WO2024182543A1 - Small molecules targeting the dna-binding region of apoe4 protein - Google Patents

Abstract

Small molecules targeting the DNA-binding region of ApoE4 protein and methods of use are described. The method may comprise administering to a subject an ApoE4-binding compound that inhibits ApoE4 from binding with a coordinated lysosomal expression and regulation (CLEAR) DNA motif.

Description

SMALL MOLECULES TARGETING THE DNA-BINDING REGION

OF APOE4 PROTEIN

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Patent Application Ser. No. 63/448,962, filed February 28, 2023, the contents of which are incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under P01 AGO 12411 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The disclosed technology is generally directed to treating neurological diseases. More particularly the technology is directed to treating neurological diseases with a ApoE4 binding compound.

BACKGROUND OF THE INVENTION

Alzheimer’s (AD) is a major cause of death in the elderly, and without effective intervention at early stages (currently unavailable), it is disruptive to family stability and costly for the national economy. The current cost to the U.S. government, plus the equivalent cost to caregivers, for care of nearly 7 million Alzheimer patients (as of 2022) is estimated to exceed 250 billion dollars per year. Drugs to cure or prevent Alzheimer’s, or to slow disease progression, as well as the means to detect its onset at early stages, are crucial needs that at present remain unmet.

BRIEF SUMMARY OF THE INVENTION

Small molecules targeting the DNA-binding region of ApoE4 protein and methods of use are described. One aspect of the technology provides for a method of treating a disease. The method may comprise administering to a subject an ApoE4-binding compound that inhibits ApoE4 from binding with a coordinated lysosomal expression and regulation (CLEAR) DNA motif. The ApoE4-binding compound can interfere with the CLEAR-DNA motif binding one or more ApoE4 residues selected from the group consisting of Arg61, Argl72, Argl78, and Argl80. The subject may possess one or two alleles of ApoE(e4). Exemplary ApoE4-binding compounds include 4-[5- (2,3-dihydro-l-benzofuran-2-ylcarbonyl)-4,5,6,7-tetrahydro-lH-imidazo[4,5-c]pyridin-2- yl]benzamide (CBA1), N-[(5-cyclopropyl-lH-pyrazol-3-yl)methyl]-N'-(3,5- dimethylphenyl)malonamide (CBA2), or 3-methyl-l-[l-({6-[(2-pyridin-3-ylethyl)amino]pyridin- 3-yl}carbonyl)piperidin-3-yl]butan-l-one (CBA3). The methods may be useful in a method for treating a neurological disease, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), frontotemporal dementia (FTD), epilepsy, or amyotrophic lateral sclerosis (ALS).

Another aspect of the technology provides for a method for enhancing expression of an autophagy protein. The method may comprise contacting ApoE4 with an ApoE4-binding compound that inhibits ApoE4 from binding to DNA comprising a coordinated lysosomal expression and regulation (CLEAR) motif. Suitably, the autophagy protein may be selected from the group consisting of LC3B, SQSTM-l/p62, LAMP2, and any combination thereof.

Another aspect of the technology provides for a method for enhancing autophagy in a cell. The method may comprise contacting a cell expressing ApoE4 with an ApoE4-binding compound that inhibits ApoE4 from binding to DNA comprising a coordinated lysosomal expression and regulation (CLEAR) motif.

Another aspect of the technology provides for a method for selecting a composition for treating a neurological disease. The method may comprise identifying whether a test compound binds to ApoE4 and determining (i) modulation of expression of an autophagy protein when the test compound is contacted with ApoE4 or cells or tissues expressing ApoE4; or (ii) modulation of autophagy in a cell expressing ApoE4 when the test compound is contacted with the cell expressing ApoE4.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. Figure 1 : Computational modeling of ApoE4-CLEAR DNA complex: Panel (a) shows predicted 3D-structure of ApoE4 protein depicting the DNA-binding region. Panel (b) shows the modeled complex between ApoE4 and CLEAR DNA motifs highlighting the DNA-binding region of ApoE4. Panel (c) shows the modeled DNA-binding region of ApoE4 depicting crucial amino acid residues of ApoE4 involved in CLEAR DNA binding. Panel (d) shows the Root Mean Square Deviation (RMSD) of ApoE4 structure from its initial atomic positions, calculated from simulation trajectories (200-1,000 ns); the dashed box encloses a region of stable conformation. Panel (e) shows a representation of the secondary structures of ApoE4, as they change over time, derived from the simulation time course; the DNA-binding region is indicated by a box.

Figure 2: Small molecules targeting the DNA-binding region of ApoE4: Panel (a) shows the structure of ApoE4 depicting a predicted ligand-binding site (a druggable cavity indicated by mesh) within the DNA-binding region of ApoE4. Panel (b) shows close-up view of this predicted ligand-binding site within the DNA-binding region of ApoE4. Panel (c) shows the predicted docking energy (affinity) of the top-ranked 800 ChemBridge molecules to the ApoE4 protein, using extreme-precision docking in the Schrodinger Glide module. Panel (d) shows binding energies predicted for the top five lead molecules. Panel (e) shows binding-pose predictions for the top five lead molecules binding to ApoE4. The ligand-binding region and interacting amino acids are highlighted in yellow.

Figure 3: Molecular Dynamic Simulation of CBA2 complexed with ApoE4: Panel (a) shows 4 successive snapshots from the docking-simulation trajectory, retrieved at 50-ns intervals to depict binding modes of CBA2 complexed with ApoE4. Panel (b) shows ligand to protein Root Mean Square Deviation (RMSD) calculated from 5000 simulation trajectory frames, corresponding to a 200-ns simulation. Average RMSD was plotted based on predicted RMSD value for each simulation frame. Panel (c) shows predicted average number and types of interactions of CBA2 with amino acid residues of ApoE4 in its DNA-binding region. Panel (d) shows predicted total number of interactions of CBA2 encountered with amino acid residues of ApoE4, over a 200-ns simulation. Panel (e) shows predicted strength of each interactions of CBA2 with individual amino acids present within the DNA-binding region of ApoE4 for 200 ns; red color indicates maximum strength/affmity.

Figure 4: CBA2 treatment restores transcription of key autophagy genes: Panel (a) shows relative levels of p62, LC3b, and LAMP2 transcripts, as determined by qRT-PCR in T98G cells expressing either AP0E3 or AP0E4. Histogram shows relative fold-change ± STDEV. Panel (b) shows relative fold-change in p62, LC3b, and LAMP2 transcript levels determined in T98G-E4 cells after CBA2 treatment. Histogram shows normalized fold-change relative to no-drug (control) treated cells ± STDEV. Significance of differences between drug treated and controls were determined by 2-tailed heteroscedastic /-test of two biological repeats with three technical repeats each. Panel (c) shows relative fold-change in p62, LC3b, andLAMP2 transcript levels determined in T98G-E3 cells after CBA2 treatment. Histogram shows mean normalized fold-change relative to no-drug (control) cells ± STDEV. Significance of differences between drug-treated and control cells was determined by 2-tailed heteroscedastic /-test (V=3). Panel (d) shows a two-dimensional diagram of CBA2. Panel (e) shows protein levels for LAMP2, SQSTM-l &2, and LC3B, as determined by western-blot quantitation in T98G-E4 cells treated with CBA2. Panel (f) shows protein-level data from western blots such as panel e, for SQSTM-1/ p62, LC3B, and LAMP-2 in the same T98G-E4 cells after 10-pM CBA2 treatment, normalized to untreated controls. Panel (g) shows quantitative real-time PCR data, indicating dose-dependent elevation of mRNA levels of SQSTM-l/p62, MAPl/LC3a, MAPl/LC3b and LAMP1 in primary astrocytes cultured from ApoE4-TR mice treated with CBA2 at the indicated doses. Error bars represent SEM. Significances of differences were determined by heteroscedastic / tests for 4 - 6 repeats per group (2 biological repeats and 2 - 3 technical repeats): *** P < 0.0001; ** P < 0.001; and * P < 0.05.

Figure 5, Panel a: CBA2 treatment alleviates Ap42 aggregate burden in a C. elegans model of AD amyloidopathy: panel (a) shows immunofluorescence detection of A0 aggregates in C. elegans expressing human APOEA ubiquitously and Ap::mCherry in neurons, 5 days of treatment with CBA2 (bottom), compared to no-drug controls (top). Panel (b) shows red fluorescence (Ap42::mCherry) intensity per worm, quantitated by Image J, from fluorescence images as shown in a, by imaging 12-15 day-5 adult worms per group. Error bars represent SEM. Significances of differences for panel b were determined from two biological repeats with at least 12-15 worms in each experiment:

< 0.01; ***P < 0.002; and Panel (c) shows the Chemotaxis Index (CI) at day 5 in a C. elegans AD model expressing either human ApoE3 or ApoE4, fed with Ap via Pulsin™- based protein transduction, treated with 10-pM CBA2 from hatch. Significance of the E3-E4 difference in panel c was determined by chi-squared test: ***P < 0.0004.

Figure 6: (a) MD-simulation trajectory analysis of top predicted lead compounds depicts the stability of ligand interactions with each amino acid in the ApoE4 protein, (b) Secondary - structure analysis of ApoE4 complexed with top lead drug compounds shows ligand-induced conformational changes to the ApoE4 structure in 200-ns simulations.

Figure 7: Panel (a) shows relative fold-change in p62, LC3b, and LAMP2 transcript levels determined in T98G-E4 cells after treatment with the top 3 predicted compounds, CBA2, CBA3, and CBA30, each at 1 or 10 pM. Histogram shows normalized fold-change relative to no-drug (untreated control) cells ± STDEV. Panel (b) shows relative fold-change in p62, LC3b, and LAMP2 transcript levels after CBA2 treatment in two replicate experiments. Histogram shows normalized fold-change relative to no-drug (untreated control) cells ± STDEV. Significances of differences were determined from two biological repeats with at least two technical repeats in each experiment: *** P < 0.0001; and ** P < 0.001.

Figure 8: Shows predicted Ligand (drug) Root Mean Square Deviation (RMSD) from its initial docking position in the ApoE4 binding pocket, as it varies during a 200-ns MD simulation. Low RMSD fluctuation after docking indicates a stable interaction of ligand to the ApoE4 structure.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are methods of treating a disease by administering a compound that inhibits ApoE4 from binding with a coordinated lysosomal expression and regulation (CLEAR) DNA motif.

Among the multiple risk factors that may contribute to the development of Alzheimer’s disease (AD), foremost is inheritance of one or two alleles of the apolipoprotein s4 gene (ApoE4, ApoEe4, or ApoE[s4]). Relative to the most common allelic combination ApoE[s3,s3], inheritance of both APOEs4 alleles (genotype ApoE[s4,s4]) confers the single greatest genetic risk factor for development of AD, increasing risk by 12- to 15-fold; inheritance of only one allele increases risk by at least 4-fold. ApoE3, the most common of the three isoforms, is considered to be the normal form. ApoE2 and apoE4 differ from apoE3 by single amino acid Cys 112 to Arg (ApoE4) or Arg 158 to Cys (ApoE2) substitutions. Further, several studies report that compared to ApoE[e3/83] carriers, AD patient carriers of ApoE[s4/s4] have conspicuous elevations in AD-associated hallmark aggregates containing identified “seed” proteins, e.g., plaque containing A01-42 and neurofibrillary tangles containing hyperphosphorylated tau protein. These reports are consistent with an ApoEe4-mediated deficiency of lysosomal autophagy. As evidence, it was found that ApoE4 expression impairs autophagy and is associated with reduced clearance of Ap aggregates. There is evidence of both an ApoE[s4,e4]-related increase in aggregates in brain tissues from Alzheimer’s patients, and a diminution in transcript levels of three mRNAs governed by transcription factor EB (TFEB) — SQSTMl/p62, MAP1LC3B, and LAMP2 — that were significantly lower in brains of AD patients carrying ApoE[e4,s4], relative to brains of AD patients who were ApoE[s3/s3] carriers. ApoE4 protein, in competition with TFEB, directly and specifically interacts with CLEAR (Coordinated Lysosomal Expression and Regulation) DNA motifs, many of which are in the DNA regions just upstream of genes contributing to lysosomal autophagy. The consequence is to diminish TFEB-mediated transcription of autophagy genes, e.g., SQSTMl/p62, MAP1LC3B, MAP1LC3A, ATG5, RUBCN, LAMP1, and LAMP2. Other autophagy genes may also be affected. Pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) show that ApoE[s4]-related decreases in CLEAR-mediated DNA expression are specific to ApoE4, and are not observed in those expressing only ApoE3 or ApoE2. Protein- DNA docking and molecular-dynamic simulation studies may be used to predict three-dimensional models in which ApoE4 and CLEAR DNA motifs interact at the atomic level.

As used herein, an ApoE4 binding compound modulates ApoE4 by inhibiting, suppressing, obstructing, or otherwise preventing its binding to a target DNA sequence. Without being bound by theory, the compound may inhibit, suppress, obstruct, or prevent the binding of ApoE4 to DNA sites (e.g., CLEAR motifs and related sequences) recognized by Transcription Factor EB (TFEB) protein. In some examples, autophagy may be suppressed by ApoE4 interfering with transcription of autophagy proteins. ApoE4 suppresses autophagy by binding to the CLEAR-DNA motifs acting as (or part of) promoter sites for the transcription of autophagy proteins. The autophagy proteins may include p62, LC3b, LC3A, ATG5, Rubicon, LAMP1, LAMP2, and any combination thereof. Other autophagy proteins may also be affected.

Inhibiting, interfering, suppressing, obstructing, or otherwise preventing the binding of ApoE4 to DNA may occur by an ApoE4-binding compound occupying a druggable site on ApoE4. The ApoE4-binding compound binds on, binds near, blocks, sterically hinders, or otherwise impedes binding by limiting access of ApoE4 residues to DNA. The residues may be arginine residues on ApoE4 such as Argl 12, Arg61, Argl72, Argl78, Argl80, or any combination thereof.

The ApoE4 binding compound may include 4-[5-(2,3-dihydro-l-benzofuran-2- ylcarbonyl)-4,5,6,7-tetrahydro-lH-imidazo[4,5-c]pyridin-2-yl]benzamide (CBA1), N-[(5- cyclopropyl-lH-pyrazol-3-yl)methyl]-N'-(3,5-dimethylphenyl)malonamide (CBA2), or 3-methyl- l-[l-({6-[(2-pyridin-3-ylethyl)amino]pyridin-3-yl}carbonyl)piperidin-3-yl]butan-l-one (CBA3). In some embodiments, the ApoE4-binding compound is N-[(5-cyclopropyl-lH-pyrazol-3- yl)methyl]-N'-(3,5-dimethylphenyl)malonamide (CBA2),

A “subject”, “subject in need”, or “a subject in need of treatment” may include a subject having one or two copies of the ApoE4 allele. In some embodiments, a subject in need may have the ApoE(s4,e4) genotype. In other embodiments, a subject in need may have the ApoE(s3,e4) or ApoE(e2,s4) genotype. In some embodiments, the subject may be experiencing a neurological disease, disorder, or condition that affect the brain and/or nerves found throughout the body and in the spinal cord. Neurological diseases may include (but are not limited to) Alzheimer’s disease, epilepsy, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis (ALS). Alternatively, the subject may experience other diseases, including amyloid-associated cardiomyopathy, that are characterized by protein aggregation and/or suppressed autophagy. A “subject in need” or “a subject in need of treatment” may include a subject having a disease, disorder, or condition characterized by obstruction or suppression of autophagy by ApoE4.

In another aspect, disclosed herein is a method for enhancing the expression of an autophagy protein by contacting ApoE4 with an ApoE4-binding compound that inhibits ApoE4 from binding with a coordinate lysosomal expression and regulation (CLEAR) DNA motif. The autophagy proteins may include p62, LC3b, and LAMP2, or any combination thereof. The ApoE4- binding compound binds on, binds near, blocks, sterically hinders, or otherwise impedes binding by limiting access of ApoE4 residues.

Also disclosed herein is a method for enhancing autophagy in a cell including contacting a cell expressing ApoE4 with an ApoE4 binding compound that inhibits ApoE4 from binding with a (CLEAR) DNA motif. In some embodiments, the ApoE4 binding compound binds on, binds near, blocks, sterically hinders, or otherwise prevents binding by limiting access to ApoE4 residues. In some embodiments, the residues are arginine residues on ApoE4. In some embodiments the arginine residues may include Argl l2, Arg61, Argl72, Argl78, Argl80, and any combination thereof.

In another aspect, disclosed herein is a method for selecting a composition for treating a neurological disease. The method includes identifying whether a test compound binds to ApoE4 and determining (i) modulation of expression of an autophagy protein or its RNA transcripts, when the test compound is contacted with ApoE4 or (ii) modulation of autophagy in a cell expressing ApoE4 when the test compound is contacted with the cell expressing ApoE4. In some embodiments, identifying the compound includes in silica screening. In some embodiments, the in silica screening involves determining whether the test compound is predicted to inhibit ApoE4 from binding with a CLEAR-DNA motif. In another embodiment, the in silica screening involves determining whether the test compound interferes with the CLEAR-DNA motif binding one or more ApoE4 residues. In certain cases, the residues are arginine residues on ApoE4. In some embodiments the arginine residues may include Argl l2, Arg61, Argl72, Argl78, Argl80, and any combination thereof.

In another aspect, the method for selecting a composition for treating a neurological disease may include determining the expression of one or more autophagy proteins or RNA transcripts encoding such protein(s). In some embodiments, the autophagy protein may include p62, LC3b, and LAMP2, and any combination thereof. In other embodiments, the method involves determining modulation of autophagy in a cell expressing ApoE4. In some embodiments, the modulation of autophagy may include inhibition of autophagy by more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90%.

Any of the aforementioned methods may be performed in vivo, including as a treatment of a subject having a disease. Additionally, the methods may be used ex vivo and/or in vitro. In some embodiments, the ApoE4-binding compound may be contacted with cells (e.g. mouse or rat astrocytes, T98G-TR4 human glioblastoma cells), tissues (e.g. brain tissue from a subject suspected of experiencing Alzheimer’s disease), organs, microorganisms, or organisms (e.g., worms such as Caenorhahdilis elegans), or in an assay or diagnostic, research, or developmental capacity.

In some embodiments, an assay for diagnostic, research, or developmental purposes may include a method involving steps: i) providing a sample including at least one or at least a portion or fraction from a cell, at least one or at least a portion or fraction from a tissue, at least one or at least a portion or fraction from an organ, at least one or at least a portion or fraction from a microorganism, at least one or a portion or fraction from an organism, or any combination thereof; (ii) optionally determining a baseline transcription of key autophagy genes (such as SQSTMl/p62, MAP1LC3B, MAP 1LC3A, ATG5, RUBCN, LAMP1, and LAMP2) in the sample; (Hi) treating the sample with a ApoE4-binding compound (such as CBA2); (iv) determining transcription of key autophagy genes (such as SQSTMl/p62, MAP1LC3B, MAP1LC3A, ATG5, RUBCN, LAMP1, and LAMP 2) with or without drug addition.

Pharmaceutical Compositions and Methods of Administration

The compounds disclosed herein may be administered as pharmaceutical compositions, and therefore pharmaceutical compositions incorporating the compounds are considered to be embodiments of the subject matter disclosed herein. Such compositions may take any physical form which is pharmaceutically acceptable; illustratively, they can be orally administered pharmaceutical compositions. Such pharmaceutical compositions contain an effective amount of a disclosed compound, which effective amount is related to the daily dose of the compound to be administered. Each dosage unit may contain the daily dose of a given compound or each dosage unit may contain a fraction of the daily dose, such as one-half or one-third of the dose. The amount of each compound to be contained in each dosage unit can depend, in part, on the identity of the particular compound chosen for the therapy and other factors, such as the indication for which it is given. The pharmaceutical compositions disclosed herein may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing well known procedures.

The compounds for use according to the methods disclosed herein may be administered as a single compound or a combination of compounds. For example, a compound that binds to ApoE4 may be administered as a single compound or in combination with another compound that disrupts DNA binding to ApoE4, or inhibits one of the biological activities of ApoE4, or that has a different pharmacological target or activity.

As indicated above, pharmaceutically acceptable salts of the compounds are contemplated and also may be utilized in the disclosed methods. The term “pharmaceutically acceptable salt” as used herein, refers to salts of the compounds which are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds as disclosed herein with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts. It will be appreciated by the skilled reader that most or all of the compounds as disclosed herein are capable of forming salts and that the salt forms of pharmaceuticals are commonly used, often because they are more readily crystallized and purified than are the free acids or bases. Acids commonly employed to form acid addition salts may include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p- bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of suitable pharmaceutically acceptable salts may include the sulfate, pyrosulfate, bi sulfate, sulfite, bi sulfate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleat-, butyne- ,1,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, alpha-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1 -sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline-earth-metal hydroxides, carbonates, bicarbonates, and the like. Bases useful in preparing such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.

The particular counter-ion forming a part of any salt of a compound disclosed herein may not be critical to the activity of the compound, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole. Undesired qualities may include unacceptable solubility or toxicity.

Pharmaceutically acceptable esters and amides of the compounds can also be employed in the compositions and methods disclosed herein. Examples of suitable esters include alkyl, aryl, and aralkyl esters, such as methyl esters, ethyl esters, propyl esters, dodecyl esters, benzyl esters, and the like. Examples of suitable amides include unsubstituted amides, monosubstituted amides, and disubstituted amides, such as methyl amide, dimethyl amide, methyl ethyl amide, and the like.

In addition, the methods disclosed herein may be practiced using solvate forms of the compounds disclosed herein or salts, esters, and/or amides, thereof. Solvate forms may include ethanol solvates, hydrates, and the like. As used herein, the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and/or to prevent or slow the appearance, or to reverse the progression or severity, of resultant symptoms of the named disease or disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration.

As used herein, the phrase “effective amount” shall mean that drug dosage that provides the specific pharmacological response for which the drug is administered in a significant number of subjects in need of such treatment. An effective amount of a drug that is administered to a particular subject in a particular instance will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art.

An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the half-time for clearance of the compound and/or its derivatives through the subject’s metabolism; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

The compounds utilized in the methods disclosed herein may be formulated as pharmaceutical compositions that include: (a) a therapeutically effective amount of one or more compounds disclosed herein; and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutical composition may include the compound in range of about 0.1 to 2000 mg (preferably about 0.5 to 500 mg, and more preferably about 1 to 100 mg). The pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to about 1000 mg/kg body weight (preferably about 0.5 to about 500 mg/kg body weight, more preferably about 50 to about 100 mg/kg body weight). In some embodiments, after the pharmaceutical composition is administered to a subject (e.g, after about 1, 2, 3, 4, 5, or 6 hours post-administration), the concentration of the compound at the site of action may be within a concentration range bounded by end-points selected from 0.001 pM, 0.005 pM, 0.01 pM, 0.5 pM, 0.1 pM, 1.0 pM, 10 pM, and 100 pM (c. ., 0.1 pM - 1.0 pM).

A typical daily dose may contain from about 0.01 mg/kg to about 100 mg/kg (such as from about 0.05 mg/kg to about 50 mg/kg and/or from about 0.1 mg/kg to about 25 mg/kg) of each compound used in the present method of treatment.

Compositions can be formulated in a unit dosage form, each dosage containing from about 1 to about 500 mg of each compound individually or in a single unit dosage form, such as from about 5 to about 300 mg, from about 10 to about 100 mg, and/or about 25 mg. The term “unit dosage form” refers to a physically discrete unit suitable as unitary dosages for a patient, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent, or excipient.

In the disclosed treatment methods, the subject may be administered a dose of a compound as low as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. In some embodiments, the subject may be administered a dose of a compound as high as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47,5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72,5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg, once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. Minimal and/or maximal doses of the compounds may include doses falling within dose ranges having as endpoints any of these disclosed doses (e.g., 2.5 mg - 200 mg).

A minimal dose level of a compound for achieving therapy in the disclosed methods of treatment may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng/kg body weight of the subject. In some embodiments, a maximal dose level of a compound for achieving therapy in the disclosed methods of treatment may not exceed about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng/kg body weight of the subject. Minimal and/or maximal dose levels of the compounds for achieving therapy in the disclosed methods of treatment may include dose levels falling within ranges having as end-points any of these disclosed dose levels (e.g. , 500 - 2000 ng/kg body weight of the subject).

The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition in solid dosage form, although any pharmaceutically acceptable dosage form can be utilized. Exemplary solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid dosage form can be, for example, a fast melt dosage form, controlled release dosage form, lyophilized dosage form, delayed release dosage form, extended release dosage form, pulsatile release dosage form, mixed immediate release and controlled release dosage form, or a combination thereof.

The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes a carrier. For example, the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.

The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Filling agents may include lactose monohydrate, lactose anhydrous, and various starches; examples of binding agents are various celluloses and crosslinked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PHI 01 and Avicel® PHI 02, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™). Suitable lubricants, including agents that act on the flowability of the powder to be compressed, may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame. Examples of flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, fruit flavors, and the like. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.

Suitable diluents may include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH 102; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch; sorbitol; sucrose; and glucose.

Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, com starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof.

Examples of effervescent agents are effervescent couples such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition for delivery via any suitable route. For example, the pharmaceutical composition may be administered via oral, intravenous, intramuscular, subcutaneous, topical, and pulmonary route. Examples of pharmaceutical compositions for oral administration include capsules, syrups, concentrates, powders and granules. In some embodiments, the compounds are formulated as a composition for administration orally (e.g., in a solvent such as 5% DMSO in oil such as vegetable oil).

The compounds utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. Pharmaceutical compositions comprising the compounds may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).

Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or nonaqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis.

Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams.

For applications to the eye or other external tissues, for example the mouth and skin, the pharmaceutical compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the compound may be employed with either a paraffinic or a water- miscible ointment base. Alternatively, the compound may be formulated in a cream with an oil- in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops where the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.

Pharmaceutical compositions adapted for nasal administration where the carrier is a solid include a coarse powder having a particle size (e.g., in the range 20 to 500 microns) which is administered in the manner in which snuff is taken (z.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose). Suitable formulations where the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient. Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tableting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavoring or coloring agents.

Oral administration is an illustrative route of administering the compounds employed in the compositions and methods disclosed herein. Other illustrative routes of administration include transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes. The route of administration may be varied in any way, limited by the physical properties of the compounds being employed and the convenience of the subject and the caregiver.

As one skilled in the art will appreciate, suitable formulations include those that are suitable for more than one route of administration. For example, the formulation can be one that is suitable for both intrathecal and intracerebral administration. Alternatively, suitable formulations include those that are suitable for only one route of administration as well as those that are suitable for one or more routes of administration, but not suitable for one or more other routes of administration. For example, the formulation can be one that is suitable for oral, transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, and/or intrathecal administration but not suitable for intracerebral administration.

The inert ingredients and manner of formulation of the pharmaceutical compositions are conventional. The usual methods of formulation used in pharmaceutical science may be used here. All of the usual types of compositions may be used, including tablets, chewable tablets, capsules, solutions, parenteral solutions, intranasal sprays or powders, troches, suppositories, transdermal patches, and suspensions. In general, compositions contain from about 0.5% to about 50% of the compound in total, depending on the desired doses and the type of composition to be used. The amount of the compound, however, is best defined as the “effective amount”, that is, the amount of the compound which provides the desired dose to the patient in need of such treatment. The activity of the compounds employed in the compositions and methods disclosed herein are not believed to depend greatly on the nature of the composition, and, therefore, the compositions can be chosen and formulated primarily or solely for convenience and economy.

Capsules are prepared by mixing the compound with a suitable diluent and filling the proper amount of the mixture in capsules. The usual diluents include inert powdered substances (such as starches), powdered cellulose (especially crystalline and microcrystalline cellulose), sugars (such as fructose, mannitol and sucrose), grain flours, and similar edible powders.

Tablets are prepared by direct compression, by wet granulation, or by dry granulation. Their formulations usually incorporate diluents, binders, lubricants, and disintegrators (in addition to the compounds). Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts (such as sodium chloride), and powdered sugar. Powdered cellulose derivatives can also be used. Typical tablet binders include substances such as starch, gelatin, and sugars (e.g., lactose, fructose, glucose, and the like). Natural and synthetic gums can also be used, including acacia, alginates, methylcellulose, polyvinylpyrrolidine, and the like. Polyethylene glycol, ethylcellulose, and waxes can also serve as binders.

Tablets can be coated with sugar, e.g., as a flavor enhancer and sealant. The compounds also may be formulated as chewable tablets, by using large amounts of pleasant-tasting substances, such as mannitol, in the formulation. Instantly dissolving tablet-like formulations can also be employed, for example, to assure that the patient consumes the dosage form and to avoid the difficulty that some patients experience in swallowing solid objects.

A lubricant can be used in the tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant can be chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid, and hydrogenated vegetable oils.

Tablets can also contain disintegrators. Disintegrators are substances that swell when wetted to break up the tablet and release the compound. They include starches, clays, celluloses, algins, and gums. As further illustration, com and potato starches, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation-exchange resins, alginic acid, guar gum, citrus pulp, sodium lauryl sulfate, and carboxymethylcellulose can be used.

Compositions can be formulated as enteric formulations, for example, to protect the active ingredient from the strongly acid contents of the stomach. Such formulations can be created by coating a solid dosage form with a film of a polymer which is insoluble in acid environments and soluble in basic environments. Illustrative films include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate.

When it is desired to administer the compound as a suppository, conventional bases can be used. Illustratively, cocoa butter is a traditional suppository base. The cocoa butter can be modified by addition of waxes to raise its melting point slightly. Water-miscible suppository bases, such as polyethylene glycols of various molecular weights, can also be used in suppository formulations.

Transdermal patches can also be used to deliver the compounds. Transdermal patches can include a resinous composition in which the compound will dissolve or partially dissolve; and a film which protects the composition and which holds the resinous composition in contact with the skin. Other, more complicated patch compositions can also be used, such as those having a membrane pierced with a plurality of pores through which the drugs are pumped by osmotic action. As one skilled in the art will also appreciate, the formulation can be prepared with materials (e.g, actives excipients, carriers (such as cyclodextrins), diluents, etc.) having properties (e.g., purity) that render the formulation suitable for administration to humans. Alternatively, the formulation can be prepared with materials having purity and/or other properties that render the formulation suitable for administration to non-human subjects, but not suitable for administration to humans.

Miscellaneous

Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

EXAMPLES

The Inventors hypothesized that targeting small molecules to the ApoE4 protein so as to block its interactions with CLEAR DNA motifs would restore transcription (via TFEB) of essential genes required for efficient clearance of pathognomonic aggregates via lysosomal autophagy. In this work, combining computational and experimental approaches, the Inventors identified several small molecules that specifically target ApoE4 but not ApoE3 protein. Each of these was tested in multiple AD-model systems for its efficacy in restoring TFEB-mediated autophagic transcription, and each was shown to impede ApoE4’s interactions with CLEAR DNA motifs.

Results

Molecular-dynamic simulations predict a druggable pocket in ApoE4

The three-dimensional structure of ApoE4 protein consists chiefly of helices and coils (Fig la), consistent with predictions that ApoE4 is a predominantly disordered protein. As the overall structural dynamics of any disordered protein are highly unstable (examples include ApoE, 14-3- 3 proteins, GFAP, and hyperphosphorylated tau), targeting such proteins with small molecules is challenging (Fig la). This may be especially the case in targeting a specific druggable pocket in ApoE4 that has a direct inhibiting effect on ApoE4 binding to CLEAR DNA (Fig. lb). Fortunately, our predicted protein-DNA interaction analysis identified key amino acid residues of ApoE4 protein, which help to stabilize its interactions with CLEAR-DNA motifs. Protein-DNA interaction analysis predicted that our potential DNA-binding groove in ApoE4 - herein after referred to as the DNA-binding region of the ApoE4 protein - depends on several arginine (positively charged) residues that interact with the negatively-charged phosphate groups of DNA. These include Argl 12, one of the two residues that differentiate ApoE4 from ApoE3 and ApoE2, which was predicted to interact with CLEAR-DNA motifs (Fig 1c). Based on these predictions, in order to impede ApoE4-DNA interactions, we sought a stable druggable pocket near or at this DNA-binding region of ApoE4 (Fig 1, b-c). First, to identify stable a conformation/region of ApoE4, we performed 1-psecond (0.001 sec.) atomistic molecular-dynamic (MD) simulations, using the Desmond simulation package. Simulation trajectory analysis predicted that, despite the overall disordered structure of ApoE4, it contains a region with a relatively stable conformation (structurally stable for >200 ns). Based on simulations of ApoE4 structure over time, the Root Mean Square Deviation (RMSD) from initial atomic positions was predicted to increase during the first ~70 ns of the simulation, and then to maintain a stable conformation for 200 - 220 ns (Fig Id, dashed box), after which RMSD became unstable and increased further (Fig Id). Analyses of the secondary structure during simulations indicated that the majority of helices in and around the ApoE4 DNA-binding region (residues 60 - 145) were stable throughout the simulation (Fig le, dotted box), indicating that this region can be used as a binding pocket for drug discovery. The coils and loops located at the N- and the C-terminal regions of the ApoE4 protein fluctuate extensively and display pronounced unfolding during the simulation (Fig le, bold arrows), a trait typical of many disordered proteins. This fluctuation, especially at the N- and C-terminal domains of the protein is responsible, at least in part, for the observed increase in RMSD values (Fig Id). An analysis of 3 -dimensional structures, captured at multiple time points during the 1 -usccond simulation, predicted that the majority of helical structures in the DNA-binding region of ApoE4 were relatively stable, especially between 100 and 200 ns (Fig Id). Collectively, we take this to indicate that the dynamics of the DNA-binding region of ApoE4 remain sufficiently stable to undergo analysis for a likely druggable pocket, to which we could target small molecules.

Small molecule binding site prediction identify druggable pocket in ApoE4

Based on the average RMSD, we selected a structural conformation after 150 ns, i.e., during the period in which the RMSD maintained a stable plateau, as our starting structure for further analysis. Using Binding Site Prediction tools from the BIOVIA Discovery Studio package, we predicted potential ligand-binding pocket(s) in the ApoE4 structure. We identified a total of 7 potential receptor cavities (druggable pockets) in ApoE4: two sites located at the C-terminal end of the DNA binding region, between several highly fluctuating coils in the ApoE4 protein, and a third site positioned near the center of the ApoE4 DNA-binding region (Fig 2b, inset). Further, MD simulation analysis implies that the helical structures immediately around this predicted druggable site three are quite stable relative to other domains of the ApoE4 protein, suggesting that this target should be suitable for in silico screening of small molecules to impede the DNA- binding activity of ApoE4 (Fig 2a & b). Amino-acid residue analysis predicts an ApoE4 druggable pocket that encompasses all ApoE4 residues deemed to be crucial for CLEAR-DNA binding. This site includes 4 arginine residues, Arg61, Argl72, Argl78, and Argl80 (Fig. 2b), which are involved in ApoE4-CLEAR DNA interactions (Fig 1c). We selected this predicted druggable site, within the DNA-binding groove of ApoE4, for further drug-discovery processes.

High-throughput Virtual Screening predicts potential lead molecules that target the DNA- binding region of ApoE4.

Analyses of brain samples from AD patients implied a mechanism of action in which ApoE4 (but not ApoE3 or ApoE2) competes with TFEB for binding to CLEAR-DNA promoter sites, thus suppressing lysosomal autophagy mediated by TFEB-directed transcription. This led us to posit that a small molecule that stably binds to ApoE4 (but not other ApoE alleles) will block ApoE4 interactions with CLEAR-DNA motifs and restore an autophagy status similar to that of ApoE3 individuals (5-fold less likely to develop AD than carriers of even a single copy of ApoE4). Such molecules would thus allow normal TFEB recognition of CLEAR-DNA binding sites, driving normal transcription of mRNAs that encode autophagy proteins. Based on our molecular modeling and MD simulation, we predicted a druggable pocket in the DNA-binding region of ApoE4 that can be targeted with small molecules to inhibit ApoE4 binding of CLEAR-DNA sites (Fig 2a & b). To identify such novel small molecules, we performed a virtual screening of the ChemBridge structural library (at that time comprising -735,000 compounds) by targeting the predicted druggable site in ApoE4 (dashed boxes in Fig 2). Using the Glide high-throughput virtual-screening protocol from the Schrodinger Suite, we screened the entire ChemBridge library (phase I). From the top-ranked molecules, based on predicted docking scores (roughly equivalent to the Gibbs free energy of protein binding), we selected those with docking scores < -7.3kcal/mol (favoring stable docking) and reassessed their docking to ApoE4 at much higher stringency (phase II), employing Schrodinger Glide docking in extreme-precision mode. Results were again ranked based on docking scores from phase-II screening (Fig 2c). In order to eliminate false positives and to identify the most avidly binding lead compounds, we performed a third stage of virtual screening (phase III), in which we calculated the solvent-based interaction energy (MM-GBSA analysis) within the Schrodinger Prime module. Results were ranked by predicted Gibbs binding free energy, and we selected the five molecules with lowest binding free energy, i. e. , the molecules with greatest stability of binding, and thus highest affinity, for ApoE4 (Fig 2d). Drug bindingpose analysis supported binding of all five predicted top candidates to the expected druggable pocket of ApoE4 protein, within the groove that interacts with CLEAR DNA (Fig 2e). Of these small molecules, the three designated CBA2, CBA23, and CBA30 (Table 1), fit best within the predicted druggable cavity of ApoE4 (Fig 2e).

Table 1. Exemplary compounds identified by virtual screening.

MD simulation of predicted top lead compounds supports CBA2 as a lead candidate to disrupt DNA binding by ApoE4. Based on our computational modeling and multistage docking analysis, we selected five compounds that showed highest binding affinity for ApoE4, at the predicted druggable site from MD analysis. These drug candidates were assessed for stability and dynamics of drug binding to ApoE4, by performing individual 200-ns molecular-dynamic simulations of ApoE4 complexes with each small molecule. Of the top five predicted compounds, CBA2 displayed remarkably stable dynamics and binding over >75% of the 200-ns simulation (Fig. 8), while CBA3 showed moderate stability over -65% of the simulation; in contrast, CBA12, 23, and 30 underwent extensive fluctuations in RMSD during the simulation, indicative of unstable binding (Fig. 8). Drug binding-pose analyses, conducted at multiple time points across the simulation trajectories, predicted stable and consistent CBA2 binding to ApoE4 throughout multiple 200-ns simulations (Fig. 3a). Ligand-to-protein RMSD analyses also showed stable plateaus between 50 - 200 ns for CBA2 complexed with ApoE4, further supporting stable CBA2 binding to ApoE4 protein (Fig. 3b). Similarly, the average number of drug-protein interactions remained stable (nearly constant) throughout each simulation (Fig. 3c & d). Analysis of stability of interactions, between CBA2 and individual amino acid residues within the ApoE4 DNA-binding region, corroborated stability of the drug-protein interaction throughout the simulation (Fig. 3e). Together, these results consistently predict stable and fully accessible binding of CBA2 to the predicted druggable pocket within the DNA-binding region of ApoE4.

CBA2 treatment of T98G-E4 cells restores transcription of key autophagy genes.

In view of our experimental observation that ApoE4 protein interaction with the CLEAR- DNA motif impedes transcription of three seminal lysosomal autophagy-mediating mRNAs - SQSTM-l/p62, MAPl/LC3a, and MAPl/LC3b - in brain tissue from APOE[c4,c4], but not from APOE[s3,e3] AD patients; and in cell cultures of T98G-ApoE4 (T98G-E4) human glioblastoma cells overexpressing ApoE4, we sought to determine if treatment of these cells with our smallmolecule drug CBA2 could restore transcription of these three autophagy-mediating mRNAs. By RT-PCR, we showed this to be true of CBA2 treatment, but only modestly with CBA3 and little to no effect of CBA30. Treatment of T98G-E3 cells with CBA2 resulted in transcript levels of key autophagy genes (SQSTMl/p62, LC3B, and LAMP2) that were 35-60% of abundances in T98G- E4 cells (Fig 4a). In T98G-E4 cells, CBA2 stimulated the expression of 3 autophagy genes by 2.6 - 2.8-fold (SQSTMl/p62), 5.6-fold (LC3B), and 1.5 - 1.6-fold (LAMP2), with relatively little dose response over the range 1 - 10 pM (Fig 4b). This induction was very allele-specific, since T98G-E3 cells were unaffected by CBA2 over the range 1 - 50 pM (Fig 4c). Treatment of T98G- E4 cells with CBA3 or CBA30 produced far less elevation (and less significance) of the same transcripts compared to untreated controls (Fig. 7). Because ApoE3 and ApoE4 differ by only one amino acid, a drug candidate targeting ApoE4 might be expected to exert off-target effects on ApoE3 protein. However, the druggable cavity in the DNA-binding region of ApoE4 is absent from ApoE3, so we did not expect the top-ranked drug candidates from our ApoE4-b inding screen to have any discernable effect on cells expressing only ApoE3. Figure 4d depicts the 2D structure of the target compound, CBA2. Furthermore, we assessed the protein levels of p62, LAMP2, and LC3B in T98G-E3 or T98G-E4 cells after CBA2 treatment. Results show a marked increase in the protein levels after lOuM CBA2 treatment compared to untreated controls in T98G-E4 cells, but unaffected in T98G-E3 cells (Fig 4e). Quantification of the western blot shows a substantial increase in the protein levels for p62, LAMP2, and LC3B treated with CBA2 (Fig 4f) To assess the efficacy of CBA2 in another type of brain cell, we treated primary astrocytes from a targeted- replacement AD mouse model expressing human ApoE4 with CBA2, using qRT-PCR to quantify mRNA levels of SQSTM-l/p62, MAPl/LC3a, and MAP l/LC3b to determine dose-response curves for CBA2 treatment, relative to untreated controls (i.e., zero dose in Fig 4g), substantiating our hypothesis that a small molecule targeting ApoE4 can elevate expression of key autophagy genes needed to clear aggregates. Based on these computational and experimental validations, we propose CBA2 as our potential lead molecule for further analysis.

CBA2 treatment protects against aggregates accruing with Ap42::mCherry, and improves chemotaxis in a C. elegans model of AD.

In human AD brain sections, we found that inheritance of one or two APOEsA alleles decreases lysosomal autophagy; while presence of AP42 plaque adds further stress to the neurons, leading to more pronounced or earlier-onset AD traits relative to APOE[E3,e3] counterparts. Interestingly, expression of human ApoE4 protein along with human AP42 in C. elegans (ApoE4::AP::mcherry worms) recapitulates this AD-like scenario. We speculated that treatment with the ApoE4-specific molecule, CBA2, would improve lysosomal autophagy in ApoE4; Afi::mcherry worms, and thereby reduce the Ap amyloid burden and associated behavioral traits. Worms treated with 10-pM CBA2 from hatch, and imaged for Ap-mCherry fluorescent foci at 7 days post-hatch, showed significantly reduced AP42:: mCherry aggregates if they co-expressed ApoE4, but the drug efficacy was considerably less in worms expressing ApoE3 (Fig 5 a&b). We had previously shown that expression of human AP42 in C. elegans neurons induces accumulation of amyloid-like aggregates, and a decline in the ability to sense and move toward a chemoattractant (1-butanol). ApoE4 transduction, i.e. its introduction by feeding the protein to worms expressing Ap in neurons, produced a 45% decline in chemotaxis compared to worms transduced with ApoE3, demonstrating the deleterious effect of ApoE4 in aggregation and its associated neurotoxicity (Fig 5c). However, CBA2 treatment of ApoE4 (but not ApoE3) worms protected them against loss of chemotaxis, and enhanced expression of autophagy genes LAMP2, SQSTMl/p62, and LC3B, supporting the therapeutic value of targeting ApoE4 with CBA2 (Fig 5c).

Discussion

In previous work, we identified and provided evidence for decreased levels of mRNAs for key autophagy proteins (SQSTMl/p62, LAMP2, LC3B) in AD patient with APOE[<^,'A,i4] genotype relative to those carrying APOE[&3,e3] . We showed that ApoE4 protein, but not ApoE3 or ApoE2, interacts directly and specifically with CLEAR DNA motifs to suppress transcription of autophagy proteins required for clearance of unwanted aggregates via lysosomal autophagy, thus demonstrating that targeting ApoE4 protein therapeutically may preserve or restore autophagy. In this work, using a multifaceted approach, we have identified a stable druggable pocket within the DNA-binding region of ApoE4 protein, and screened this pocket for binding of small molecules to inhibit ApoE4 binding to CLEAR motifs. We have previously modelled the full-length structure of ApoE4 protein and predicted its stable interaction with CLEAR DNA motifs. Based on these models, i.e., ApoE4-CLEAR DNA complex, we identified regions/amino acid residues in ApoE4 protein that are essential participants in CLEAR DNA interactions. Interestingly, this region, i.e., the DNA-binding region of ApoE4, is enriched for arginine residues capable of electrostatic attraction to the negatively-charged phosphates of the DNA backbone, and thus supporting the role of this groove in binding DNA.

Microsecond-scale molecular-dynamic simulations of ApoE4 protein identified a stable region/pocket within the putative DNA-binding groove of ApoE4 protein. We posited that impeding the DNA-binding region by targeting a small molecule to bind within it would inhibit interactions of ApoE4 to CLEAR DNA motifs. High-throughput virtual screening of 750,000 small molecules from the ChemBridge structural library predicted and ranked their affinities for ApoE4 protein. We narrowed the field of candidate drugs by 3 successive cycles of computational screening at progressively higher stringency, followed by molecular-dynamic simulations to calculate the Gibbs binding free energy of the top-ranked five compounds (CBA2, CBA3, CBA12, CBA23 and CBA30). Of these top candidates, only CBA2 and (to a lesser extent) CBA3 were predicted to stably bind within the DNA-binding region of the ApoE4 protein based on molecular- dynamic simulations. These molecules were then pursued for experimental validation in biological systems.

Treatment of T98G-E4 cells with drug candidate CBA2 resulted in the best restoration of mRNA levels of SQSTM-l/p62, MAPl/LC3a, and MAPl/LC3b, which was highly significant relative to untreated control cells; CBA3 was less effective in elevating these transcripts and changes were less significant. This validates our hypothesis that targeting ApoE4 with small molecules can restore transcription of genes crucial for lysosomal autophagy, suppressed in the presence of ApoE4. Since our molecules are directed toward the DNA-binding region of ApoE4, we infer that CBA2 can directly block the ApoE4 DNA-binding site, while altering the structure of the DNA-binding region, together resulting in inhibition of ApoE4 interaction with CLEAR DNA motifs. Further validations in primary astrocyte cultures from a targeted-replacement mouse model (ApoE4-TR) supported CBA2 as a potent inhibitor of ApoE4, resulting in significant restoration of mRNA levels of SQSTM-l/p62, MAPl/LC3a, and MAPl/LC3b in this mouse model.

Others showed that human ApoE4 carriers have lower autophagic flux than those expressing only ApoE3. Moreover, early endosomes (part of an autophagy-related pathway) are enlarged in brains of early-stage AD patients carrying an APOEs4 allele. Ap is trafficked through the endosomal system to the lysosome, but lysosomal clearance of A is deficient in the presence of ApoE4 relative to ApoE3. We previously reported that neuronal expression of human AP42 leads to amyloid-like deposits in C. elegans neurons, impairing chemotaxis in these worms progressively as they age. This decline is somewhat more pronounced in worms carrying the human APOE4 gene, thus mimicking the influence of APOE4 genes in sporadic AD. As these C. elegans strains express human AP42 fused to mCherry in neurons, they provide a convenient reporter to allow the relative assessment of Ap aggregate load via fluorescence microscopy after each drug treatment. Treatment of these worms with CBA2 also elicited significant increases in the protein levels for key autophagy proteins SQSTM-l/p62, LC3B, and LAMP2, compared to placebo or untreated- control worms. In agreement with our observations in brain tissues of human AD patients, the presence of ApoE4 alters the mRNA expression of SQSTM-l/p62, MAPl/LC3a, and MAPl/LC3b significantly relative to ApoE3 worms, suggesting that these worm models parallel the mechanism of action of ApoE4-targeted lead molecules. Further, we noticed that treatment of C. elegans with CBA2 also conferred significant protection against A aggregates relative to untreated or placebo- treated worms, demonstrating the role of diminished autophagy in APOE A individuals, and the need for drugs that restore autophagy to provide protection against protein aggregation.

The Examples demonstrate that compounds blocking the DNA-binding region of ApoE4 protein can restore the autophagy response to levels observed in the presence only of ApoE3 alleles. This discovery allows for development of therapeutic interventions to restore individuals carrying one or two alleles of APOEtA to the lower AD susceptibility of those lacking APOEtA.

Claims

1. A method of treating a disease comprising administering to a subject an ApoE4-binding compound that inhibits ApoE4 from binding with a coordinated lysosomal expression and regulation (CLEAR) DNA motif.

2. The method of claim 1, wherein the subject possesses one or two alleles of ApoE4.

3. The method of any one of claims 1-2, wherein the ApoE4 binding compound interferes with the CLEAR-DNA motif binding one or more ApoE4 residues selected from the group consisting of Arg61, Argl72, Argl78, and Argl80.

4. The method of any one of claims 1-2, wherein the ApoE4 binding compound is selected from the group consisting of: N-[(5-cyclopropyl-lH-pyrazol-3-yl)methyl]-N'-(3,5- dimethylphenyljmalonamide (CBA2) or 3-methyl-l -[1 -({6-[(2-pyridin-3- ylethyl)amino]pyridin-3-yl}carbonyl)piperidin-3-yl]butan-l-one (CBA3).

5. The method of claim 4, wherein the ApoE4-binding compound is N-[(5-cyclopropyl-lH- pyrazol-3-yl)methyl]-N'-(3,5-dimethylphenyl)malonamide (CBA2).

6. The method of any one of the preceding claims, wherein the disease is a neurological disease.

7. The method of claim 6, wherein the neurological disease is Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), frontotemporal dementia (FTD), epilepsy, or amyotrophic lateral sclerosis (ALS).

8. The method of any one of claims 6-7, wherein the neurological disease is characterized by suppressed autophagy.

9. The method of any one of claims 6-8, wherein the neurological disease is characterized by protein aggregation.

10. The method of any one of the preceding claims, wherein the subject is administered a pharmaceutical composition comprising an effective amount of the ApoE4-binding compound.

11. A method for enhancing expression of an autophagy protein comprising contacting ApoE4 with an ApoE4-binding compound that inhibits ApoE4 from binding to DNA comprising a coordinated lysosomal expression and regulation (CLEAR) motif.

12. The method of claim 11, wherein the autophagy protein is selected from the group consisting of LC3B, SQSTM-l/p62, LAMP2, and any combination thereof

13. A method for enhancing autophagy in a cell comprising contacting a cell expressing ApoE4 with an ApoE4-binding compound that inhibits ApoE4 from binding to DNA comprising a coordinated lysosomal expression and regulation (CLEAR) motif.

14. The method of any one of claims 11-13, wherein the ApoE4-binding compound interferes with the CLEAR-DNA motif binding one or more ApoE4 residues selected from the group consisting of Arg61, Argl72, Argl78, and Argl80.

15. The method of any one of claims 11-14, wherein the ApoE4 binding compound is selected from the group consisting of: N-[(5-cyclopropyl-lH-pyrazol-3-yl)methyl]-N'- (3,5-dimethylphenyl)malonamide (CBA2) or 3-methyl-l-[l-({6-[(2-pyridin-3- ylethyl)amino]pyridin-3-yl}carbonyl)piperidin-3-yl]butan-l-one (CBA3).

16. The method of claim 15, wherein the ApoE4-binding compound is N-[(5-cyclopropyl- lH-pyrazol-3-yl)methyl]-N'-(3,5-dimethylphenyl)malonamide (CBA2).

17. The method of any one of claims 11-16, wherein the method is performed in vitro or ex vivo.

18. The method of any one of claims 11-16, wherein the method is performed in vivo.

19. A method for selecting a composition for treating a neurological disease, the method comprising identifying whether a test compound binds to ApoE4 and determining (i) modulation of expression of an autophagy protein when the test compound is contacted with ApoE4 or cells or tissues expressing ApoE4; or (ii) modulation of autophagy in a cell expressing ApoE4 when the test compound is contacted with the cell expressing ApoE4.

20. The method of claim 19, wherein identifying the compound comprises in silico screening to determine whether the test compound inhibits ApoE4 from binding with a CLEAR- DNA motif.

21. The method of claim 20, wherein identifying the compound comprises in silico screening to determine whether the test compound interferes with the CLEAR-DNA motif binding one or more ApoE4 residues selected from the group consisting of Arg61, Argl72, Argl78, and Argl80.

22. The method of any one of claims 19-21, wherein the method comprises determining expression of the autophagy protein.

23. The method of claim 22, wherein the autophagy protein is selected from the group consisting of LC3B, SQSTM-l/p62, LAMP2, and any combination thereof.

24. The method of any one of claims 19-23, wherein the method comprises determining modulation of autophagy in the cell expressing ApoE4.

25. The method of any one of claims 19-24, wherein the determining step is performed in vitro or ex vivo.

26. The method of any one of claims 19-24, wherein the determining step is performed in vivo.