 worms were bleached, and the eggs were incubated on PLATE 1. On day two, the UA196 worms were transferred (using M9 buffer) on PLATE 2 (without ligand). On day five, 100 UA196 worms were used to determine the ROS level (as described earlier) prior to the treatment with ligands on day five. In tandem, approximately 200 worms were transferred to PLATE 3 (with ligand) using M9 buffer (10-20 pL) and the plate was incubated at 20 °C in the incubator. On day 8, 100 UA196 worms from PLATE 2 (without ligand) and PLATE 3 (with ligand) were used to determine the ROS level (as described earlier). For each condition, three biological replicates were performed, and 3 technical replicates were used for each biological replicate. The data were expressed as mean and the error bars report the s.e.m. (n = 3 independent experiments and each n included 3 technical replicates).

[0154] Pharmacokinetics and distribution of SK-129 in brain tissues

[0155] In vivo pharmacokinetics were performed using a previously published methods. Test mice (male and female, C57BL/6J) were injected intravenously with a single dose of SK-129 (20 mg/kg) (n = 3). Blood was drawn at various time points over 72 hours from the saphenous vein, collected in chilled, K3-EDTA microcentrifuge tubes (Greiner Bio-One), and centrifuged for 10 minutes (1700g, 4°C) to isolate the plasma. The resulting separated plasma stored at -86 °C until LC-MS/MS analysis. Thereafter, 100 piL of collected plasma was spiked with SK-129 (1 pg/mL), and mixed with 100 pL of Tris buffer (1 M, pH 8). To extract the SK-129, the mixture was thrice diluted in 3 mL chloroform/methanol (9:1, v/v), vortexed (10 minutes), and centrifuged (3500g, 20 °C, 10 minutes). This was followed by collection and evaporation of the organic phase to dryness under a N2 stream. The dry residue was then dissolved in 60 pL of methanol and centrifuged (3500g, 20°C, 10 minutes), and supernatant was collected and filtered using a 0.2 pm syringe filter. Finally, 10 pL of supernatant was assayed by LC-MS/MS analysis.

[0156] Similarly, whole brains (n = 3 per time point) were collected at different time points over 72 hours and thoroughly rinsed with cold phosphate-buffered saline prior to freezing on dry ice. Whole brains were weighed and diluted (3 mL) with 70:30 isopropanokwater (v/v). The mixture was subjected to mechanical homogenization employing a Bead Mill Homogenizer (Bead Ruptor 96, Omni International, Kennesaw, GA, USA) and 1.0 mm Silica Beads followed by centrifugation (3500g, 20 °C, 10 minutes). The brain homogenate supernatant was diluted (4x) in ice-cold acetonitrile to precipitate the protein. Then the samples were centrifuged (3500g, 20 °C, 10 minutes) and the supernatants were diluted in 3 mL of chloroform/methanol (9:1, v/v), vortexed (10 minutes), and centrifuged (3500g, 20 °C, 10 minutes). This was followed by collection and evaporation of the organic phase to dryness under a N2 stream. The dry residue was then dissolved in 60 pL of methanol and centrifuged (3500g, 20 °C, 10 minutes), and supernatant was collected and filtered using a 0.2 pm syringe filter. Finally, 10 pL of supernatant was assayed by LC-MS/MS analysis.

[0157] LC-MS/MS analysis

[0158] Quantification of SK-129 was performed on an Agilent 1290 Infinity ultrahigh- performance liquid chromatography (UHPLC) system coupled with a Bruker EVOQ triple quadrupole mass spectrometer. Agilent C-18 column (1.8-pm particle size) was used with an inner diameter of 2.1 mm and length of 15 mm. Mobile phases included 10 mM ammonium acetate for solvent A and 10 mM ammonium acetate in methanol for solvent B. A sample volume of 10 pL for both calibration standard and unknown was injected onto the column. The samples were eluted from the column using a linear gradient starting from 25% B that progressed to 65% B in 3 minutes. A 2-minute wash at 85% B was used to keep the column sensitivity high and prevent carry-over, and 3-minute equilibration with 25% B completed the gradient. Column, temperature maintained at 40 °C, was attached to the UHPLC with a flow rate maintained at 400 pL per minute.

[0159] Selected/Multiple reaction monitoring (S/MRM) analyses were carried out on an EVOQ ESI-tri pie quadrupole mass spectrometer (Bruker) operated in negative ion mode. A calibration standard was used for creation of the transitions. Collision energy (CE) was optimized for each transition tested. The final method for S/MRM included the following transitions and specifications: 502.3/444.1 (CE 16V), 502.3/473.2 (CE 6V) and 502.3/185.0 (CE 53) where the precursor 502.30 corresponds to SK-129. The transition 502.3/444.1 (CE 16V) was set as quantifier ions while the other two as qualifier ion. The rest of the setting for the EVOQ triple quad mass spectrometer were as follows: spray voltage 4500 V, cone temperature 350°C, cone gas 25 units, heated probe temperature 150°C, probe gas 10 units, exhaust gas on, and nebulizer gas 10 units. Residual SK-129 concentration was determined by means of the linear least square regression model after external calibration with calibration standards (n = 4). Calibration standards and samples were run in triplicates, with two blanks run before and after each sample run. The R2 of the calibration curve was observed to be > 0.98.

[0160] Animal and treatment groups

[0161] All studies involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of New York University Abu Dhabi (23-0003). Male and female B6;C3- Tg(Prnp-SNCA*A53T)83Vle/J mice (short: TgM83+/- mice) and C57BL/6J from The Jackson Laboratory (Bar Harbor, ME, USA) were used in this study. The mice were 4-8 weeks old at the start of the experiment and were housed under environmentally controlled standard conditions with a 12 hours light/dark cycle and free access to food and water. The mice were housed in a controlled environment, adhering to standard conditions. These conditions included a 12-hour light and dark cycle to regulate their circadian rhythms. Furthermore, the mice had continuous access to both food and water to ensure their well-being throughout the study.

[0162] Stereotactic injections

[0163] The stereotactic injections were performed using the previously published protocol.

The study employed male and female mice aged between 6 and 8 weeks as experimental subjects. The aim was to examine their response to the challenge with alpha-synuclein fibrils (aS-fibers), particularly when aS-fibers were administered in combination with either SK-129 or SK-Q3.

[0164] To administer these substances, TgM83+/- mice were anaesthetized with isoflurane and underwent stereotactic injection. The injection site was within the substantia nigra, with coordinates following commonly established methods (A -3-4, L + 1-4, H +4 0, Slotnik & Leonard, 1975). A volume of 200 nanoliters (n L) of a solution comprising 100 pM Synuclein monomer and 1% brain homogenate was injected intracranially, referred to as aS-fibers. Simultaneously, 200 nL of 100 pM synuclein monomer with 1% brain homogenate and 200 pM of SK-129 (n = 10) were injected intracranially, referred as Group 2.

[0165] Following the injection, the needle was left in place for an additional 5 minutes to ensure complete absorption of the solution. This meticulous procedure was essential to provide the necessary exposure for the study's specific objectives, facilitating a comprehensive evaluation of the mice's responses to the administered substances.

[0166] Intravenous injections of different small molecules. After the intracranial injection of Synuclein monomer and 1% brain homogenate treatment, the mice were divided into three distinct groups, denoted Group 1 (n = 10). Following groups received the treatments once in every two days for 21 days.

[0167] aS-fibers: Mice in this group were administered with Saline.

[0168] aS-fibers+SK-Q3: For this group, mice were intravenously administered with 20 mg/kg of SK-Q3

[0169] aS-fibers+SK-129: For this group, mice were intravenously administered with 20 mg/kg of SK-129

[0170] The entire experiment extended over a duration of 9 months, during which the mice were consistently monitored for any changes in body weight and their overall survival status. These evaluations were conducted at specific intervals, approximately once every 10 days. Upon reaching the 180- and 270- days mark, the 4 mice from each group were humanely euthanized, and their brain tissues were meticulously collected by perfusion with a solution of phosphate buffered saline (PBS), followed by fixation with 4% paraformaldehyde. Then the collected tissues were processed for immunohistochemistry analysis.

[0171] Immunohistochemistry and immunofluorescence imaging

[0172] Mice were deeply anaesthetized and sequentially perfused with saline and 4% paraformaldehyde for immunohistochemistry staining. Brain tissues were removed, infiltrated with paraffin and cut into 4 pm sections. Paraffin-embedded tissue was dewaxed by 2 minutes consecutive incubations in Xylene, Histoclear, 100% ethanol, 95% ethanol, 70% ethanol, and distilled water. After rehydration, samples were incubated in 10% hydrogen peroxide in PBS, to reduce background. To unmask the antigenic sites, we performed antigen retrieval by heating the sections at 94 °C for 20 minutes in antigen retrieval solution (80 mM citric acid, 20 mM sodium citrate, pH 6.0). Then the sections were incubated in 3% hydrogen peroxide for 10 minutes to eliminate endogenous peroxidase activity. Sections were blocked in 3% BSA (with 0.3 % Triton X-100) at room temperature for 30 minutes. Then the slices were incubated with primary antibody (pS129, 1:500) overnight at 4°C and then rinsed three times in PBS. Signals were developed using the DAB staining kit following the previously published protocol. Images were captured using a Nikon LV-Dia microscope.

[0173] For double immuno-fluorescence staining from previously published protocol, primary antibodies were used for immunofluorescence staining: pS129 (1:500) and AT8 (1:1000) and a mixture of Alexa Fluor 488- and 594-coupled secondary antibodies (1:500) was applied. After staining in DAPI solution, images were captured using an Leica Stellaris 8 confocal microscope. [0174] Confocal and FLIM imaging of in vitro-generated aS+tau condensates

[0175] Phase separation experiments with aS and tau were carried out by mixing 20 pM aS (19 pM unlabeled + 1 pM AF488-labelled), with or without a pre-incubation of 30 minutes with SK- 129 (at double the concentration of aS), with 10 pM of WT tau or AggDef-tau (9 pM unlabeled + 1 pM Atto647N-labeled) in PBS pH 7.4, 10% PEG (8,000 kDa) and 0.02% (w/V) sodium azide. Buffer, additives and PEG were mixed before adding the proteins in all cases. 200 pL of LLPS samples were spotted onto confocal 96-well glass bottom plates (Ibidi GmbH, Grafelfing, Germany), sealed with adhesive foil and incubated for the indicated time at 25 °C.

[0176] The presence of protein coacervates was confirmed by fluorescence confocal microscopy on a Leica DMI6000B inverted microscope (Leica Microsystems, Wetzlar, Germany) coupled to a ViRTEx Realtime Experiment Control unit (VisiTron Systems GmbH, Puchheim, Germany) and a LDI-4 laser diode illuminator (89 North, Williston, VT, USA). Samples were illuminated with two laser lines of 470 and 640 nm for the excitation of AF488-labelled aS and Atto647N- labelled tau, respectively, and the emission was passed through a X-Light V2 spinning disk unit (CrestOptics, Rome, Italy).

[0177] At least 3 images of 340x340 pm were collected for each sample with the VisiView imaging software (Visitron Systems GmbH, Puchheim, Germany) focusing on the indicated height over the well bottom, and analyzed with ImageJ (NIH, Bethesda, MD, USA).

[0178] Fluorescence lifetime imaging microscopy (FLIM) experiments were performed with a MicroTime 200 time-resolved fluorescence confocal microscope (PicoQuant, Berlin, Germany) with a time-correlated single photon counting (TCSPC) unit. Samples were illuminated with two laser lines of 491 and 637 nm in pulsed interleaved excitation (PIE) mode, and the emission beam was passed through a 50 pm pinhole, splitted by a 50/50 prism and passed through 520/35 and 690/70 bandpass filters prior to single photon avalanche diodes (SPADs) detectors.

[0179] At least 6 images of 80x80 pm were taken for each condition with a dwell time of 0.4 s/pixel, focusing on the indicated height over the well bottom. Acquisition field of zoomed images varies between 5x5 and 20x20 pm. To avoid pile-up effects, the laser power was adjusted for each experiment so that the total recorded photons were at least 104, and the maximum photon recording for a single pixel was 500. The total lifetime decay of each image was fitted to 2-3 components through a tail-fitting algorithm included in the SymphoTime64 (PicoQuant GmbH, Berlin, Germany) software. The false color rainbow indicates the fluorescence lifetime averaged by intensity of each pixel, and it was maintained the same for all images of each emission channel.

[0180] In vitro single-molecule fluorescence analysis of salt-resistant aS+Tau assemblies

[0181] After an incubation time of 13 to 15 hours, the bottom of each LLPS-containing well was gently scrapped with a pipette tip to resuspend the sedimented coacervates, and gently mixed to ensure sample homogeneity. 20 pit of each sample were mixed with 980 pL of a buffer containing 10 mM HEPES (pH 7.4), 500 mM NaCI, named isolation buffer, achieving a 1:50 dilution of the resuspended LLPS reaction where electrostatic coacervates are disrupted due to high salt and non-crowding PEG concentration. All samples were pumped into a pi-Slide VI 0.1 confocal glass bottom microfluidics chip (Ibidi, Grafelfing, Germany) at a flow rate of 0.1 pL/s.

[0182] Single-molecule fluorescence (SMF) measurements were carried out with the aforementioned MicroTime 200 setup by placing the confocal effective volume 20 pm above the glass bottom of the microfluidics channel. Laser powers were adjusted to 6.0 pW for the 481 nm line and 6.2 pW for the 637 nm line, and at least 3 time-traces of 10 minutes each were measured to obtain statistically relevant data.

[0183] The resulting SMF time traces were exported with a binning of 0.2 ms, and fluorescence bursts due to high-order protein assembles were isolated by an intensity threshold that was updated every second for both channels in order to avoid artifacts due to monomer background variability. The threshold was defined as

[0184] Threshold = x_dec + 10a_dec Eq. (1)

[0185] where >T_dec and o_dec are the mean and the standard deviation of the higher decile of the intensity distribution.

[0186] The apparent assemble size (A) and the stoichiometry (S) of each assemble were calculated as

[0187] A = F_DDN_D + F_AAN_A Eq. (2)

[0188] S = — - _Eq. (3)

^FDA + Y?D D ^ND + ^FAA NA

[0189] Where F_DD, F_AA and F_DA are the maximum fluorescence intensities of the direct excitation of AF488-aS, the direct excitation of Atto647N-tau, and the indirect excitation of Atto647N-tau, respectively, N_D and N_A are the total number of monomers of aS and tau, respectively, per fluorescently labeled monomer, and y is the detection efficiency (y=0.517) of the instrumental setup. Assembly distribution heatmaps were plotted using OriginPro 2018. [0190] Effect of SK-129 on aS aggregation potentiated degeneration of DA neurons in a C. elegans PD model

[0191] The aS aggregation mediated degeneration of DA neurons is a pathological hallmark of PD. We tested the efficacy of SK-129 to inhibit the intraneuronal aggregation of aS in DA neurons in a well-established C. elegans PD model (UA196 worms). The UA196 worms simultaneously express human WT aS and GFP in six DA neurons, which are located within the anterior region of UA196 worms. During the aging of UA196 worms, the aS aggregation leads to the progressive neurodegeneration of DA neurons as shown by others as well.

[0192] For all experiments, the UA196 worms were incubated at 20°C in an incubator with constant humidity and oxygen. The aS aggregation potentiated degeneration of the DA neurons is represented by the gradual loss of cell bodies as observed from day 3 through day 15. The degeneration of DA neurons also induces fragmentation and blebbing of neurites from day 3 to day 5. For each biological replicate, we used 10 worms with a maximum number of six intact DA neurons (total neurons = 60). During the progression of neurodegeneration, the average number of healthy DA neurons on day 3, 5, 10, and 15, was 59, 42.1, 20.5, and 15.2, respectively.

[0193] The worms were treated with SK-129 (50 pM) on day two and day four to test its effect on the neurodegeneration of DA neurons in UA196 worms. In the presence of SK-129, we did not observe a significant decline in the number of healthy DA neurons as they were 58.2, 56.8, and 56.5 on day 5, 10, and 15, respectively. SK-129 effectively rescued the neurodegeneration in a dose-dependent manner. SK-129 was also intact in the biological milieu of C. elegans, as confirmed by high resolution mass spectrometer. SK-129 was a far better ligand than the literature-reported ligands to rescue the degeneration of DA neurons in UA196 worms. The number of healthy neurons in UA196 worms on day 15 in the presence of Bexarotene, Tyrosol, Valporic acid, and EGCG (50 pM each) was 22.5, 25.5, 22.5, and 34.5, respectively. Most of these ligands were ineffective; however, EGCG was a moderate ligand in rescuing the degeneration of DA neurons in UA196 worms.

[0194] Effect of SK-129 on the motility of UA196 worms

[0195] In UA196 worms, the neurodegeneration of DA neurons has been associated with the loss of motor functions, which significantly decreases the motility of worms. Therefore, we assessed the effect of SK-129 on the motility of UA196 worms. The UA196 worms were treated with SK-129 (50 pM on days 2 and 4), and the motility of UA196 worms was tested using a WMicroTracker ARENA plate reader. The maximum motility was measured on day 4 of the control worms. The motility of UA196 worms significantly declined during the aging process compared to the control worms. In sharp contrast, there was a significant improvement in the motility of UA196 worms in the presence of SK-129. The motility of SK-129 treated UA196 worms was very close to the motility of N2 worms.

[0196] Effect of SK-129 on the ROS level in UA196 worms. The generation of ROS has been associated with lipids, proteins, and DNA oxidation, which is linked to the etiology of PD. The aS aggregation in UA196 worms significantly enhances the production of intraworm ROS level, which can determined using a fluorescent probe (CM-H2DCFDA) as shown previously. The probe reacts with ROS in UA196 worms, and the reporter signal intensity (green fluorescence) increases up to 2 hours. In the presence of SK-129 (50 pM on day 2 and 4), there was a significant decline in the amount of the ROS signal intensity. The decrease in the ROS level is likely due to the inhibition of the intracellular aS aggregation by SK-129.

[0197] Effect of OPs on Behavioral deficits in UA196 worms

[0198] The degeneration of DA neurons results in a decrease in the dopamine level, which leads to behavioral deficits in C. elegans, such as food-sensing behavior. We used a well- established chemotaxis assay to assess the effect of SK-129 on the behavioral deficits of UA196 worms. In the chemotaxis assay, the healthy worms distinguish between their food as an attractant (E. coli) and a repellent chemical (ethanol). We compared the behavioral deficits of UA196 (±50 pM SK-129 on day 2 and 4) and N2 worms on day three and day 10. For each experiment, the worms were placed at the center of the petri dish, which contains two quadrants with E. coli and two quadrants with ethanol. We used the ARENA plate reader to measure the chemotaxis index (Cl) over time with values from -1.0 to +1.0. The Cl value closer to +1 and -1 represents the total time spent by the worms in quadrants with attractant or repellent, respectively. On day three, UA196 (+50 pM SK-129) and N2 worms did not demonstrate any behavioral deficit reflected by their Cl values of approximately 1 and approximately -1 for E. coli and ethanol, respectively. The Cl values suggest that all the worms spent most of their time in the E. coli quadrants, reflected by a value of approximately 1 (E. coli) and approximately -1 (ethanol) during the course of the experiment. On day 10, the Cl values for UA196 worms were close to zero, suggesting that UA196 worms do not display any preference for ethanol or E. coli, which is most likely due to the behavioral deficits. In marked contrast, the Cl values of SK-129 treated UA196 worms were approximately 1 (E. coli) and approximately -1 (ethanol), similar to the N2 worms. Thus, SK-129 was a potent inhibitor of intracellular aS aggregation and was able to rescue neurodegeneration and behavioral deficits in UA196 worms.

[0199] Effect of SK-129 on dopamine in UA196 worms

[0200] The neurodegeneration in UA196 worms is associated with impaired motility, likely due to the decrease in the dopamine level. We treated UA196 worms with 2 mM dopamine on day 2 (±50 pM SK-129 on day 2 and day 4) and compared their motility with the N2 worms treated with 2 mM dopamine (day 2). There was no noticeable change in the motility of N2 worms in the absence and presence of dopamine, most likely due to the lack of degeneration of DA neurons. In marked contrast, we observed a significant improvement in the motility of UA196 worms when treated with dopamine, which is likely due to the compensation provided by the external dose of dopamine for the decrease in the dopamine synthesis due to the loss of DA neurons. Similar to N2 worms, we did not observe a significant increase in the motility of UA196 worms (+SK-129) in the presence of dopamine due to the rescue of the degeneration of DA neurons and continuous dopamine synthesis.

[0201] Effect of SK-129 on the neurodegeneration in a post-disease onset PD model

[0202] SK-129 potently rescues the DA neurons and rescues various PD phenotypes in UA196 worms. We added SK-129 to UA196 worms at an early stage of the PD phenotypes, which potentially mimics the preventative therapeutic strategy. As the current therapeutic landscape for PD relies on post-diagnosed disease phenotypes, SK-129 was tested in a post-disease onset PD model. In the post-disease model, neurotoxicity and various PD phenotypes were facilitated by multiple mechanisms, including the de novo aS aggregation and fibers-catalyzed aS aggregation (prion-like spread of aS fibers). We have also shown that SK-129 is a potent inhibitor of the de novo aS aggregation and fibers-catalyzed aS aggregation.

[0203] A total neuronal loss of approximately 30% or day five in C. elegans has been suggested to be a post-disease onset model, as shown by us and others. We have also demonstrated that approximately 30% worms degenerate on day five in UA196 worms. We treated UA196 worms with SK-129 (50 pM) on day 5 to test its effect on a post-disease onset model of UA196 worms. The healthy DA neurons on day 3, day 5, day 10, and day 15 were 58.5, 41.5, 21, and 13.2, respectively. When treated with SK-129 (on day 5), the number of healthy DA neurons was 38.4 and 31.8 (on day 15) at 50 pM and 25 pM, respectively, which suggests a complete rescue of the degeneration of DA neurons in a dose-dependent manner. The other reported ligands were not very effective in rescuing the degeneration of DA neurons under matched conditions. The number of healthy neurons on day 15 in the presence of Bexarotene, Tyrosol, Valporic acid, and EGCG were 13.3, 13.5, 13.3, and 21.5, respectively. One of the likely reasons for such a high efficacy of SK-129 is likely due to its ability to inhibit both the de novo aS aggregation and fibers- catalyzed aggregation of aS.

[0204] Effect of SK-129 on the ROS level in a PD model

[0205] SK-129 was also tested for its effect on the ROS level in the post-disease model of

UA196 worms. The ROS level was measured on day 5 and then 50 pM SK-129 was added to UA196 worms. The ROS level was measured on day 8 in the absence and presence of SK-129. Expectedly, there was a significant increase in the ROS level in UA196 worms because of aS aggregation and degeneration of DA neurons. In marked contrast, the level of the ROS significantly declined in the presence of SK-129. Clearly, SK-129 is a potent inhibitor of aS aggregation and rescues PD phenotypes in both early and late-stage PD models and mimics the clinical landscape for current therapeutic efforts that rely on the post-diagnosis of PD73,78,79.

[0206] SK-129 rescues aS aggregation mediated PD phenotypes in induced pluripotent stem cell (iPSC) patient-derived DA neurons

[0207] We further investigated the effect of SK-129 in iPSC-derived neurons from patients with triple SNCA genes. The iPSC-derived neurons have been shown to develop aS aggregation mediated PD phenotypes, including the decrease of dopamine levels and calcium dysregulation. We developed DA neurons from iPSCs of PD patients with triple SNCA gene according to the published protocol. The DA neurons were treated with SK-129 (10 pM) on day 28 of postdifferentiation and the degeneration of DA neurons was measured using the tyrosine hydroxylase staining and the calcium imaging on day 42 of post differentiation. We observed a significant decline in the TH+ staining of DA neurons in both untreated and the vehicle (solution without SK-129) conditions. In marked contrast, there was a significant rescue of the TH+ staining in the presence of 10 pM SK-129. We also monitored the KCI-induced Ca2+ influx spike in the DA neurons in the absence and presence of SK-129. There was a significant decline in the Ca2+ influx spike in the DA neurons in both untreated and vehicle conditions. However, the Ca2+ influx spike was significantly improved in the presence of SK-129. We also determined that SK-129 can permeate the neuronal membrane. We treated the DA neurons with SK-129F (10 nM) and used confocal imaging after 24 hours. We observed a significant amount of SK-129F in the cytoplasm of the DA neurons, indicating that it efficiently crosses the cell membrane of DA neurons. Thus, SK-129 was effective in rescuing PD phenotypes in an iPSC-derived neuronal PD model.

[0208] SK-129 binds specifically to aS oligomers

[0209] We used fluorescence polarization (FP) titrations to gain insight into its binding affinity for various conformations of aS, including monomers, oligomers, and fibers. For FP titrations, increasing doses of aS conformers were titrated in a solution of the fluorescent analog of SK-129 (SK-129F, 100 nM) until no more changes were observed in the FP signal. The fitting of the plots of the FP titrations yielded Kd's of SK-129 of 1284±54 nM, 214+34 nM, and 641±22 nM for aS monomers, aS oligomers, and aS fibers. The data suggest that SK-129 binds with approximately 6-fold and approximately 3-fold higher affinity with the toxic aS oligomers and aS fibers than the functional monomeric aS. It is important to note that SK-129 will likely bind with the toxic states of aS without interfering with the functional form of aS (monomer).

[0210] Testing of SK-129's pharmaceutical properties

[0211] The pharmaceutical properties are important parameters to further advance the SK-129 towards PD and other synucleinopathies. Six to 8-week-old control mice (B6C3F1/J mice) were used to evaluate long-term blood and brain exposure levels of SK-129 using multiple times points up to 72 hours. A single dose of SK-129 was administered intravenously (IV, 20 mg/kg) and the mice (n=3/time point) were humanely euthanized (CO2 inhalation) at the end of each time point and whole blood was collected via heart puncture, following the earlier published protocols. We used this dose of SK-129 to compare it with other reported ligands at the same dose for neurodegenerative disorders.

[0212] Plasma was processed by centrifugation at 3000xg for 15 minutes at 4°C. Brain samples were collected after the blood perfusion with saline buffer (lxPBS) and then homogenized in the saline buffer. SK-129 was extracted from both plasma and brain homogenates and characterized using LC-MS/MS as previously described in publication. SK-129 demonstrated rapid absorption (Tl/2=1.78 hours) with a maximum concentration reaching 139.8±26.7 pg/mL. SK-129 crosses the blood-brain barrier (BBB) with a maximum concentration of 13.1±0.3 pg/mL.

[0213] SK-129 has a much higher concentration in the blood plasma (approximately 5 fold) and

BBB (approximately 2-10 fold) in comparison to other potential drugs that inhibit aS aggregation or LB formation in mouse models. Moreover, we detected a concentration of 13.1±0.3 pg/mL of SK-129 in the brain tissue for more than 72 hours post-injection (>20 fold higher than other ligands at 72 hours)37,38 suggesting a potential high efficacy for its target in the brain due to longer time in the brain tissue. Additionally, the in vitro physicochemical properties of SK-129 are comparable to ligands that efficiently cross the BBB. The maximum concentration ratio of brain/blood plasma for SK-129 is high (9.4%), allowing a high amount of SK-129 in the brain tissue for potent antagonist activity.

[0214] We also tested the stability of SK-129 in mice plasma, brain homogenate, and human liver microsome using published protocols. Briefly, 100 pM SK-129 was added to the mouse plasma mixture (0.5 mg/mL, lxPBS, pH 7.4) and incubated at 37 ⁹C at 0, 15, 30, 60 minutes, and 24 hours with constant shaking (1000 rpm). The solution mixtures were removed at their respective time points and a solution of cold acetonitrile was added to the mixtures to precipitate the proteins. The samples were centrifuged, and the supernatants were analyzed using UHPLC and MS to determine the peak area ratio. SK-129 was very stable up to 24 hours in the mouse plasma (91.9±4.8%).

[0215] We also determined the fraction unbound of SK-129 in the mouse plasma using a published protocol. The fraction unbound of SK-129 to mice plasma proteins was 4.4±0.4%. We determined the stability and % unbound of SK-129 (in mice brain) were 46.7±3.1% and 29.9±3.6%, respectively.

[0216] We also determined the stability and fraction unbound of SK-129 in the human liver microsome. We used similar experiment conditions to those we used for the mouse plasma. The stability and % unbound of SK-129 were 94.5+5.3% and 7.1±0.8%, respectively. SK-129 was very stable in mice plasma and human liver microsome, but it was less stable in mice brain. In contrast, the % unbound of SK-129 was approximately 4 to 6 fold higher free concentration in the case of mice brain. Overall, SK-129 demonstrates good pharmaceutical properties in mice (plasma and brain) and human liver microsomes.

[0217] Effect of SK-129 on a prion-like spread mouse model of PD

[0218] We tested the effect of SK-129 on aS aggregation mediated PD phenotypes in a mouse model. We used the M83 mouse model [male and female, (B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J] from the Jackson Laboratory to study the effects of SK-129 on various PD phenotypes. The M83 transgenic mice express mutant A53T aS and develop PD phenotypes. Additionally, PD neuropathology can be accelerated in M83 mice when inoculated with pre-formed aS aggregates. It has been shown that the injection of aS aggregates decreases the survival time (median approximately 182 days) in comparison to the natural survival time (median approximately 359 days) of M83 mice and also accelerates various PD phenotypes potentially due to the templating of the intracellular A53T aS by the preformed aS aggregates. We have also shown that SK-129 is an effective inhibitor of PD neuropathology accelerated by the preformed aS aggregates in biophysical, cellular, and C. elegans (late-stage model) PD models.

[0219] We extracted aS aggregates from post-mortem PD brain tissue using a known procedure, which has shown the acceleration of PD phenotypes in cellular and neuronal models. Therefore, we intend to use this model to monitor the effect of SK-129 on the preformed aS aggregates treated M83 mouse model [known as M(83)A], The 8-week-old M83 mice were anesthetized and stereotactically injected with 100 pM aS aggregates (200 nl_) in the substantia nigra (SN, coordinates: AP -3-4, ML ± 1-4, DH -4-0), similar to the previously reported protocols.

[0220] Two weeks following aS aggregates injection, the M83 (A) were randomly divided into two groups. One group was injected intravenously with vehicle (IV, 100 pL/mice, saline buffer+2% DMSO) and the second group was injected with SK-129 (IV, 20 mg/kg, 100 pL/mice, saline buffer+2% DMSO) for 21 days (every second day, 10 doses). We used the doses of SK-129 on alternate days because its concentration in the brain was maximum for more than two days. Therefore, to keep the highest concentration of SK-129 in the brain for its optimal efficacy, we used the doses of SK-129 on alternate days.

[0221] The survival rate and the body weight of mice were monitored every 10 days until 270 days of age. The median survival time for M83(A) was approximately 175 days and less than 20% of mice survived after 270 days. The M83(A) displayed quadriparesis, arched back, and were unable to stand up and support their body weight. This is in close agreement with earlier studies of the M83(A) model. The survival of M83(A) was 100% in the presence of SK-129 (20 mg/kg) up to270 days, similar to the control mice. The SK-129-treated M83(A) appeared to be healthy (similar to the control mice) and did not display any signs of quadriparesis or arched back. There was a significant weight loss in M83(A) mice. However, we did not observe weight loss in SK-129 treated M83(A) mice, and their weights were similar to those of the control mice.

[0222] We used IHC to assess the effects of SK-129 on various brain regions of M83(A). We used the brain sections of M83(A) mice in the absence and presence of SK-129 at two time points, including the six month and nine-month old mice. We detected the pathological deposits of aS-129 and P62 proteins in six- and nine-month old mice, which are key pathological hallmarks of PD. The M83(A) mice displayed deposits of both aS-129 and P62 proteins throughout the motor cortex, striatum, hypothalamus, hippocampus, amygdala, and SN for both six month and nine-month old mice. In the presence of SK-129, we did not observe any significant aggregates of aS-129 and P62 proteins in brain regions of M83(A).

[0223] We also stained for the activation of microglia, which is a consequence of the neuroinflammation due to aS aggregation. We observed activation of microglia (Ibal staining) in the absence of SK-129; however, we did not observe any significant activation of microglia in the presence of SK-129. The LB biomarkers were quantified by calculating the number of stained regions in a defined area and at least 10 regions were used from each mouse. Our data demonstrate that SK-129 is an effective ligand in rescuing PD phenotypes in the M83(A) model.

[0224] SK-129 is a potent inhibitor of aS+Tau co-aggregation

[0225] The co-aggregation of aS+Tau proteins is another key pathological biomarker, and the co-aggregates of aS+Tau are frequently identified in PD and LBD. Therefore, it is essential to test the antagonist activity of SK-129 against the co-aggregation of aS+Tau to expand the efficacy across the broad spectrum of synucleinopathies.

[0226] We tested the antagonist activity of SK-129 on the co-aggregation of aS+Tau proteins using a ThT aggregation assay. The ThT-based kinetic assay demonstrated that aS (1 mg/mL, 70 pM) aggregation is significantly accelerated in the presence of Tau protein (1 mg/mL, 27.5 pM). Additionally, there was a significant increase in the ThT signal of aS aggregation in the presence of Tau protein, which has been reported in the literature as well. Under matched conditions, we did not observe any aggregation of Tau protein. The TEM image of the aS+Tau coaggregation confirms the formation of aggregates. The aS aggregates were more ribbon-like; however, there were more twists in the co-aggregates of aS+Tau and they were more compact. We did not observe any formation of co-aggregates of aS+Tau in the presence of SK-129 at an equimolar ratio (70 pM), validated using both ThT and TEM.

[0227] We also tested the antagonist activity of a ligand, which is in the clinical trial for PD (UCB0599). The ligand UCB0599 was not effective in inhibiting the co-aggregation of aS+Tau. We also tested the cytotoxicity induced by the co-aggregation of aS+Tau (±SK-129 or ±UCB0599). The cell viability was decreased to 12.2% of the control cells in the presence of 5pM co-aggregate of aS+Tau. The cell viability of the solution of co-aggregates of aS+Tau in the presence of SK-129 and UCB0599 were 94.6% and 17.9%, respectively. SK-129 is a potent inhibitor and UCB0599 is a poor inhibitor of the co-aggregation of aS+Tau.

[0228] In addition to the aggregation of aS and the co-aggregation of aS+Tau, another source of cytotoxicity is the fibers-catalyzed aggregation of both aS and aS+Tau. We monitored the effect of SK-129 on the preformed fibers (of aS and aS+Tau) templated aggregation of aS. The aggregation of aS (70 pM) was significantly accelerated by the preformed fibers of aS (20% concentration of 70 pM) and aS+Tau (20% of 70 pM). In the presence of SK-129 (70 pM), we did not observe any formation of fibers as validated by the ThT signal. It is important to note that SK-129 is a unique ligand that can inhibit the de novo and the fibers catalyzed aggregation of aS and aS+Tau.

[0229] We used cellular assay to test that the inhibition of the co-aggregation of aS+Tau by SK- 129 do not generate fiber-competent cytotoxic structures. We utilized a well-established model of HEK293 cells that stably express monomeric YFP-labeled aS-A53T mutant in the cytosol. The endogenous monomeric aS-A53T-YFP can be templated into fibers by transfecting with preformed aS and aS+Tau fibers in the presence of Lipofectamine 3000. We transfected the HEK cells with the aggregated solution of aS and aS+Tau fibers (5pM in monomeric concentration) in the absence and presence of SK-129. In the presence aS and aS+Tau fibers, there was an abundance of intracellular fluorescent condensates/puncta.

[0230] Studies have shown that Tau protein complexes with aS and forms the liquid condensates via coacervation, which eventually terminates into the aggregates (punctas) of aS+Tau. We counted the number of condensates/puncta in HEK cells treated with the preformed fibers of aS and aS+Tau for 48 hours in the absence and presence of SK-129. Compared to the control, we observed condensates/punctas in HEK cells in the presence of aS. We observed a much higher number of condensates/punctas in the presence of aS+Tau fibers and they were much smaller in size than the aS fibers treated cells, corroborating with the earlier published work. The morphology of the condensates was almost spherical, and the punctas were abnormally shaped non-spherical structures, as shown by others as well. We did not observe any significant number of condensates/punctas in the presence of SK-129.

[0231] We also tested the ability of SK-129 to inhibit the formation of condensates/punctas by the preformed fibers of aS+Tau in the cellular milieu. We treated the HEK cells (expressing aS- A53T-YFP) with 10 pM SK-129 for 12 hours, which is the time required for the internalization of SK-129, as shown by us previously. Subsequently, we treated the cells with the preformed fibers of aS/aS+Tau (5 pM monomer concentration) and incubated the cell for 48 hours. Compared to the untreated cells, we observed a complete reduction in condensates/punctas in HEK cells for both aS/aS+Tau in the presence of SK-129. [0232] To study the effect of SK-129 on the condensates of aS+Tau, we used a recently established experimental system, which demonstrates the colocalization of aS+Tau, the formation of condensates, and eventual termination into amyloids. We used SK-129F to test its ability to form a complex with aS in the aS+Tau condensates. Indeed, when added to the demixed solutions of aS and Tau containing aS+Tau condensates, SK-129F colocalizes and concentrates in the interior of the condensates for a long period of time. The fluorescence analysis of the colocalization suggests the formation of the complex of SK-129 with aS+Tau. We then followed the maturation of the aS+Tau condensates in the presence of SK-129 by FLIM.

[0233] We previously showed that amyloid aggregation inside the condensates can be identified by means of a reduction of the fluorescence lifetime of the fluorophores attached to the proteins as a consequence of a pronounce fluorescence quenching in the protein solid state (Gracia et al., Nat Commun 2022, 13(1):4586. doi: 10.1038/s41467-022-32350-9.). Using FLIM, we demonstrated the formation of amyloid-like aggregates inside aS+Tau liquid condensates. These aggregates are resistant to high salt concentrations (unlike the liquid condensates) and smaller than the average size of the aS+Tau condensates, and enriched in Tau molecules, as indicated by single-molecule fluorescence burst analysis, in agreement with the higher propensity of Tau to aggregate.

[0234] In line with these findings, when a well-established amyloid aggregation deficient Tau variant, referred here to as AggDef Tau variant (Seidler, P. M. et al. Structure-based inhibitors of tau aggregation. Nat. Chem. 10, 170-176 (2018)), was used to generate the heterotypic condensates, no salt-resistant aggregates were detected upon the aging of the condensates. However, when SK-129 was present, the aS/Tau condensates suffer a rapid and drastic change in their maturation process towards a gel-like state, unable to transition into amyloid-like aggregates. These gel-like condensates become resistant to high salt concentrations, and their fluorescence burst analysis reflects the size and the stoichiometry of the initially formed liquid condensates (with a mean stoichiometry of 1:1 between Tau and aS), characteristics of gel-like electrostatic aS/Tau coacervates.

[0235] Thus, SK-129 inhibits the formation of amyloid-like co-aggregates of aS+Tau and we have shown that the aS+Tau complexes formed with SK-129 are not cytotoxic in nature.

[0236] Effect of SK-129 on the preformed condensates/punctas of aS+Tau

[0237] The preformed condensates/punctas of aS+Tau have been shown to participate in the spreading of synucleinopathies via the seed-catalyzed mechanism (prion-like spread). Therefore, we tested the efficacy of SK-129 to modulate the seeding of condensates/punctas of aS+Tau using biophysical and cellular conditions. The complex of aS+Tau (70 pM+27.5 pM, Img/mL) was aggregated for 96 hours and then incubated with SK-129 (70 pM) for 24 hours. We observed a significant decrease in the ThT signal, indicative of the modulation of the preformed aggregates of aS+Tau by SK-129 as its ThT signal was close to the control ThT signal. The ThT signals of the aggregated aS+Tau by SK-129 (premixed or postfixed) are in close proximity, which suggests that SK-129 inhibits the aggregation of aS+Tau and modulates the preformed aggregates of aS+Tau. Compared to the fibers of aS+Tau, SK-129 treated fibers of aS+Tau were different in morphology and much smaller and with less twist in the fibers.

[0238] We also tested the SK-129 treated preformed fibers of aS+Tau for their ability to template intracellular monomeric aS in HEK cells (expressing aS-A53T-YFP). The HEK cells were treated with various conditions, including the preformed fibers of aS, aS+Tau, and aS+Tau (treated with SK-129) for 48 hours. The number of condensates/punctas in HEK cells increased from control to aS to aS+Tau. In marked contrast, we did not observe a significant number of condensates/punctas for the SK-129-treated preformed fibers of aS+Tau. The substantial decrease in the condensates/punctas of SK-129-treated preformed fibers of aS+Tau is a consequence of the modulation of the preformed fibers of aS+Tau and the binding of SK-129 to aS+Tau fibers, which significantly decreases the seeding ability of preformed aS+Tau fibers.

[0239] We further tested the effect of SK-129 on the seeding ability of preformed aS+Tau fibers in HEK cells (expressing aS-A53T-YFP). For this experiment, we treated HEK cells with preformed aS+Tau fibers (5 pM) for 24 hours, followed by adding SK-129 (10 pM) to the cells for 24 hours. We observed a significant number of condensates/puncta in cells treated with preformed fibers of aS+Tau. However, there was a significant decline in the condensates/puncta in cells of SK- 129 treated aS+Tau fibers, similar to the control. We also observed a substantial decline in the condensates/puncta in cells of SK-129 treated preformed aS fibers, similar to the control.

[0240] We used confocal spectroscopy to further validate these results. For this experiment, we added SK-129F to the preformed complex of aS+Tau. We observed the colocalization of SK- 129F with the preformed complex of aS+Tau, indicative of the formation of the complex between SK-129 and aS+Tau for a long period of time. To test the effect of SK-129, we added SK-129 (1 pM) to the preformed complex of aS+Tau after 1 hour. After 5 hours, we observed the formation of the co-aggregates of aS+Tau. However, in the presence of SK-129, we detected the formation of gel-like structures instead of the co-aggregates of aS+Tau. We have shown earlier that the structures of aS+Tau formed after treatment with SK-129 are neither able to seed nor cytotoxic. We also analyzed the SN of the M83(A) mice (9 months old) for the coaggregates of aS+Tau using immunostaining assays. We observed the colocalization of the phosphorylated aS+Tau in the SN of M83(A) mice. In marked contrast, we did not observe a significant colocalization of the phosphorylated aS+Tau in M83(A) mice treated with SK-129 (20 mg/kg, IV). These results demonstrate that SK-129 is a potent antagonist of aS aggregation and co-aggregation of aS+Tau.

[0241] Discussion of the experimental results

[0242] Synucleinopathies include an umbrella of pathologies, including PD, LBD, and MSA. Synucleinopathies are debilitating diseases without any known cure, making them a pressing need to develop therapeutic interventions for them. The central pathological lesion entails the aggregation of aS, which can be facilitated by multiple mechanisms, including the de novo aggregation of aS, the prion-like spread of aS fibers, and the co-aggregation of aS+Tau. There are numerous ligands that have been identified as potent inhibitors of aS aggregation and rescued various disease phenotypes in various model systems. However, most of these ligands have not advanced to the preclinical stage because of multiple limitations, including poor pharmaceutical properties, poor ability to cross the BBB, complex structures that limit their modifications for optimization of efficacy and pharmaceutical properties, lack of information about the mode of action of these ligands against the toxic targets, and the heterogeneity of the PD and other synucleinopathies.

[0243] We have used a distinct approach of foldamers to identify potent antagonists of aS aggregation. The foldamer specifically binds with the neurotoxic aS oligomers with nanomolar affinity, which is multiple-fold higher affinity than with the aS monomers, suggesting that it will potently modulate the toxic aS oligomerization without interfering with the function of aS. We also demonstrated in iPSC-derived neurons that the foldamer was very potent in inhibiting aS aggregation mediated phenotypes without interfering with the native function of the monomeric aS. The foldamer was very effective in rescuing aS aggregation mediated disease phenotypes in cellular, multiple C. elegans models, iPSC-derived neurons, and an aggressive mouse model. The foldamer demonstrated tolerable pharmaceutical properties, including stability in mouse plasma, mouse brain, and human liver microsomes. It was very efficient in crossing the BBB, a prerequisite property for the potential drugs for neurodegenerative disorders. [0244] The foldamer was also a very potent antagonist of the co-aggregation of aS+Tau in multiple models. The co-aggregation of aS+Tau is another key pathological event in synucleinopathies (PD and LBD), which complicates the disease pathology due to multiple mechanisms and accounts for the heterogeneity of the disease. To the best of our knowledge, there is no report of a ligand that can potently inhibit the co-aggregation of aS+Tau; therefore, ligands that potently inhibit aS aggregation may not likely inhibit the co-aggregation of aS+Tau as shown by us. Therefore, these ligands may not account for the heterogeneity of the disease and may be partly successful in advancing toward clinical trials.

[0245] Using our foldamer approach, we have demonstrated that the specific targeting of the aS oligomers potently inhibits aggregation of aS, the prion-like spread, and the co-aggregation of aS+Tau, which would partly account for the heterogeneity of the synucleinopathies. Foldamers are unique scaffolds as they can be conveniently tuned for both the optimization of efficacy and pharmaceutical properties, which will be a future effort in our study for the further advancement of foldamers to the clinical trials for synucleinopathies.

[0246] Example 2 - Alzheimer's Disease (AD)

[0247] Similar to the studies presented above with respect to PD, ligands that demonstrate the combined properties of both small-molecule and peptide intervention can be promising therapeutics towards the modulation of APPIs. As discussed above, foldamers are a class of synthetic protein mimetics that are dynamic and can mimic the complex surface topology, chemical space, and secondary structure of proteins. Specifically, OQ based foldamers have been shown previously to modulate the aggregation of Amyloid-P ( AP), a-Synuclein, and islet amyloid polypeptides in Alzheimer's disease (AD), Parkinson's disease, and Type-ll Diabetes, respectively. OQ foldamers exhibit the properties of both small-molecules and peptides and are synthetically tunable to optimize the side-chain interactions with various protein targets, all features essential to specific abrogation of APPIs.

[0248] Using Alzheimer's disease Caenorhabditis elegans (C. elegans) worm models (GMC101 and CL2355 worm strains) and a set of in vivo assays, we have identified a potent OQ foldamer- based small molecule (SK-131) which inhibits amyloid beta plaque formation. SK-131 was found to be a potent antagonist of AP1.42 aggregation in a Thioflavin T (ThT) kinetics assay, even in the presence of Zn₂+ ions. Furthermore, SK-131 was able to inhibit intracellular aggregation of APi. 42, rescuing the onset of Alzheimer's disease phenotypes in the worms including aggregation- induced paralysis. Moreover, the reactive oxygen species (ROS) level, which is elevated due to aggregate formation, was significantly reduced in the GMC101 C. elegans, indicating the effectiveness of SK-131 as a potent inhibitor of plaque formation. Also, SK-131 exhibited a significant potency in rescuing behavioral deficits in the CL2355 strain caused by plaque formation in neurons. Altogether, these results show that SK-131 is a very effective antagonist of APi-42 aggregation and further validate the use of C. elegans as suitable in vivo multicellular models in studies pursuing potent therapeutics for AD.

[0249] It has been shown that soluble Ap oligomers are a key neurotoxic species, therefore the disruption of the oligomeric interactions to prevent fibril formation is a promising therapeutic strategy. Though there have been great efforts in the prevention of disease progression, there are no known treatment options for individuals affected by the disease, therefore modulation of the aggregation of AP is a promising therapeutic target. Previously, a di-anionic OQ tetramer (SK-131) has been identified in in vitro trials to interact with AP42 in a structure and sequencespecific manner, preventing the formation of insoluble Ap fibrils and the toxic downstream effects in a simple cell model.

[0250] In this report, we carried out for the first time in vivo studies to validate the efficiency of the OQ ligand in the prevention of AP1.42 fibril formation, as well as a significant reduction in the onset of aggregation-mediated disease phenotypes. Our in vivo studies are carried out utilizing Alzheimer's C. elegans models to demonstrate the effect of SK-131 on the intracellular aggregation of Ap₄₂. It is shown in this work that SK-131 is capable of significantly reducing the intracellular aggregation of AP1.42 and the aggregation-mediated increase in intracellular ROS levels. Accordingly, we have also demonstrated a significant reduction in aggregation-induced paralysis in GMC101 strains. Lastly, our studies probe the behavioral deficits in CL2355 C. elegans demonstrating a marked rescue of aggregation-mediated AD phenotypes in vivo. Through this work, we have identified SK-131 to be a potent therapeutic inhibitor of AP aggregation and validate the use of several C. elegans in vivo experiments as a model for the development and identification of potential therapeutic ligands of abrogating disease-related APPIs.

[0251] Thioflavin T Aggregation Kinetics

[0252] After demonstrating the reproducibility of SK-131 to inhibit AP1.42 aggregation by reducing the ThT fluorescence from 100% to 4% in the absence and presence of SK-131, we further tested our molecule's antagonist activity at substoichiometric ratios. At 0.1 molar equivalents of SK-131, the ThT fluorescence was reduced to 23% compared to the AP1.42 control. [0253] We then tested whether the antagonist activity of our OQ could stand up to the accelerating effects of Zn₂+ ions. It is understood that the Zn₂+ metal ions coordinate the histidine residues of A i.₄₂, accelerating aggregate nucleation and further fibril formation. In the presence of 1 equivalent of SK-131 and equimolar concentration of Zn₂+, the ThT fluorescence was reduced to 8% compared to the A|3I.₄₂ which was aggregated with Zn₂+. The aggregation of API.₄₂ in the with equimolar Zn₂+ was reduced to 40% when SK-131 was present at 0.1 molar equivalents.

[0254] Having tested SK-131 against Ap_{i 42} aggregation in vitro using ThT aggregation and seen a remarkable efficacy in vitro, here we embarked on testing the inhibitory potency of SK-131 against intracellular Ap_4.42 aggregation. We used the nematode Caenorhabditis elegans (C. elegans), which has been used extensively in studying protein aggregation-associated neurodegenerative diseases.

[0255] We used two AD C. elegans models for this study: the GMC101 strain which expresses API.₄₂ in body wall muscle cells, and the CL2355 strain which forms Ap_4.42 aggregates in the neurons. The formation of APi.₄₂ aggregates in the muscle cells of GMC101 worms induces several AD phenotypes including paralysis (reduction in motility rate), a significant increase in reactive oxygen species (ROS) level, as well as amyloid plaque formation. The CL2355 transgenic strain expresses human API.₄₂ in neuronal cells which can form aggregates upon temperature change (from 16 °C to room temperature) resulting in neurotoxicity and consequent chemotaxis defective behaviors. These nematodes have been used extensively used as in vivo models for studies aimed at understanding the mechanism of disease etiology and more importantly as tools for the testing of ligands against APi.₄₂ aggregation-mediated disease phenotypes. A cascade of assays has been developed and employed for the screening of ligands to identify potent inhibitors of intracellular API.₄₂ aggregation. We have made use of these assays in previous studies to screen for potent inhibitors of protein aggregation.

[0256] Effect of SK-131 on API-₄₂ Aggregation in GMC101 C. elegans

[0257] We tested the potency of SK-131 against intracellular aggregation and rescuing of aggregation-mediated disease phenotypes. The GMC101 worms were age-synchronized by bleaching on day 1 and grown on FUDR-containing NGM plates inhibiting reproduction thus keeping them of the same age. Using a molecular fluorescent probe, NIAD-4, and confocal microscopy, we determined the extent of aggregation. [0258] FIG. 16 shows a bar graph illustrating the fluorescence intensity measurements for treated and untreated GMC101 C. elegans worms, in accordance with an embodiment. The

GMC101 is a worm strain exhibiting A aggregation, mimicking a AD model. The treated worms were provided with SK131 (50 pM).

[0259] We observed a gradual increase in AfJi.42 aggregate formation from day 1 of adulthood (total day 4) to day 6 of adulthood (total day 10). The worms were treated per a reported protocol with 5 pM of NIAD-4 dye, which binds to API.₄₂ and makes them visible through fluorescence microscopy. We observed a significant increase in aggregate formation on day 6 of adulthood post-temperature upshift (to 25 °C). A relatively higher corresponding fluorescence intensity quantified by ImageJ was observed for post-temperature upshift worms. We then treated the GMC101 worms twice with 50 pM SK-131 first on day 3 (total Days) and day 5 (Day 2 of adulthood), then upshifted temperature on day 6 and quantified aggregate formation for adulthood day 6 (day 10). Contrary to the untreated GMC101, a relatively deficient aggregate formation was observed for the treated worms as depicted by the low NIAD4-stained fluorescence signal and the corresponding intensity measurement. The relatively low aggregate formation in the presence of the ligand is a testament to the potency of SK-131 against API_₄₂ aggregate formation intracellularly.

[0260] Effect of SK-131 on 42 aggregation-induced paralysis of GMC101 worms

[0261] Our next step was determining whether SK-131 is potent enough to rescue AP1.42 aggregation-induced reduction in worm motility. As stated earlier, GMC101 strain expresses AP1.42 in body wall muscle cells where these peptides can oligomerize and aggregate resulting in chronic age-progressive paralysis of worms. Hence, we tested the antagonist activity of SK-131 against AP1.42 aggregation-induced paralysis. The worms were synchronized according to the previously explained protocol and plated in a 24-well plate. We used a Phylum Tech ARENA plate reader to assess the motility rate of the worms. The Arena plate reader is a well- established instrument used in C. elegans studies to measure aging-related defects such as motility rate and neuro sensitivity (chemotaxis behavior).

[0262] FIG. 17 shows a bar graph illustrating the motility rate of N2 and GMC101 C. elegans worms with and without 50 pM SK-131. We observed a significant decrease in the motility rate of the GMC101 worms over time from day 2 to day 10 of adulthood compared to that of the N2 worms, in agreement with previous studies. In marked contrast, the GMC101 worms, treated with 50 pM SK-131 showed significant improvement in motility with values closer to those of the N2 worms. The rescuing effect on motility by SK-131 observed further suggests potency against AP1.42 peptide aggregation. Moreover, the significant improvement in the motility of the treated GMC101 worms indicates the rescuing potency of SK-131 against intracellular aggregation-induced paralysis. This data suggests that SK-131 is an effective ligand against intracellular AP1.42 aggregation.

[0263] Effect of SK-131 on ROS level in GMC101 strain

[0264] Next, we tested to determine the rescuing potency of SK-131 against intracellular ROS level elevation caused by AP142 aggregation. Numerous studies have shown the association of ROS level elevation with AP1.42 aggregate formation. It has been shown that the binding of redox-potent metals such as Fe, Cu, etc. leads to Ap aggregate-metal complex formation which enhances the generation of ROS in the periphery of membranes, a phenomenon known to be deleterious to neurons structural integrity. Furthermore, the generation of ROS can lead to the oxidation of essential biomolecules like lipids, proteins, and DNA which are known factors in AD etiology. We, therefore, tested for the inhibitory potency of SK-131 against ROS generation resulting from intracellular Ap aggregation. We used a molecular probe 2,7-dichlorofluorescein diacetate (H2DCFDA), which penetrates the intestinal walls of the worms and gives a strong fluorescent signal upon reacting with ROS, with fluorescence increasing over time up to 2 hours.

[0265] FIG. 18 shows a graph illustrating the intracellular reactive oxygen species (ROS) levels, demonstrating the rescuing effect of SK-131 on aggregation-induced ROS elevation, in certain embodiments. The GMC101 worms, without treatment with the molecule, showed significant elevation in ROS level. On the other hand, for GMC101 worms treated with 50 pM SK-131, we observed a significantly decreased level of ROS formation relative to the negative control worms (untreated GMC101). The N2 worms (non-disease) showed an insignificantly low ROS fluorescent signal as expected similar to previous reports by others

[0266] Effect of SK-131 on the behavioral deficit in CL2355 strain

[0267] We also tested SK-131 to investigate its potency to rescue Ap aggregation-mediated neurodegeneration. Studies have shown that the clustering of AP peptide into bilayers of neuron membranes, aggregate formation in membrane periphery that leads to ROS generation, and subsequent redox reactivity with some biomolecules impair membrane structural integrity eventually leading to neurodegeneration characteristic of AD. Severe loss of neurons results in a significant decline in brain functions including memory function. Thus, assumedly, SK-131 may rescue neurodegeneration and aggregation-induced behavioral deficits. [0268] We used the CL2355 C. elegans strain which, as mentioned earlier, expresses AP1.42 in neurons and aggregates upon temperature upshift, affecting neurocognitively and chemotaxis behavior. We used a chemotaxis assay to assess the rescuing effect of SK-131 on the behavioral deficits of CI2355 worms. In this assay, a six-well plate with 3 ml of agar plated in each well was used. Each well was divided into four quadrants where two opposite quadrants (left diagonal or right diagonal) were treated with a toxic chemical (ethanol, repellent) or food (E. coli, attractant) for worms. For each experiment, 50 worms were placed at the center of the dish, and the ARENA plate reader was used to measure the chemotaxis index (Cl) over time with values from - 1.0 to +1.0 which are indicative of the time spent by worms in ethanol or E. coli quadrants. The kinetics of the Cl over was monitored on Day 4 and Day 8 for 2 hours for various worms, including N2, CL2355, and CL2355 plus molecule.

[0269] The plotted Cl data for the assay on day 4 suggests that all the worms (including untreated disease strain) spent most of their time in the E. coli quadrants reflected by a value of approximately 1 over 2 hours. None of the worms displayed behavioral deficits on day four.

[0270] In marked contrast, on day 8, the kinetics of the CL2355 Cl indicated no display of preference for ethanol (repellent) nor E. coli (attractant), only directionless movements of the worms for the whole-time course of the experiment indicating the behavioral deficits caused by the aggregation-induced neuron loss. However, the CL2355 worms treated with SK-131 strongly favored E. coli over ethanol, similar to the wild-type worms, indicated by the chemotaxis indices closer to +1. SK-131 was able to rescue the behavioral deficits of CL2355

[0271] SK-131 Traverses the blood-brain barrier

[0272] As discussed above, the blood-brain barrier (BBB), a highly specialized barrier that controls the movement of substances between the circulatory system and the central nervous system (CNS), functions as an essential protective measure, preventing the entry of potentially detrimental molecules into the brain while facilitating the transport of vital nutrients and gases. In as much as it serves an indispensable protective role, the BBB also presents a significant obstacle to delivering therapeutic agents to treat neurological disorders. vStudies of many molecules exhibiting significant potency through in vitro biophysical and biochemical assays reached bottlenecks due to failure to cross the BBB. vHence, we did a complete pharmacokinetic study in mice to determine the ability of SK-131 to move across the BBB.

[0273] We administered SK-131 (0.3 mg/Kg) compound to mice via intravenous injection following established protocols. Following the intravenous injection of mice with SK-131, blood samples were collected at specific intervals, and their concentrations were analyzed using the liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) technique.

[0274] FIGS. 19 and 20 show results of in vivo pharmacokinetics studies of SK-131 in mice at different time points.

[0275] In particular, FIG. 19 shows a graph of the biodistribution of SK-131 in plasma through 72 hours post administration.

[0276] FIG. 20 shows a graph of the biodistribution of SK-131 in the brain tissue through 72 hours post administration.

[0277] FIGS. 21 and 22 show results of the absorption studies of SK-131 in the human liver.

[0278] In particular, FIG. 21 shows the absorption of SK-131 in the human liver over time.

[0279] FIG. 22 shows the difference in the area beneath the curve in FIG. 21 for the measured absorption in the human liver versus a control sample.

[0280] Within 15 minutes of the intravenous injection, a maximum concentration of 3.3 ± 0.5 pg of SK-131 was detected in the plasma. This rapid distribution suggests a swift entry of SK-131 into the systemic circulation. Furthermore, the in vivo circulation half-life of SK-131 was determined to be 0.25 ± 0.10 hours, which is notably comparable to that of Galantamine, a well- known acetylcholinesterase inhibitor utilized for managing symptoms of Alzheimer's disease (ti/₂ = 43 minutes). This similarity in circulation half-life implies the potential therapeutic relevance of SK-131 in the context of Alzheimer's disease management, with a prolonged presence in circulation expected to enhance its accumulation in brain tissues over time.

[0281] Simultaneously, brain tissue samples were collected at various time points postinjection, and their SK-131 concentrations were quantified using LC-MRM-MS. Notably, within 72 hours of intravenous injection, a substantial concentration of 1.3 pg of SK-131 was observed in the brain tissues. This finding underscores the ability of SK-131 to effectively traverse the blood-brain barrier, indicating its potential efficacy in targeting Alzheimer's disease symptoms at the neurological level.

[0282] C. eleqans strains and culture methods

[0283] The following strains along with Escherichia coli OP50 (E. coli, a uracil-requiring mutant) were acquired from the Caenorhabditis Genomics Center (CGC) in Minneapolis, MN, and used in this study: N2 (wild-type C. elegans Bristol Strain); GMC101 ( [0284] [(Punc-54::A-beta::unc-54 3Prime UTR; Pmtl2::GFP)] C. elegans model of AD which expresses AfJi.42 aggregates in body wall muscle cells upon temperature upshift to 25° C, and CL2355 (smg-l(cc546); pCL45 [Psnb-l::human Amyloid beta 1-42: :3' UTR (long); Pmtl-2::GFP])

AD model which expresses AP1.42 in neurons with aggregation induced upon temperature upshift from 16° C to room temperature that leads to age-dependent neurotoxicity effects. These animals were reared at standard conditions on nematode growth media (NGM) agar plates (60 mm, Cyto One, Ocala, FL) with 350 pL plated Escherichia coli OP50 (E. coli, uracil requiring mutant) as the food source. They were maintained according to established protocols in an incubator at 20° C. The NGM agar plates, M9 buffer, and OP50 solution at 0.5 optical density (OD600nm) were prepared using previously established methods.

[0285] GMC101 strain treated with SK-131 motility rate assay

[0286] The motility assay followed established protocols with some modifications. To synchronize the mature N2 and GMC101 strains, the bleaching process was employed, which involves egg laying and incubating the eggs at 20°C for 30 hours on NGM culture plates with OP50 as the food source . On the third day, when the worms were at larva stage 3, they were transferred (using M9 buffer) from the 60 mm NGM plates to 35 mm NGM plates containing 75 pM fluorodeoxyuridine (FUDR) to prevent worm reproduction and ensure that the worms used in the assay were of equal age.

[0287] The first dose of SK-131 (50 pM diluted in M9 buffer from a 10 mM DMSO stock solution) was administered to the GMC101 strain after transfer. The worms were then incubated at 21°C for 48 hours. On the fourth day, a fresh stock of OP50 was prepared by diluting 1 pL of OP50 in 5 mL of Luria Broth (LB) Miller media (Neogen, Lansing, Ml) and incubating it in a shaking incubator (Eppendorf, Hamburg, Germany) at 37°C and ~200 for ~24 hours. The assay was conducted using a sterile 24-well plate containing 700 pL of liquid media (prepared according to the published recipe) per well. SK-131 (50 pM in M9 buffer) was reconstituted in the same liquid media for the GMC101 strain of C. elegans for the second treatment dose. The conditions for this experiment included the N2 strain (positive control), the GMC101 strain (untreated negative control), and the GMC101 strain treated with SK-131, as previously described. There were four technical replicates (four wells) for each worm condition, and a total of 50 worms per well were transferred manually with a worm pick into the 24-well plate. [0288] Before each paralysis assay, the 24-well plate was placed on a shaker for approximately

5 minutes to stimulate worm activity in the liquid media. The assay was initiated on day 6, and

20 activity scores per well were collected using the micro Tracker Arena plate reader at 21° C for

1 hour per day over 10 days. Three biological replicates were conducted, and the average activity scores were reported, with the error bars representing the standard error of the mean (SEM).

[0289] Quantification of AB Aggregates in GMC101 Worms Using NIAD-4 Dye

[0290] The initial preparation steps (synchronization, transfer, etc.) for the GMC101 worms for aggregate quantification were similar to the procedure followed for the motility assay. After treatment of the worms on day 3 and day 5 with SK-131 (50 pM in M9 buffer, prepared from a 10 mM DMSO stock) the GMC101 worms were then transferred to a 25° C incubator and kept there for 48 hours to induce aggregation. On day 9 (and day 3 for the pre-aggregation induction quantification), the worms were transferred using a worm pick into NIAD-4 solution (5 pM in M9 buffer) and incubated at room temperature for 4h at 150 rpm on a shaker (Thermo Scientific, Waltham, MA). This incubation ensured that the worms were thoroughly stained. The worms were subsequently transferred to NGM plates containing FUDR and incubated again at 20 °C for 24 hours to allow the worms to recover through metabolism. At least 10 worms per condition were transferred with a worm pick to a cover slide containing an anesthetic (40 mM sodium azide) and mounted on glass microscope slides (Fisher Scientific, Pittsburgh, PA) containing 2% agarose pads for imaging.

[0291] The images of the worms were collected using an Olympus Fluoview FV3000 confocal/2- photon microscope (40x Plan-Apo/1.3 NA objective with DIC capability) on day 4 and day 10. The fluorescence intensity for the inclusions of amyloid protein aggregates (in the muscle cells) was quantified using ImageJ software. For each condition, three biological replicates were performed, and the average number of aggregates was reported with the error bars representing the SEM.

[0292] Measuring ROS level in GMC101 strain

[0293] We measured ROS levels using a previously published modified protocol. Briefly, the worms (wild-type N2 strain and disease GMC101 strain) were synchronized on day 1 as explained earlier. On the third day, the worms were transferred to 35 mm FUDR-containing plates to prevent reproduction and ensure the age uniformity of the worms used in the assay. The disease GMC101 strain was given two doses of SK-131 (50 pM in M9 buffer, prepared from a 10 mM DMSO stock), with the first dose given on day 3 and the second dose on day 5. The GMC101 worms were then transferred to a 25° C incubator and kept there till the day of the assay to induce aggregation.

[0294] On day 9, the worms were carefully washed off the plates with M9 (to avoid transferring E. coli) and transferred to 1.5 mL centrifugal tubes. The worms were centrifuged at 17000 g, at 25° C and the supernatant was carefully removed. This process was repeated twice to wash off E. coli from the worms. The worm sample solution density was then measured by pipetting 10 pL to a glass slide, manually counting to determine the number of worms, and adjusting to 50 worms per 10 pL where necessary.

[0295] The assay was performed by plating the worms in a 96-well plate (costar black round bottom) and employing the use of the fluorescent probe 2,7-dichlorofluorescein diacetate (CM- H2DCFDA). Before doing the assay, a 50 pM in M9 working solution of the probe was prepared from DMSO stock. The assay was performed according to these conditions with four technical replicates per condition: 1) Blank (50 pL M9 + 50 pL ROS probe); 2) N2; 3) GMC101; and 4) GMC101+SK-131 (40 pL M9 + 10 pL worms +50 pL ROS probe). The ROS fluorescence signal was then measured using a Tecan instrument every 30 minutes for 2 hours. This assay was repeated three times to ensure the reliability of the data.

[0296] Measuring Behavioral Deficit in CL2355 strain

[0297] The behavioral deficit induced by aggregation and the rescuing effect of SK-131 was done according to established protocols with minor modifications. The worms were synchronized as usual on day 1 and incubated on a 60 mm NGM plate at 16° C for 48 hours. On the third day, they were transferred to the 35 mm FUDR-containing plates, and the first dose (50 pM) of the molecule was administered to the CL2355 strain. The second dose was given on day 5. The CL2355 strains were transferred to a higher temperature environment (at room temperature) on day 6 and incubated there for 48 hours giving them enough time for aggregation to be induced.

[0298] Also on day 6, the chemotaxis plate (plates containing agar) and chemotaxis buffer were prepared using a published recipe. Briefly, for the chemotaxis plates, 2 g of agar was dissolved in 100 mL Milli Q. water and sterilized by autoclaving. To the sterilized agar, 500 pL of 1 M KH2PO4 buffer along with 1 M CaCL and 1 M MgSO₄ (100 pL each) was added, mixed thoroughly by shaking, then transferred to the 6-well plate (3 mL per well). The chemotaxis buffer was prepared also by autoclaving Milli Q. water and subsequently adding 1 M KH2PO4 (500 pL), 1 M CaCI₂ (100 pL), and 1 M MgSO₄ (100 pL).

[0299] On the day of the assay (day 8), the worms were washed off the NGM plates carefully with the Chemotaxis buffer and transferred to 1.7 mL Eppendorf tubes where the worms were allowed to settle at the bottom of the tubes by gravity. The buffer was pipetted out and replaced (repeated twice) to wash off E. coli attached to the worms. The density of the worms was then determined and adjusted to 50 worms per 10 pL.

[0300] For each experiment, 50 worms were placed at the center of each well, and ethanol and E. coli were placed at the polar ends of the petri dish. We used the ARENA plate reader to measure the chemotaxis index (Cl) over time with values from -1.0 to +1.0. The kinetics of the Cl index over time were generated based on the time spent by worms in ethanol or E. coli quadrants.

[0301] Identification of SK-131 in the Cell Lysate of GMC101 Worms

[0302] The identification of the SK-131 molecule in the cell lysate of the GMC101 worms was carried out using a procedure developed by us. Briefly, the GMC101 worms were synchronized and treated with 50 pM SK-131, as described above with respect to the paralysis assay.

[0303] On day 10, the worms were transferred into 1.7 mL microcentrifuge tubes using M9 buffer (1 mL). The samples were centrifuged for 2 minutes at 2500 rpm and 20 °C. Subsequently, the worms were washed five times with PBS buffer. The worms were incubated at -80 °C for 24 hours. The worms were dissolved in lysis buffer for 1 hourh followed by sonication for 30 minutes. Subsequently, the solution was dried using an ana lyophilizer and redissolved in a 2 mL solution of water and methanol (50:50, v/v). The solution was then centrifuged for 10 minutes at 15,000 rpm. Subsequently, the supernatant was transferred and used for the LC- analysis. We repeated the experiment four times with a minimum of 200 GMC101 worms.

[0304] Pharmacokinetic studies (Mouse Plasma and Blood-Brain Barrier Study)

[0305] In vivo pharmacokinetic studies were conducted at New York University Abu Dhabi, UAE. Both male and female mice (C57BL/6, 6-8 weeks old, Jackson Laboratories, Farmington, CT, USA) were housed individually in a controlled environment of 21 ± 3°C, relative humidity 50 ± 20%, 12 hours light, 12 hours dark and were randomly divided to 3 mice per time point.

[0306] Mice were administered with the SK-131 (0.3 mg/Kg) compound via intravenous injection following established protocols. Subsequently, blood samples collected from mice (n = 3) were subjected to various time points (0, 0.25, 0.5, 1, 2, 4, 8, 16, 24, 48, and 72 hours), in line with reported literature. At each time point, before the blood sampling, mice were anesthetized for 5 minutes using 2% isoflurane in a vaporizer with an oxygen flow of 0.8 L/min (Euthanex EZ-SA800 Animal Anesthesia System, Palmer, PA, USA). Blood samples (30—80 pL) were collected using a sparse sampling method via the right or left retro-orbital vein3 using heparinized capillary tubes. To preserve sample integrity, plasma was promptly separated via centrifugation at 12000xg for 1 minute at room temperature and stored frozen at -20 °C in Protein LoBind tubes until LC-MRM analysis.

[0307] Concurrently, brain tissue samples were harvested at different time points (0, 6, 12, 24, 48 and 72 hours) and homogenized in a homogenization buffer (250 mM sucrose, 150 mM NaCI, 1 mM EDTA, 50 mM HEPES, pH 7.0 protease and phosphatase inhibitors) for 60 seconds using an IKA T25 Digital Ultra-Turrax® homogenizer (IKA, Konigswinter, Germany). The ratio of buffer to the tissue was 10:1 (mL/g) (v/m). The tip of the homogenizer was rinsed with methanol, water, and homogenization buffer before each homogenization. The homogenate was then centrifuged at 20,000x g for 30 minutes at 4 °C (5804/5804 R Centrifuge, Eppendorf, Enfield, CT, USA). Prepared homogenates were immediately frozen and stored at -80 °C.

[0308] A volume of 40 pL containing either plasma or brain homogenate was utilized, supplemented with 10 pL of methanol to achieve a total volume of 50 pL, mirroring the composition of standard curve samples. Subsequently, 50 pL of mouse plasma, or brain homogenate, was combined with 5 pL of an internal standard working solution (100 ng/mL), resulting in a total volume of 55 pL. Then, 500 pL of ethyl acetate was introduced to the 55 pL samples, followed by vortexing for 60 seconds and subsequent centrifugation at 10,000x g for 15 minutes. The supernatant (400 pL) was then carefully collected and dried using a nitrogen blow-down evaporator (Labconco RapidVap N2/48 Evaporations System, Kansas City, MO, USA). The resulting dry residue was reconstituted with 200 pL of methanol/water (4:1, v/v) and subjected to quantitative liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) analysis.

[0309] Incubation with Pooled Human Liver Microsomes

[0310] A solution of IX PBS (94.5 pL) containing 1 pL SK-131 (10 mM, DMSO) and 2.5 pL of the pooled human liver microsomes (Gibco by Thermo Fisher Scientific, Frederick, MD, USA Catalog

# HMMCPL, LOT PL050H-A) was placed into a Thermomixer (Eppendorf Fl.5, Sigma Aldrich, Burlington, MA) set to 37 °C and 1,000 rpm shaking. After 24 hours, the proteins were precipitated out of solution with the addition of 100 pL of a solution containing 90% acetonitrile (ACN), and 10% water with 0.1% formic acid. The solution was centrifuged for 8 minutes at 26,000 x G, and the supernatant was lyophilized overnight. The resulting cake was then redissolved in ACN with 0.1% trifluoroacetic acid (TFA), briefly vortexed, and sonicated for 10 minutes. After centrifuging for 8 minutes at 26,000 x G, the supernatant was run on a high- performance liquid chromatography (HPLC) system (Dionex Ultimate 3000, Thermo Scientific, Waltham, MA) equipped with an Accucore C18 column (Thermo Scientific, Catalog # 17126- 154630) running ACN containing 0.1% TFA for 20 minutes at 0.5 mL/min. The corresponding peak was collected and subjected to HRMS.

[0311] The area of the peaks was calculated using Eq. 4 below, where Al is equal to the measured absorbance at elution time point 1 (tl) and A2 is equal to the measured absorbance at time point 2 (t2).

[0313] Quantitative LC-M RM-MS analysis

[0314] Quantification of SK-131 was performed on an Agilent 1290 Infinity UHPLC system coupled with a Bruker EVOQ triple quadrupole mass spectrometer. Agilent C-18 column (1.8-pm particle size) was used with an inner diameter of 2.1 mm and length of 100 mm. Mobile phases consisted of 10 mM ammonium acetate (solvent A) and 10 mM ammonium acetate in methanol (solvent B). A sample volume of 5 pL for both standards and unknown samples was injected into the column. The samples were eluted from the column using a linear gradient starting from 55% B that progressed to 85% B in 3 minutes. A 2-minute wash at 95% B was used to keep the column sensitivity high and prevent carry-over, and a 3-minute equilibration with 55% B completed the gradient. Column temperature was maintained at 40 °C. The column was attached to the UHPLC with a flow rate maintained at 400 pL min ¹.

[0315] Selected/Multiple reaction monitoring (S/MRM) analyses were carried out on an EVOQ ESI-tri pie quadrupole mass spectrometer (Bruker). The system was operated in negative ion mode. SK-131 calibration standard was used for the creation of the transitions. Collision energy (CE) was optimized for each transition tested. The final method for S/MRM included the following transitions and specifications: 1061.5/944.8 (CE 46V), 1061.5/1002.9 (CE 41V), and 1061.5/943.3 (CE 63V), where the precursor 1061.5 corresponds to SK-131. The rest of the settings for the EVOQ triple quad mass spectrometer were as follows: spray voltage 4500 V (negative ion), cone temperature 350 °C, cone gas 25 units, heated probe temperature 150°C, probe gas 10 units, exhaust gas on, and nebulizer gas 10 units.

[0316] Residual SK-131 concentration was determined by means of the linear least square regression model after external calibration with calibration standards (n=5). Calibration standards and samples were run in triplicates, with two blanks run before and after each sample run. The R2 of the calibration curve was observed to be >0.99.

[0317] Overall, our studies have identified lead therapeutics for synucleinopathies and presented a target (aS oligomers) and a template to account for the heterogeneity of the pathology.

[0318] As used herein, the recitation of "at least one of A, B and C" is intended to mean "either A, B, C or any combination of A, B and C." The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the disclosure. Thus, the present disclosure is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

[0319] The terms and expressions employed herein are used as terms and expressions of description and not of limitation, and there is no intention, in the use of such terms and expressions, of excluding any equivalents of the features shown and described or portions thereof. Each of the various elements disclosed herein may be achieved in a variety of manners. This disclosure should be understood to encompass each such variation, be it a variation of an embodiment of any apparatus embodiment, a method or process embodiment, or even merely a variation of any element of these. Particularly, it should be understood that the words for each element may be expressed by equivalent apparatus terms or method terms— even if only the function or result is the same. Such equivalent, broader, or even more generic terms should be considered to be encompassed in the description of each element or action. Such terms can be substituted where desired to make explicit the implicitly broad coverage to which this invention is entitled.

[0320] As but one example, it should be understood that all action may be expressed as a means for taking that action or as an element which causes that action. Similarly, each physical element disclosed should be understood to encompass a disclosure of the action which that physical element facilitates. Regarding this last aspect, by way of example only, the disclosure of a "protrusion" should be understood to encompass disclosure of the act of "protruding"— whether explicitly discussed or not— and, conversely, were there only disclosure of the act of "protruding", such a disclosure should be understood to encompass disclosure of a "protrusion". Such changes and alternative terms are to be understood to be explicitly included in the description.

[0321] References

[0322] 1. Spillantini MG, et al. a-Synuclein in Lewy bodies. Nature, 1997, 388, 839-40.

[0323] 2. Dettmer U, et al. New insights into cellular a-synuclein homeostasis in health and disease. Curr Opin NeurobioL 2016, 36, 15-22.

[0324] 3. Chiti F, et al. Protein Misfolding, Functional Amyloid, and Human Disease. Annu Rev

Biochem. 2006, 75, 333-66.

[0325] 4. Dawson TM, Dawson L. Molecular Pathways of Neurodegeneration in Parkinson's

Disease. Science. 2003, 302, 819-22.

[0326] 5. Goedert M. a-synuclein and neurodegenerative diseases. Nat Rev Neurosci. 2001, 2,

492-501.

[0327] 6. Pujols J, et al. Chemical Chaperones as Novel Drugs for Parkinson's Disease. Trends

Mol Med. 2020, 26, 408-421.

[0328] 7. Pujols J, et al. Small molecule inhibits a-synuclein aggregation, disrupts amyloid fibrils, and prevents degeneration of dopaminergic neurons. Proc Natl Acad Sci U S A. 2018;115(41):10481-10486.

[0329] 8. Pineda A, Burre J. Modulating membrane binding of a-synuclein as a therapeutic strategy. Proc Natl Acad Sci U S A. 2017, 114, 1223-1225.

[0330] 9. Perni M, et al. A natural product inhibits the initiation of a-synuclein aggregation and suppresses its toxicity. Proc Natl Acad Sci U S A. 2017, 114, E1009-E1017.

[0331] 10. Sangwan S, et al. Inhibition of synucleinopathic seeding by rationally designed inhibitors. Elife. 2020, 9, e46775.

[0332] 11. Ahmed J, et al. Foldamers reveal and validate novel therapeutic targets associated with toxic a-synuclein self-assembly. Nature Commun. 2022, 13, 2273.

[0333] 12. Staats R, et al. Screening of small molecules using the inhibition of oligomer formation in a-synuclein aggregation as a selection parameter. Commun Chem. 2020, 3, 191. [0334] 13. Santos J, et al. a-Helical peptidic scaffolds to target a-synuclein toxic species with nanomolar affinity. Nature Commun. 2021, 12, 3752.

[0335] 14. Kumar S, et al. Foldamer-Mediated Structural Rearrangement Attenuates A|3

Oligomerization and Cytotoxicity. J Am Chem Soc. 2017, 139, 17098-17108.

[0336] 15. Kumar S, et al. Foldamer-mediated manipulation of a pre-amyloid toxin. Nature

Commun. 2016, 7:11412.

[0337] 16. Giasson Bl, et al. Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein. Neuron. 2002, 34, 521-33.

[0338] 17. Kim S, et al. Inhibition of a-synuclein aggregation by MT101-5 is neuroprotective in mouse models of Parkinson's disease. Biomed Pharmacother. 2022, 154, 113637.

[0339] 18. Tatenhorst L, et al. Fasudil attenuates aggregation of a-synuclein in models of

Parkinson's disease. Acta Neuropathol Commun. 2016, 4, 39.

[0340] 19. Betemps D, et al. Alpha-synuclein spreading in M83 mice brain revealed by detection of pathological a-synuclein by enhanced ELISA. Acta Neuropathol Commun. 2014, 2, 29.

[0341] 20. Mougenot AL, et al. Prion-like acceleration of a synucleinopathy in a transgenic mouse model. Neurobiol Aging. 2012, 33, 2225-8.

[0342] 21. Wrasidlo W, et al. A de novo compound targeting a-synuclein improves deficits in models of Parkinson's disease. Brain. 2016, 139, 3217-3236.

[0343] 22. Kuwahara T, et al. Familial Parkinson mutant alpha-synuclein causes dopamine neuron dysfunction in transgenic Caenorhabditis elegans. J Biol Chem. 2006, 281, 334-40.

[0344] 23. Garcia-Moreno JC, et al. Tyrosol, a simple phenol from EVOO, targets multiple pathogenic mechanisms of neurodegeneration in a C. elegans model of Parkinson's disease. Neurobiol Aging. 2019; 82:60-68.

[0345] 24. Sillman NH, et al. A 2D Fragment-Assisted Protein Mimetic Approach to Rescue a-

Synuclein Aggregation Mediated Early and Post-Disease Parkinson's Phenotypes. Nature Commun. under revision, https://www.biorxiv.org/content/10.1101/2022.07.ll.499659vl.

[0346] 25. Liu, X. et al. Discovery of XL01126: A Potent, Fast, Cooperative, Selective, Orally

Bioavailable, and Blood-Brain Barrier Penetrant PROTAC Degrader of Leucine-Rich Repeat Kinase 2. J Am Chem Soc. 2022, 144, 16930-16952.

[0347] 26. Kim YS, et al. Incubation periods and survival times for mice injected stereotaxically with three scrapie strains in different brain regions. J Gen ViroL 1987, 68, 695-702. [0348] 27. Hu X, et al. Optimizing side chains for crystal growth from water: a case study of aromatic amide foldamers. Chem Sci. 2017, 8, 3741-3749.

Claims

1. A synthetic protein molecule comprising: a scaffolding framework; and at least one ligand attached to the scaffolding framework, wherein the scaffolding framework is selected for mimicking a conformation of a naturally occurring monomer protein, wherein a plurality of the naturally occurring monomer protein so selected exhibit a tendency for aggregation thereof to form a plurality of oligomers, and wherein the at least one ligand is selected for an affinity for the plurality of oligomers so as to inhibit aggregation of the plurality of oligomers.

2. The synthetic protein molecule of claim 1, wherein the synthetic protein molecule is a foldamer.

3. The synthetic protein molecule of claim 1, wherein the scaffolding framework is selected for mimicking at least one of helicity, molecule composition, length, curvature, spirality, compatibility with at least one sidechains to be selected as the at least one ligand, and compatibility with ligands with helical nature.

4. The synthetic protein molecule of claim 1, wherein the scaffolding framework is a foldamer- based scaffolding.

5. The synthetic protein molecule of claim 4, wherein the scaffolding framework includes an oligoquinoline scaffolding.

6. The synthetic protein molecule of claim 5, wherein the oligoquinoline scaffolding is a foldamer.

7. The synthetic protein molecule of claim 6, wherein the oligoquinoline scaffolding includes at least one of SK-129 and SK-131.

8. The synthetic protein molecule of claim 1, wherein the naturally occurring monomer protein is at least one of an alpha-Synuclein monomer and amyloid beta 1-42.

9. A method for producing a synthetic protein molecule, the method comprising: selecting a naturally occurring monomer protein, the naturally occurring monomer protein exhibiting a tendency for aggregation to form a plurality of oligomers; selecting a scaffolding framework mimicking a conformation of the naturally occurring monomer protein; and attaching at least one ligand to the scaffolding framework to synthesize the synthetic protein molecule, wherein attaching the at least one ligand to the scaffolding framework includes selecting the at least one ligand for an affinity for the plurality of oligomers so as to inhibit aggregation of the plurality of oligomers.

10. The method of claim 9, wherein the synthetic protein molecule so produced is a foldamer.

11. The method of claim 9, wherein selecting the scaffolding framework includes choosing the scaffolding framework based on at least one of helicity, molecule composition, length, curvature, spirality, compatibility with at least one side chain selected as the at least one ligand, and compatibility with ligands with helical nature.

12. The method of claim 9, wherein selecting the scaffolding framework includes selecting an oligoquinoline scaffolding.

13. The method of claim 12, wherein selecting the oligoquinoline scaffolding includes choosing a foldamer-based scaffolding.

14. The method of claim 13, wherein selecting the oligoquinoline scaffolding further includes choosing at least one of SK-129 and SK-131.

15. The method of claim 9, wherein selecting the naturally occurring monomer protein includes choosing at least one of an alpha-Synuclein monomer and amyloid beta 1-42.