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WO2020043812A1 - Procédé de détection d'un composé modulateur pour le ciblage chimique d'interactions protéine-protéine - Google Patents

Procédé de détection d'un composé modulateur pour le ciblage chimique d'interactions protéine-protéine Download PDF

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Publication number
WO2020043812A1
WO2020043812A1 PCT/EP2019/073058 EP2019073058W WO2020043812A1 WO 2020043812 A1 WO2020043812 A1 WO 2020043812A1 EP 2019073058 W EP2019073058 W EP 2019073058W WO 2020043812 A1 WO2020043812 A1 WO 2020043812A1
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Prior art keywords
protein
seq
variant
interaction
reporter polypeptide
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Inventor
Timurs MACULINS
Andreas Ernst
Mateusz PUTYRSKI
Maria KUZIKOV
Michael Parnham
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Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
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Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention relates to a method for detecting a modulator compound of a protein-protein interaction between a first and a second protein, a modulator compound detected by any of the methods described herein for use in the prophylaxis and/or treatment of a disease, an engineered high affinity variant used in the method of the present invention and a kit-of-parts comprising an engineered high affinity variant according to the present invention and a test system that is capable of detecting the extent of modulation of the modulator compound on said prate in- prate in interaction between the first and the second protein. Further, the present invention relates to the use of said methods and also relates to compositions comprising said modulator compound.
  • PPIs Protein-Protein interactions
  • HTS high-throughput screening
  • Dixon et a /. 1 describes protein-fragment complementation assays, looking at protein- protein interactions (PPIs) in cells, without any identification of modulators or a compound, which influences the investigated protein-protein interaction.
  • PPIs protein- protein interactions
  • Rahighi et al 2 reported on the binding capability of the UBAN (ubiquitin binding in ABIN and NEMO proteins) motif of NEMO to linear ubiquitin chains, without presenting any specific assay therefore or any assay, which allows the screening for compounds, which modulate or inhibit the respective protein-protein interaction.
  • Wiechmann et al. 3 investigated the small ubiquitin-like modifiers (SUMOs) and found that the E2-conjugated enzyme Ubc9 catalyzes the conjugation of SUMOs, meaning that the inhibition of the enzyme Ubc9 impairs SUMO chain formation.
  • SUMOs small ubiquitin-like modifiers
  • the present invention is the first report of a method that provides proof-of- principle evidence for the application of protein engineering in the development of robust cell- based assays. Due to this method according to the present invention, one can envisage a wide application of this strategy in HTS, accelerating drug discovery programs in the future.
  • the present invention relates to a method for detecting a modulator compound of a protein-protein interaction between a first and a second protein, comprising the steps of:
  • step (b) comparing the measurements made in step (b), wherein an alteration of the signal of said reporter polypeptide in step a) ii) to the signal of said reporter polypeptide in step a) i) indicates the presence of a modulator compound of said protein-protein interaction, and
  • step c) quantifying the extent of modulation of the protein-protein interaction by the test modulator compound by step c).
  • the alteration of the signal of said reporter polypeptide in step a) ii) to the signal in step a) i) is a reduction or deletion of the signal of the reporter polypeptide.
  • step a) of the method is within a cell, cell lysate, within a reaction mixture or outside a cell, preferably within a cell.
  • the first protein of said protein-protein interaction is fused to a first part of the reporter polypeptide and the second protein of said protein-protein interaction is fused to a second part of the reporter polypeptide.
  • the modulator compound is a compound being capable of inhibiting said protein-protein interaction.
  • the modulator compound is a compound being capable of inhibiting said protein-protein interaction, preferably a protein-protein interaction of the nuclear factor kappa B signaling pathway, more preferably the protein-protein interaction between NEMO and linear ubiquitin chains and most preferably the interaction between the UBAN domain of NEMO (SEQ ID NO: 2) and an Ubiquitin variant (SEQ ID NO: 6) as defined herein.
  • fusing the first protein of said protein-protein interaction with the reporter polypeptide or part thereof is carried out by recruiting the first protein via an interaction between a DNA binding domain and an upstream activator sequence of the reporter polypeptide or part thereof to the reporter polypeptide or part thereof.
  • the first protein of said protein-protein interaction is recruited via an interaction between a DNA binding domain and an upstream activator sequence of the reporter polypeptide or part thereof to the reporter polypeptide or part thereof.
  • the binding of the DNA binding domain to the upstream activator sequence of the reporter polypeptide and the binding of a transcription activation domain to a promoter sequence of the reporter polypeptide causes expression of the reporter polypeptide.
  • a second fusion is build between the second protein of said protein-protein interaction and the transcription activation domain. The second fusion is build for the method of the present invention or for the test system described herein.
  • the protein-protein interaction is the interaction between a protein of interest or a part thereof and an engineered high affinity variant, preferably the interaction between the UBAN domain of NEMO and an Ub variant, the interaction between UBC9 and a small ubiquitin-like modifier 2 (SUM02) variant, the interaction between the ATG3 enzyme and a GATE16 variant, or the interaction between OPTN and a LC3B variant.
  • an engineered high affinity variant preferably the interaction between the UBAN domain of NEMO and an Ub variant, the interaction between UBC9 and a small ubiquitin-like modifier 2 (SUM02) variant, the interaction between the ATG3 enzyme and a GATE16 variant, or the interaction between OPTN and a LC3B variant.
  • the first protein of the protein-protein interaction is selected from the group consisting of UBAN domain of NEMO (SEQ ID NO: 2), UBC9 enzyme (SEQ ID NO: 3), ATG3 enzyme (SEQ ID NO: 4) and OPTN (SEQ ID NO: 5), preferably the UBAN domain of NEMO (SEQ ID NO: 2)
  • the second protein of the protein-protein interaction is selected from the group consisting of an Ub variant (SEQ ID NO: 6), a small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), a GATE 16 variant (SEQ ID NO: 8) and a LC3B variant (SEQ ID NO: 9), preferably an Ub variant (SEQ ID NO: 6).
  • the first protein of the protein-protein interaction is selected from the group consisting of UBAN domain of NEMO (SEQ ID NO: 18), UBC9 enzyme (SEQ ID NO: 3), ATG3 enzyme (SEQ ID NO: 4) and OPTN (SEQ ID NO: 5), preferably the UBAN domain of NEMO (SEQ ID NO: 18), and/or the second protein of the protein-protein interaction is selected from the group consisting of an Ub variant (SEQ ID NO: 6), a small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), a GATE 16 variant (SEQ ID NO: 8) and a LC3B variant (SEQ ID NO: 9), preferably an Ub variant (SEQ ID NO: 6).
  • the reporter polypeptide according to the present invention is selected from the group consisting of a split ubiquitin, a fluorescent protein, preferably the green, red or yellow fluorescent protein, more preferably a split green fluorescent protein, a beta-lactamase, a beta-galactosidase, chloramphenicol acetyltransferase (CAT) and a luciferase, preferably Firefly luciferase, Renilla luciferase or split nanoluciferase.
  • a split ubiquitin a fluorescent protein, preferably the green, red or yellow fluorescent protein, more preferably a split green fluorescent protein, a beta-lactamase, a beta-galactosidase, chloramphenicol acetyltransferase (CAT) and a luciferase, preferably Firefly luciferase, Renilla luciferase or split nanoluciferase.
  • CAT chloramphenicol
  • the method is a high-throughput screening assay, preferably a cell-based high-throughput screening assay, for detecting the modulator compound of the prate in -prate in interaction.
  • the present invention also relates to a modulator compound detected by any of the methods described herein for use in the prophylaxis and/or treatment of a disease selected from the group consisting of cancer, chronic inflammatory diseases, autoimmune diseases, neurodegenerative diseases, arthritides and infectious diseases.
  • the present invention also relates to a kit comprising a modulator compound as described herein.
  • the present invention is also directed to a protein or engineered high affinity variant selected from the group consisting of an Ub variant (SEQ ID NO: 6), a small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), a GATE 16 variant (SEQ ID NO: 8) and a LC3B variant (SEQ ID NO: 9), preferably an Ub variant (SEQ ID NO: 6).
  • a protein or engineered high affinity variant selected from the group consisting of an Ub variant (SEQ ID NO: 6), a small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), a GATE 16 variant (SEQ ID NO: 8) and a LC3B variant (SEQ ID NO: 9), preferably an Ub variant (SEQ ID NO: 6).
  • the present invention also relates to a kit comprising a protein or engineered high affinity variant selected from the group consisting of an Ub variant (SEQ ID NO: 6), a small ubiquitin- like modifier 2 (SUM02) variant (SEQ ID NO: 7), a GATE16 variant (SEQ ID NO: 8) and a LC3B variant (SEQ ID NO: 9), preferably an Ub variant (SEQ ID NO: 6) as described herein.
  • a protein or engineered high affinity variant selected from the group consisting of an Ub variant (SEQ ID NO: 6), a small ubiquitin- like modifier 2 (SUM02) variant (SEQ ID NO: 7), a GATE16 variant (SEQ ID NO: 8) and a LC3B variant (SEQ ID NO: 9), preferably an Ub variant (SEQ ID NO: 6) as described herein.
  • the present invention relates to a kit-of-parts comprising an engineered high affinity variant as described herein and a test system that is capable of detecting the extent of modulation of the modulator compound on a protein-protein interaction between a first protein and a second protein, wherein the second protein is the engineered high affinity variant.
  • the test system further comprises a reporter polypeptide, wherein the reporter polypeptide is selected from the group consisting of a split ubiquitin, a fluorescent protein, preferably the green, red or yellow fluorescent protein, more preferably a split green fluorescent protein, beta-lactamase, beta-galactosidase, chloramphenicol acetyltransferase (CAT) and a luciferase, preferably Firefly luciferase or Renilla luciferase or split nanoluciferase.
  • a reporter polypeptide is selected from the group consisting of a split ubiquitin, a fluorescent protein, preferably the green, red or yellow fluorescent protein, more preferably a split green fluorescent protein, beta-lactamase, beta-galactosidase, chloramphenicol acetyltransferase (CAT) and a luciferase, preferably Firefly luciferase or Renilla luciferase or split
  • the inventors found for the first time the generation of a specific high affinity variant or engineered affinity variant that targets the interaction interface between the UBAN domain of NEMO and linear ubiquitin chains, an interaction that is critical for NF-kB signalling pathway activation in response to TNF receptor stimulation 2 .
  • the UBAN domain of NEMO is usually engaged in binding to linear ubiquitin chains, wherein the mentioned variant specifically targets the molecular surface of the UBAN domain of NEMO that is responsible for this binding.
  • the inventors demonstrate that high affinity variants that were developed against NEMO, UBC9, ATG3 and OPTN protein targets significantly improve cell-based assay robustness and thus, enable chemical compound screening against PPIs of these proteins. These variants are used to illustrate the applicability of the method of the present invention against multiple functionally unrelated protein targets.
  • Figure 1 shows a theoretical description of the present invention.
  • Figure 1A shows the schematic representation of a cellular assay based on the Dual Luciferase Reporter system (DLR) (Promega).
  • DLR Dual Luciferase Reporter system
  • Vectors modified to express a target protein (Target, relating to the first protein of the protein-protein interaction according to the present invention) fused to the DNA- binding domain (DBD) and an engineered high affinity variant (Variant, relating to the second protein of the protein-protein interaction according to the present invention) generated against the target protein fused to the VP16 activation (ACT) domain are co- transfected into mammalian cells along with the firefly luciferase reporter vector (SEQ ID NO: 12), which contains five Gal4 binding sites upstream of the minimal TATA box.
  • DLR Dual Luciferase Reporter system
  • FIG. 1B shows the schematic representation of a cellular assay based on the NanoBiT system (Promega).
  • the system vectors are modified to express a target protein, relating to the first protein of the protein-protein interaction according to the present invention, and an engineered high affinity variant, relating to the second protein of the protein- protein interaction according to the present invention, as fusions to either small bit (SmBiT), relating to a first part of the reporter polypeptide of the present invention, or large bit (LgBiT), relating to a second part of the reporter polypeptide of the present invention, of split nanoluciferase (Active NANO), relating to the reporter polypeptide according to the present invention, are co-transfected into mammalian cells.
  • small bit relating to a first part of the reporter polypeptide of the present invention
  • LgBiT large bit
  • Active NANO split nanoluciferase
  • Figure 2 shows the identification of the best high affinity Ub variant.
  • Figure 2A shows a competition assay of generated Ub variants to the UBAN domain of NEMO (UBAN NEMO . SEQ ID NO: 2).
  • IC 50 values were determined in a competition ELISA assay as the concentration of mouse UBAN MEMO (SEQ ID NO: 18) in solution that blocked 50% of Ub variant binding to immobilized mouse UBAN NEMO (SEQ ID NO: 18).
  • Figure 2B shows the sequence alignment of wild type (wt) Ub (SEQ ID NO: 14) and Ubv-A (SEQ ID NO: 6), corresponding to the best high affinity Ub variant according to the present invention.
  • the amino acid sequence alignment shows only those positions that were diversified in the Ub variant library. Positions that were conserved as wt sequence are indicated by dashes.
  • Affinity estimate is based on phage ELISA while competing with mUBAN NEM0 (SEQ ID NO: 18) in solution for binding to immobilized GST- mUBAN NEM o-
  • Figure 3 shows the specificity and selectivity validation of Ubv-A (SEQ ID NO: 6).
  • Figure 3A shows that HEK293 cells were transiently co-transfected with control or vectors expressing HA-tagged Ubv-A (SEQ ID NO: 6) in combination with either wild type (wt) (SEQ ID NO: 2) or F312A human UBAN NEMO (SEQ ID NO: 19).
  • Ubv-A specifically (SEQ ID NO: 6) co- immunoprecipitated wt (SEQ ID NO: 2) and not F312A mutant UBAN NEMO (SEQ ID NO: 19).
  • Figure 3B indicates that GST fusions were immobilized on the ELISA plate, followed by incubation with the phage expressing Ubv-A (SEQ ID NO: 6) and detection of bound phages spectrophotometrically (optical density at 450 nm). Background binding to BSA was subtracted from the signal.
  • Figure 3C shows surface plasmon resonance data of the interaction between UBAN of (i) NEMO (SEQ ID NO: 18), (ii) OPTN (SEQ ID NO. 22) and (iii) ABIN1 (SEQ ID NO: 25) with Ubv-A (SEQ ID NO: 6). GST-fused UBAN nemo .
  • UBAN 0 PT N and UBAN A BI N I were immobilized on a GST antibody-coated CMD200m sensor chip.
  • Ubv-A (SEQ ID NO: 6) molecules were loaded over the chip. Each measurement was performed at the indicated concentrations, and the fitted curves were used to calculate equilibrium dissociation constant (K D ) values.
  • Figure 4 shows proof-of-concept using the assay technology according to Figure 1A (Dual Luciferase reporter system, DLR-system).
  • Figure 4A shows that HEK293 cells were co- transfected with modified DLR assay vectors encoding the first fusion containing DBD- hUBAN NEM0 and the second fusion containing ACT-2xUbv-A of the present invention in combination with the Firefly reporter vector. Following 20-hour incubation cells were lysed and then assayed for Firefly and Renilla luciferase. Firefly/Renilla ratio served as normalization control for transfection efficiency between samples (as recommended by Promega assay protocols).
  • the background level of the DLR assay is measured in the presence of DBD or ACT empty vectors.
  • Figure 4B shows that HEK293 cells were co-transfected with modified DLR assay vectors encoding a fusion of DBD to either control SUM02 (SEQ ID NO: 15) or high affinity SUM02 variant (SEQ ID NO: 7) and a fusion containing ACT fused to UBC9 (SEQ ID NO: 3) in combination with the Firefly reporter vector.
  • Figure 5 shows proof-of-concept being based on the NanoBiT assay technology.
  • Figure 5A shows that HEK293 cells were reverse transfected with modified NanoBiT assay vectors encoding fusions of SmBiT to hUBAN NEM0 (SEQ ID NO: 2) and LgBiT to Ubv-A (SEQ ID NO: 6). Following 20-hour incubation cells were assayed for nanoluciferase (NANO) activity according to manufacturer’s protocol (Promega). The background assay activation by LgBiT fusion was determined by transfection of cells with LgBiT-Ubv-A with SmBiT fusion to HaloTag.
  • NANO nanoluciferase
  • Figure 6 shows a method validation in high-throughput screening.
  • Figure 6A shows that HEK293, CHO or A549 cell lines were reverse transfected with modified NanoBiT constructs by either LgBiT-Ubv-A and SmBiT-wt UBAN nem0 or LgBiT-Ubv-A and SmBiT-HaloTag construct combinations, denoted as assay or control, respectively.
  • Transfected cells were then dispensed into 384-well plate and incubated for 20 hours. Nanoluciferase activity was assayed 30 minutes following substrate addition to the wells to determine relative luminescence units (RLU).
  • RLU relative luminescence units
  • A549 cells were reverse transfected with NanoBiT constructs expressing LgBiT-Ubv-A and SmBiT-wt UBAN NEMO , dispensed into 384-well plates on two separate occasions (shown as repeat 1 and repeat 2). Following 20-hour incubation, compounds were dispensed at 10 mM final concentration (f.c.) and nanoluciferase activity was determined two hours after compound addition. The response for each individual test compound was then quantified relative to the reference Aloe Emodin inhibitor (f.c. 50 mM) and depicted as percent inhibition.
  • the present invention relates to a method for detecting a modulator compound of a protein-protein interaction (PPI) between a first and a second protein, comprising the steps of: a) fusing the first protein of said protein-protein interaction with a reporter polypeptide or part thereof, and fusing the second protein of said protein-protein interaction with a transcription activation domain or a part of the reporter polypeptide,
  • PPI protein-protein interaction
  • step c) comparing the measurements made in step (b), wherein an alteration of the signal of said reporter polypeptide in step a) ii) to the signal of said reporter polypeptide in step a) i) indicates the presence of a modulator compound of said protein-protein interaction, and d) quantifying the extent of modulation of the protein-protein interaction by the test modulator compound by step c).
  • the method of the present invention provides means by which it is possible to identify whether or not a test modulator compound is a modulator compound of a protein-protein interaction (PPI) of interest.
  • the method also allows quantification of the extent to which a test modulator compound is able to modulate PPIs, preferably the extent to which a modulator compound is able to inhibit a given PPI. This in turn allows a comparison to be made between the ability of different test modulator compounds or different doses of the same test modulator compound, to modulate PPIs.
  • the method does not only enable the detection of a modulator compound of the protein- protein interaction between a first and a second protein, but also enables to identify or screen for such a modulator compound.
  • detecting “identifying” and“screening” can be used interchangeably.
  • the term“protein” means a molecule in which two or more amino acids are linked by (a) peptide bond(s), and modified products thereof.
  • the term is a concept including not only full-length proteins, but also so-called oligopeptides and polypeptides.
  • the modification of the protein include phosphorylation, glycosylation, palmitoylation, prenylation (for example, geranylgeranylation), methylation, acetylation, ubiquitination, SUMOylation, hydroxylation, and amidation.
  • the term“protein” also comprises parts of proteins as defined or described herein.
  • the first protein of the protein-protein interaction is selected from the group consisting of the UBAN domain of NEMO (SEQ ID NO: 2), the UBC9 enzyme (SEQ ID NO: 3), the ATG3 enzyme (SEQ ID NO: 4) and OPTN (SEQ ID NO: 5).
  • the first protein of the protein-protein interaction is the UBAN domain of NEMO (SEQ ID NO: 2).
  • the second protein of the protein-protein interaction is selected from the group consisting of an Ub variant (SEQ ID NO: 6), a small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), a GATE 16 variant (SEQ ID NO: 8) and a LC3B variant (SEQ ID NO: 9).
  • the second protein of the protein-protein interaction is an Ub variant (SEQ ID NO: 6).
  • The“protein- protein interaction between a (the) first protein and a (the) second protein” includes not only direct interactions, but also indirect interactions such as an interaction for forming a complex. It might even be possible that in such a complex another molecule (protein, nucleic acid, sugar, lipid, low-molecular-weight compound, or the like) is interposed between the first protein and the second protein.
  • the present invention relates in one specific embodiment to a method for detecting a modulator compound of the protein-protein interaction (PPI) between the UBAN domain of NEMO according to SEQ ID NO: 2 and an Ub variant according to SEQ ID NO: 6, comprising the steps of:
  • step c) comparing the measurements made in step (b), wherein an alteration of the signal of said reporter polypeptide in step a) ii) to the signal of said reporter polypeptide in step a) i) indicates the presence of a modulator compound of said protein-protein interaction, and d) quantifying the extent of modulation of the protein-protein interaction by the test modulator compound by step c).
  • the present invention also relates in one specific embodiment to a method for detecting a modulator compound of the protein-protein interaction (PPI) between the UBC9 enzyme according to SEQ ID NO: 3 and a small ubiquitin-like modifier 2 (SUM02) variant according to SEQ ID NO: 7, comprising the steps of:
  • step c) comparing the measurements made in step (b), wherein an alteration of the signal of said reporter polypeptide in step a) ii) to the signal of said reporter polypeptide in step a) i) indicates the presence of a modulator compound of said protein-protein interaction, and d) quantifying the extent of modulation of the protein-protein interaction by the test modulator compound by step c).
  • the present invention also relates in one specific embodiment to a method for detecting a modulator compound of the protein-protein interaction (PPI) between the ATG3 enzyme according to SEQ ID NO: 4 and a GATE16 variant according to SEQ ID NO: 8, comprising the steps of:
  • step c) comparing the measurements made in step (b), wherein an alteration of the signal of said reporter polypeptide in step a) ii) to the signal of said reporter polypeptide in step a) i) indicates the presence of a modulator compound of said protein-protein interaction, and d) quantifying the extent of modulation of the protein-protein interaction by the test modulator compound by step c).
  • the present invention also relates in one specific embodiment to a method for detecting a modulator compound of the protein-protein interaction (PPI) between the OPTN protein according to SEQ ID NO: 5 and a LC3B variant according to SEQ ID NO: 9, comprising the steps of:
  • the OPTN enzyme according to SEQ ID NO: 5 and the LC3B variant according to SEQ ID NO: 9 are capable of binding to each other;
  • step c) comparing the measurements made in step (b), wherein an alteration of the signal of said reporter polypeptide in step a) ii) to the signal of said reporter polypeptide in step a) i) indicates the presence of a modulator compound of said protein-protein interaction, and d) quantifying the extent of modulation of the protein-protein interaction by the test modulator compound by step c).
  • Fusing the first protein of said protein-protein interaction with a reporter polypeptide or part thereof can be called, in the context of the present invention, a“first fusion”.
  • Fusing the second protein of said protein -protein interaction with a transcription activation domain or a reporter polypeptide or part thereof can be called, in the context of the present invention, a“second fusion”.
  • the“first protein of the protein- protein interaction” or the“second protein of the protein -protein interaction” only comprises a part of a respective protein for building the first or second fusion, meaning that the part thereof is about 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%,
  • The“reporter polypeptide” as used in the context of the present invention can be any protein, which is encoded by a reporter gene, wherein a reporter gene can be any genetic material, whose utilization by the transcriptional and/or translational apparatus derived from a cell (e.g., intact cell or cell-free extract) can be monitored, for example, any DNA sequence that is fused to a promoter sequence of interest so as to measure the relative activity of the promoter sequence.
  • the “reporter polypeptide” can be any luminescence-generating polypeptide, which is any polypeptide that is capable to generate bioluminescence.
  • the“reporter polypeptide” according to the present invention can be selected from the group consisting of a split ubiquitin, a fluorescent protein, more preferably the green, red or yellow fluorescent protein, most preferably a split green fluorescent protein, beta-lactamase, beta- galactosidase, chloramphenicol acetyltransferase (CAT) and a luciferase, more preferably Firefly luciferase, Renilla luciferase or split nanoluciferase.
  • Luciferases catalyze light emission in the presence of their substrates, luciferins, and this property has made them attractive for various types of assay systems.
  • luciferases There are different types of luciferases that occur in species including beetles, bacteria, worms, fungi, and squid with several of them cloned and tested for molecular biology research. Each of these luciferases has different characteristics, which makes them attractive for certain applications, but not optimal for others. For high-throughput applications and assays, an optimal luciferase would display the following characteristics: (1 ) enzyme stability over a variety of conditions; (2) highlight output for increased sensitivity; (3) non-invasive monitoring of enzymatic activity at different time points in real-time; and (4) the catalysis of stable light emission for minimal variability between thousands of screened wells.
  • the alteration of the signal of said reporter polypeptide in step a) ii) to the signal of said reporter polypeptide in step a) i) is a reduction or deletion of the signal of the reporter polypeptide.
  • the signal of the reporter polypeptide is any signal of a reporter polypeptide as defined above.
  • the signal of the reporter polypeptide is a luminescence signal.
  • The“alteration of the signal of the reporter polypeptide” in the context of the present invention can be any reduction of the signal received in step a) ii) of the methods as described above, meaning that the signal of the reporter polypeptide measured in step a) i) is only about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%,
  • the test modulator compound according to the present invention is not a modulator compound, meaning that the test modulator compound does not, in such a case, modulate the protein-protein interaction between the first and the second protein of said protein-protein interaction.
  • step a) of any method of the present invention as described herein is carried out within a cell, cell lysate, within a reaction mixture or outside a cell, preferably within a cell.
  • the first protein of said protein-protein interaction is fused to a first part of the reporter polypeptide and the second protein of said protein-protein interaction is fused to a second part of the reporter polypeptide.
  • the first part of the reporter polypeptide can be 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,
  • the second part of the reporter polypeptide can be 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%,
  • the reporter polypeptide can be a split ubiquitin, a split luciferase or a split fluorescence protein, preferably a split luciferase.
  • a split luciferase is deactivated, when divided into an N-terminal and a C-terminal side fragment, being split in a particular position. Then both fragments show no luciferase activity. Luciferase activity can be measured, when both fragments build a functional complementation.
  • the N-terminal fragment and C-terminal fragment of the split luciferase is also known as "split luciferase" collectively, wherein usually the smaller fragment thereof is called the “SmBiT” and the larger fragment thereof is called the “LgBiT”.
  • the SmBiT of the split luciferase can be 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%,
  • the LgBiT of the split luciferase can be 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%,
  • test modulator compound when then a test modulator compound is able to bind to the first or the second protein of the protein- protein interaction, the protein-protein interaction cannot or in a reduced way take place so that the split luciferase fragments cannot get into close proximity with each other.
  • the test modulator compound can then be named a modulator compound or can have the function of a modulator compound.
  • Coupled is not particularly limited, and a covalent bond, ionic bond, hydrogen bond, van der Waals forces, a hydrophobic bond or a non-covalent bond may also be encompassed by these terms.
  • the term“quantifying” or“quantifying the extent of the modulation of the protein-protein interaction” as used in the context of the present invention means measuring a reporter polypeptide activity, especially measuring a reduction in reporter polypeptide activity and determining an extent to which a modulator reduces reporter polypeptide activity.
  • the term “measuring the/ a signal of the reporter polypeptide” includes“measuring reporter polypeptide activity”,“measuring a reduction in reporter polypeptide activity” and can be used synonymously in the context of the present invention.
  • the modulator compound is a compound being capable to modulate the protein-protein interaction, preferably is capable of inhibiting said protein-protein interaction.
  • the modulator compound is a compound being capable of inhibiting a protein-protein interaction, preferably a protein-protein interaction of the nuclear factor kappa B signaling pathway, more preferably the protein-protein interaction between NEMO and linear ubiquitin chains and most preferably the interaction between the UBAN domain of NEMO (SEQ ID NO: 2) and an Ubiquitin variant (SEQ ID NO: 6).
  • a modulator compound according to the present invention can be an inhibitor.
  • An “inhibitor” as used herein is defined as a compound/molecule reducing or blocking the activity of a target molecule and/or signaling pathway. The inhibitor may achieve this effect by reducing or blocking the transcription of the gene encoding the protein to be inhibited and/or reducing/blocking the translation of the mRNA encoding the protein to be inhibited. It can also be that the protein to be inhibited performs its biochemical function with decreased efficiency in the presence of the inhibitor or that the protein to be inhibited performs its cellular function with reduced efficiency in the presence of the inhibitor.
  • the term “inhibitor” encompasses both molecules/compounds that have a directly reducing/blocking effect on the specific signaling pathway, but also molecules that are indirectly inhibiting, e.g. by interacting, for example, with molecules that positively regulate (e.g. activate) said pathway.
  • the inhibitor can also be an antagonist of the pathway (receptor) to be inhibited.
  • Methods for testing if a compound/molecule is capable to reduce or block the activity of a target molecule and/or signaling pathway are known to the skilled artesian.
  • An“inhibitor” within the context of the present invention can also include a modulator compound that affects the activity of the reporter polypeptide and therefore reduces reporter polypeptide activation or activity.
  • the term“affects” in this context of the present invention means that the activity of the reporter polypeptide is reduced for about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,
  • the modulator compound/molecule that can be used as an inhibitor can be any compound/molecule, which can reduce or block the respective pathway or which inhibits an activator of the signaling (pathway) to be inhibited.
  • exemplary inhibitors can include suitable binding proteins as described herein, which are directed e.g. against activators of a certain pathway.
  • the inhibitor can also be a nucleic acid molecule, such as an RNA, siRNA, miRNA or a non-proteinaceous aptamer as described herein. Also the nucleic acid molecules may be used to suppress an activator of a pathway to be inhibited.
  • the inhibitor is a small molecule or protein/polypeptide.
  • a small molecule can have a low molecular weight of less than 900 daltons (da), less than 800 da, less than 700 da, less than 600 da or less than 500 da.
  • the size of a small molecule can be determined by methods well known in the art, e.g., mass spectrometry.
  • the inhibitor can also be an antagonist of the pathway/signaling pathway to be inhibited.
  • An inhibitor may reduce or decrease the pathway to be inhibited by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,
  • a block of the pathway to be inhibited is present when the pathway is inhibited by 100%, when compared to the activity of the pathway without the addition (or before the addition) of the inhibitor.
  • such a modulator compound according to the present invention can also be an activator.
  • activator is defined as a compound/molecule enhancing or achieving the activity of a target molecule and/or signaling pathway. The activator may achieve this effect by enhancing or inducing the transcription of the gene encoding the protein to be activated and/or enhancing the translation of the mRNA encoding the protein to be activated. It can also be that the protein to be activated performs its biochemical function with enhanced efficiency in the presence of the activator or that the protein to be activated performs its cellular function with enhanced efficiency in the presence of the activator.
  • the term "activator” encompasses both molecules/compounds that have a directly activating effect on the specific signaling pathway but also molecules that are indirectly activating, e.g. by interacting, for example, with molecules that negatively regulate (e.g. suppress) said pathway.
  • the activator can also be an agonist of the signaling pathway (receptor) to be activated.
  • Methods for testing if a compound/molecule is capable to induce or enhance the activity of a target molecule and/or pathway are known to the skilled artesian.
  • the compound/molecule that can be used as an activator can be any compound/molecule, which can activate the respective pathway or which inhibits a suppressor of the pathway to be activated.
  • Exemplary activators can include suitable binding proteins directed e.g. against suppressors of a certain pathway.
  • fusing the first protein of said prate in- prate in interaction with the reporter polypeptide or part thereof is carried out by recruiting the first protein via an interaction between a DNA binding domain and an upstream activator sequence of the reporter polypeptide or part thereof to the reporter polypeptide or part thereof.
  • a DNA binding domain include, but are not limited to, Gal4 DNA- binding domain (DBD) (SEQ ID NO: 10).
  • DBD Gal4 DNA- binding domain
  • an upstream activator sequence include, but are not limited to, Gal4 DBD binding site repeats (SEQ ID NO: 11 ).
  • the binding of the DNA binding domain to the upstream activator sequence and the binding of the transcription activation domain of the reporter polypeptide to a reporter sequence of the reporter polypeptide causes expression of the reporter polypeptide.
  • a reporter sequence include, without limitation, Firefly luciferase (SEQ ID NO: 12).
  • a transcription activation domain include, but are not limited to, VP16 (SEQ ID NO: 13).
  • the protein-protein interaction is the interaction between a protein of interest or a part thereof and an engineered high affinity variant, preferably the interaction between the UBAN domain of NEMO (SEQ ID NO: 2) and an Ub variant (SEQ ID NO: 6), the interaction between the UBC9 enzyme (SEQ ID NO: 3) and a small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), the interaction between the ATG3 enzyme (SEQ ID NO: 4) and a GATE16 variant (SEQ ID NO: 8), and the interaction between OPTN (SEQ ID NO: 5) and a LC3B variant (SEQ ID NO: 9).
  • an engineered high affinity variant preferably the interaction between the UBAN domain of NEMO (SEQ ID NO: 2) and an Ub variant (SEQ ID NO: 6), the interaction between the UBC9 enzyme (SEQ ID NO: 3) and a small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), the interaction between the ATG3
  • protein of interest is intended to mean a protein to be prepared, and includes physiologically active proteins such as growth hormone, proteins of signalling pathways, interferon, interleukin, granulocyte colony stimulating factor, erythropoietin, and membrane receptors. Additionally, the term“protein of interest”, in the context of the present invention, means any protein desired for investigation, which is intended for detection of a protein-protein interaction and which is able to build a respective protein-protein intercation with a further protein or interaction partner. Unless specifically mentioned otherwise, the terms, “protein,” “peptide,” and “polypeptide” are used interchangeably.
  • the engineered high affinity variant as used in the context of the present invention corresponds to the second protein of the protein-protein interaction according to the methods as described herein, wherein the engineered high affinity variant is then preferably selected from the group consisting of an Ub variant (SEQ ID NO: 6), a small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), a GATE16 variant (SEQ ID NO: 8) and a LC3B variant (SEQ ID NO: 9), more preferably an Ub variant (SEQ ID NO: 6).
  • UBAN can mean the NEMO-like protein optineurin and is therefore termed UBD in ABIN proteins and NEMO (UBAN).
  • “UBAN” may comprise or have the sequence of SEQ ID NO: 2 or be a sequence, which is 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or even 100% identical to SEQ ID NO. 2. This amino acid sequence corresponds to amino acids 246 - 337 of human NEMO.
  • “NEMO”, as used herein, can mean the regulatory subunit of the IkB-kinase (IKK) in the NF-kB activation. This highly conserved regulatory protein“NEMO” is also known as IKKg or FIP-3. IKK activity relies on the interaction between the kinase and NEMO.
  • “NEMO” may comprise or have the sequence of SEQ ID NO: 1 according to UniProtKB Q9Y6K9 or be a sequence, which is 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or even 100% identical to SEQ ID NO: 1.
  • “NEMO” may also comprise or have the sequence of SEQ ID NO: 18 according to UniProtKB 088522 or be a sequence, which is 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or even 100% identical to SEQ ID NO: 18, which corresponds to mouse NEMO.
  • “Ubiquitin”, as used herein, can mean a small but extremely important protein that induces the "death" to other proteins or change the activity of other proteins. Ubiquitin consists of 76 amino acids. In the normal course of events, proteins are inactivated or their activity is changed by the attachment of ubiquitin to them, a process called ubiquitination.
  • Ubiquitin acts as a tag by which the protein-transport machinery ferries a protein to the proteasome for degradation. Antagonizing this process are enzymes that remove ubiquitin from proteins.
  • ubiquitin may comprise or have the sequence of SEQ ID NO: 14 or be a sequence, which is 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 14.
  • “UBC9” as used herein can mean the E2-conjugating enzyme, which catalyzes the conjugation of small ubiquitin-like modifiers (SUMOs) to e-amino groups of lysine residues in target proteins.
  • “UBC9” may comprise or have the sequence of SEQ ID NO: 3 according to UniProtKB P63279 or be a sequence, which is 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3.
  • “SUM02” may comprise or have the sequence of SEQ ID NO: 15 according to UniProtKB P61956 or be a sequence, which is 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 15.
  • ATG3 can mean an ubiquitin-like-conjugating enzyme.
  • ATG3 is an E2 conjugating enzyme, which is required for the cytoplasm to vacuole transport (Cvt), autophagy and mitochondrial homeostasis.
  • ATG3 is responsible for the E2-like covalent binding of phosphatidylethanolamine to the C-terminal Gly of ATG8-like proteins (GABARAP, GABARAPL1 , GABARAPL2 or MAP1 LC3A).
  • “ATG3” may comprise or have the sequence of SEQ ID NO: 4 according to UniProtKB Q9NT62 or be a sequence, which is 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or even 100% identical to SEQ ID NO: 4.
  • GATE 16 can mean Golgi-associated ATPase Enhancer of 16 kDa (GATE-16), also known as Apg8p2 and GABARAPL2. It is a 117 amino acid (aa) polypeptide and a member of the Autophagy-related 8 (Atg8) family of proteins. GATE-16/Apg8p2 has 100% amino acid sequence identity with its mouse and rat orthologs, and is orthologous to the yeast Atg8. GATE-16/Apg8p2 is best known for its role in autophagy.
  • GATE-16/Apg8p2 covalently attaches to phosphatidylethanolamine the phagophore (autophagosome precursor) membrane using an Ubiquitin-like conjugation system that includes Ubiquitin-activating (E1 )-, Ubiquitin- conjugating (E2)-, and Ubiquitin Ligase (E3)-like enzymes. It can be involved in the later stages of autophagosome formation. It may also be involved in cargo recruitment to autophagosomes.
  • E1 Ubiquitin-activating
  • E2 Ubiquitin- conjugating
  • E3 Ubiquitin Ligase
  • “GATE16” may comprise or have the sequence of SEQ ID NO: 16 according to UniProtKB P60520 or be a sequence, which is 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or even 100% identical to SEQ ID NO: 16.
  • OPTN can mean optineurin and can play an important role in the maintenance of the Golgi complex, in membrane trafficking, in exocytosis, through its interaction with myosin VI and Rab8.
  • OPTN links myosin VI to the Golgi complex and plays an important role in Golgi ribbon formation. It can play a role in the activation of innate immune response during viral infection. Mechanistically, it may recruit TBK1 at the Golgi apparatus, promoting its trans-phosphorylation after RLR or TLR3 stimulation. In turn, activated TBK1 phosphorylates its downstream partner IRF3 to produce IFN-beta.
  • OPTN may comprise or have the sequence of SEQ ID NO: 5 according to UniProtKB Q96CV9 or be a sequence, which is 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or even 100% identical to SEQ ID NO: 5.
  • LC3B can mean an ubiquitin-like protein that is a constituent of the ATG8-conjugation system, one of two evolutionarily conserved phosphatidylethanolamine conjugation systems necessary for the formation of the autophagosome.
  • the human ATG8 system includes seven ubiquitin-like light chain proteins (LCPs) that are homologs of yeast LC3: MAP1 LC3A, -B, -C, GABARAP, GABARAPL1 , -2, and -3.
  • LCPs ubiquitin-like light chain proteins
  • the exposed C-terminus is conjugated to the head group amine of phosphatidylethanolamine through an amide bond by a sequence of ubiquitination-like reactions that involves an E1 (ATG7), an E2 (ATG3), and an E3 (a complex including ATG5, ATG12, and ATG16L).
  • “LC3B” may comprise or have the sequence of SEQ ID NO: 17 according to UniProtKB Q9GZQ8 or be a sequence, which is 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 17.
  • such an“engineered high affinity variant” means a modified protein variant (e.g. the“variant” shown in Figure 1A and B).
  • the term“engineered” means in the context of the present invention and as it is used in“engineered high affinity variant” described herein to contain amino acid mutations that enhance the affinity of a specific target protein to an epitope. Amino acid mutations are selected from a combinatorial library of modified protein variants using phage display. However, other methods are also possible such as yeast display, ribosome display, mRNA display or computational methods.
  • variant as used in the context of the invention means that any protein like ubiquitin variant (Ubv-A), LC3B or SUM02 variants harbour affinity enhancing mutations with regard to the protein-protein interaction of a first and a second protein compared to the protein without these mutations. It is a variant of itself with altered properties, such as enhanced affinity, compared to the protein without these mutations.
  • modified as used in the context of the invention, means the introduction of amino acid mutations into a protein so that the original protein is altered in its primary structure.
  • the word “mutated” as being known to the skilled artesian can be used synonymously with the terms“modified” and“engineered” as used herein.
  • engineered affinity variant corresponds to the“second protein of the protein-protein interaction” as defined herein.“Affinity” and“epitope” are known to a skilled artesian and does not have to be defined in more concrete terms herein.
  • the present invention is also directed to the engineered high affinity variant as described herein, wherein the high affinity variant is selected from the group consisting of the Ub variant (SEQ ID NO: 6), the small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), the GATE 16 variant (SEQ ID NO: 8), and the LC3B variant (SEQ ID NO: 9).
  • the present invention is also directed to the engineered high affinity variant as described herein, wherein the engineered high affinity variant is selected from the group consisting of the small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), the GATE16 variant (SEQ ID NO: 8), and the LC3B variant (SEQ ID NO: 9).
  • the present invention is also directed to proteins selected from the group consisting of the Ub variant (SEQ ID NO: 6), the small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), the GATE16 variant (SEQ ID NO: 8), and the LC3B variant (SEQ ID NO: 9).
  • the present invention is also directed to proteins selected from the group consisting of the small ubiquitin- like modifier 2 (SUM02) variant (SEQ ID NO: 7), the GATE16 variant (SEQ ID NO: 8), and the LC3B variant (SEQ ID NO: 9).
  • the present invention is directed to proteins selected from the group consisting of the Ub variant (SEQ ID NO: 6), the small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), the GATE16 variant (SEQ ID NO: 8), and the LC3B variant (SEQ ID NO: 9) for use in any of the methods as described herein. Further, the present invention is directed to proteins selected from the group consisting of the small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), the GATE16 variant (SEQ ID NO: 8), and the LC3B variant (SEQ ID NO: 9) for use in any of the methods as described herein.
  • the method is a high-throughput screening assay, preferably a cell-based high-throughput screening assay, for the modulator compound of the protein-protein interaction.
  • a high-throughput screening assay preferably a cell-based high-throughput screening assay, for the modulator compound of the protein-protein interaction.
  • a person skilled in the art knows the term“high-throughput screening assay” and is aware thereof, when such an assay is given. No further definition is herein necessary for the person skilled in the art. The same applies for the term “cell-based high-throughput screening assay” or “cellular high-throughput screening assay”.
  • the present invention also relates to a modulator compound detected by any of the methods described herein for use in the prophylaxis and/or treatment of a disease selected from the group consisting of cancer, chronic inflammatory diseases, autoimmune diseases, neurodegenerative diseases, arthritides and infectious diseases.
  • a disease selected from the group consisting of cancer, chronic inflammatory diseases, autoimmune diseases, neurodegenerative diseases and arthritides, more preferably cancer, chronic inflammatory diseases, autoimmune diseases, and arthritides and most preferably chronic inflammatory diseases, autoimmune diseases, and arthritides.
  • the modulator compound is a Ca antagonist.
  • the present invention also relates to a method as described herein for use in the prophylaxis and/or treatment of a disease selected from the group consisting of cancer, chronic inflammatory diseases, autoimmune diseases, neurodegenerative diseases, arthritides and infectious diseases.
  • a disease selected from the group consisting of cancer, chronic inflammatory diseases, autoimmune diseases, neurodegenerative diseases and arthritides, more preferably cancer, chronic inflammatory diseases, autoimmune diseases, and arthritides and most preferably chronic inflammatory diseases, autoimmune diseases, and arthritides.
  • the present invention also relates to a kit comprising an engineered high affinity variant as described herein.
  • the engineered high affinity variant corresponds to the second protein of the protein-protein interaction as described herein and vice versa.
  • the engineered high affinity variant is selected from the group consisting of the Ub variant (SEQ ID NO: 6), the small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), the GATE16 variant (SEQ ID NO: 8), and the LC3B variant (SEQ ID NO: 9).
  • the engineered high affinity variant is selected from the group consisting of the small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), the GATE 16 variant (SEQ ID NO: 8), and the LC3B variant (SEQ ID NO: 9).
  • the present invention also provides a composition comprising a protein selected from the group consisting of the Ub variant (SEQ ID NO: 6), the small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), the GATE16 variant (SEQ ID NO: 8), and the LC3B variant (SEQ ID NO: 9).
  • kits comprising one or more components (e.g., assay system components) described herein are also encompassed by the present invention. Such kits will typically contain printed instructions as to the use of the components contained therein.
  • the present invention relates to a kit-of-parts comprising an engineered affinity variant as described herein and a test system that is capable of detecting the extent of modulation of a modulator compound on a protein-protein interaction between a first protein and a second protein as defined herein.
  • the second protein is the engineered high affinity variant as defined herein.
  • the engineered affinity variant is selected from the group consisting of the Ub variant (SEQ ID NO: 6), the small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), the GATE16 variant (SEQ ID NO: 8), and the LC3B variant (SEQ ID NO: 9).
  • the test system further comprises a reporter polypeptide, wherein the reporter polypeptide is selected from the group consisting of a split ubiquitin, a fluorescent protein, preferably the green, red or yellow fluorescent protein, more preferably a split green fluorescent protein, beta-lactamase, beta-galactosidase, chloramphenicol acetyl- transferase (CAT) and a luciferase, preferably Firefly luciferase or Renilla luciferase or split nanoluciferase.
  • a reporter polypeptide is selected from the group consisting of a split ubiquitin, a fluorescent protein, preferably the green, red or yellow fluorescent protein, more preferably a split green fluorescent protein, beta-lactamase, beta-galactosidase, chloramphenicol acetyl- transferase (CAT) and a luciferase, preferably Firefly luciferase or Renilla luciferase or split
  • the present invention further provides a test system that is capable of detecting the extent of modulation of the modulator compound on a protein-protein interaction between a first and a second protein as described herein.
  • the protein-protein interaction is the interaction between a protein of interest and an engineered high affinity variant, more preferably the interaction between the UBAN domain of NEMO (SEQ ID NO: 2) and a Ub variant (SEQ ID NO: 6), the interaction between UBC9 (SEQ ID NO: 3) and a small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), the interaction between the ATG3 enzyme (SEQ ID NO: 4) and a GATE16 variant (SEQ ID NO: 8), and the interaction between OPTN (SEQ ID NO: 5) and a LC3B variant (SEQ ID NO: 9).
  • the first protein of the protein-protein interaction is selected from the group consisting of UBAN domain of NEMO (SEQ ID NO: 2), UBC9 enzyme (SEQ ID NO: 3), ATG3 enzyme (SEQ ID NO: 4) and OPTN (SEQ ID NO: 5), preferably the UBAN domain of NEMO (SEQ ID NO: 2)
  • the second protein of the protein-protein interaction is selected from the group consisting of an Ub variant (SEQ ID NO: 6), a small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), a GATE 16 variant (SEQ ID NO: 8) and a LC3B variant (SEQ ID NO: 9), preferably an Ub variant (SEQ ID NO: 6), wherein the second protein of the protein- protein interaction is the engineered high affinity variant.
  • the present invention also relates to a composition comprising an engineered high affinity variant as described herein.
  • the composition comprises a protein or an engineered high affinity variant selected from the group consisting of an Ub variant (SEQ ID NO: 6), a small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), a GATE16 variant (SEQ ID NO: 8) and a LC3B variant (SEQ ID NO: 9), preferably an Ub variant (SEQ ID NO: 6).
  • the present invention also comprises the use of an engineered high affinity variant as described herein in any of the methods as mentioned herein.
  • the present invention encompasses the use of a protein or an engineered high affinity variant selected from the group consisting of an Ub variant (SEQ ID NO: 6), a small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), a GATE 16 variant (SEQ ID NO: 8) and a LC3B variant (SEQ ID NO: 9), preferably the use of an Ub variant (SEQ ID NO: 6), in any of the methods as described herein.
  • the present invention comprises also the use of a method as described or defined herein.
  • a use of a method as described herein can be for detecting a modulator compound of a protein-protein interaction between a first and a second protein as described or defined herein.
  • the present invention also relates to a test system for detecting the extent of modulation of the modulator compound as described herein on a protein -protein interaction between a first protein and a second protein as described herein.
  • the test system comprises the following components: a first fusion between a first protein of a protein-protein interaction as defined herein, a second fusion between a second protein of a protein-protein interaction as defined herein and a reporter polypeptide.
  • the test modulator compound can then be applied to the test system as described herein.
  • the first protein of the protein-protein interaction is selected from the group consisting of the UBAN domain of NEMO (SEQ ID NO: 2), the UBC9 enzyme (SEQ ID NO: 3), the ATG3 enzyme (SEQ ID NO: 4) and OPTN (SEQ ID NO: 5).
  • the first protein of the protein-protein interaction is the UBAN domain of NEMO (SEQ ID NO: 2).
  • the second protein of the protein-protein interaction is selected from the group consisting of an Ub variant (SEQ ID NO: 6), a small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), a GATE16 variant (SEQ ID NO: 8) and a LC3B variant (SEQ ID NO: 9).
  • the second protein of the protein-protein interaction is an Ub variant (SEQ ID NO: 6).
  • the test system further comprises a reporter polypeptide, wherein the reporter polypeptide is selected from the group consisting of a split ubiquitin, a fluorescent protein, preferably the green, red or yellow fluorescent protein, more preferably a split green fluorescent protein, beta-lactamase, beta-galactosidase, chloramphenicol acetyltransferase (CAT) and a luciferase, preferably Firefly luciferase or Renilla luciferase or split nanoluciferase.
  • a reporter polypeptide is selected from the group consisting of a split ubiquitin, a fluorescent protein, preferably the green, red or yellow fluorescent protein, more preferably a split green fluorescent protein, beta-lactamase, beta-galactosidase, chloramphenicol acetyltransferase (CAT) and a luciferase, preferably Firefly luciferase or Renilla luciferase or split
  • the term "at least" preceding a series of elements is to be understood to refer to every element in the series.
  • the term“at least one” refers to one or more such as two, three, four, five, six, seven, eight, nine, ten and more.
  • the term “about” means plus or minus 10%, preferably plus or minus 5%, more preferably plus or minus 2%, most preferably plus or minus 1 %.
  • Example 1 Generation and characterization of a Ub variant that binds to the UBAN domain of NEMO with high affinity
  • the inventors selected the interaction between the UBAN domain of NEMO (UBAN N EMO) (SEQ ID NO: 2) with linear ubiquitin (Ub) chains (linear Ub ) as primary target protein- protein interaction (PPI) due to its crucial role in the activation of the NF-kB signalling pathway.
  • UBAN N EMO UBAN N EMO
  • PPI primary target protein- protein interaction
  • High affinity Ub variants that bind to the target protein, UBAN NE MO were developed using protein engineering, specifically phage display technology.
  • the phage-displayed Ub variant (Ubv) library was generated using soft randomization strategy and mutations were introduced to the regions of Ub that are engaged in the UBAN NEM O (SEQ ID NO: 2) and linear Ubiquitin interaction (named Regions 1 , 2 and 3 in Figure 2B) 4 . Then the phage display library was screened against mouse UBANNEMO domain (mUBAN NEM o) (SEQ ID NO: 18), which shares high conservation with the human UBAN NEMO (hUBAN NEMO ) (SEQ ID NO: 2).
  • UBAN domain is highly conserved and can be also found in ABIN and optineurin (OPTN) proteins. Since the phage display strategy was designed to select high affinity variants against UBAN NEMO, Ubv-A (SEQ ID NO: 6) was then tested for selectivity in binding between UBAN domains of NEMO (SEQ ID NO: 2), ABIN1 (SEQ ID NO: 20) and OPTN (SEQ ID NO: 5).
  • Ubv-A (SEQ ID NO: 6) preferentially interacted with UBAN NEMO (SEQ ID NO:2) with a dissociation constant of 0.29 mM, which is ⁇ 7 and ⁇ 20 times smaller values of that of OPTN (SEQ ID NO: 5) and ABIN1 (SEQ ID NO: 20), respectively, indicating that Ubv-A (SEQ ID NO: 6) selectively binds the linear UBAN domain of NEMO (SEQ ID NO: 2) ( Figure 3B).
  • Ubv-A (SEQ ID NO: 6) was further validated by phage ELISA using wild type or mutant UBAN-domains of NEMO (aa 250-339, SEQ ID NO: 18), NEMO (aa 250-339, V293A Y301A K302A, SEQ ID NO: 21 ), OPTN (aa 454-514, SEQ ID NO: 22), OPTN (aa 454-577, SEQ ID NO: 23), OPTN (aa 454-514, D474N, SEQ ID NO: 24), ABIN1 (aa 465- 525, SEQ ID NO: 25), ABIN1 (aa 465-525, D485A, SEQ ID NO: 26), ABIN1 (aa 465-525, D485N and F486A mutant, SEQ ID NO: 27).
  • Ub binding proteins HOIL SEQ ID NO: 28, corresponding to UniProtKB Q9BYM8
  • HOIP SEQ ID NO: 29, corresponding to UniProtKB Q96EP0
  • USP21 SEQ ID NO: 30, corresponding to UniProtKB Q9UK80
  • USP7 SEQ ID NO: 31 , corresponding to UniProtKB Q93009
  • OTUD7A SEQ ID NO: 32, corresponding to UniProtKB Q8TE49
  • USP14 SEQ ID NO: 33, corresponding to UniProtKB P54578
  • USP5 SEQ ID NO: 34, corresponding to UniProtKB P45974
  • Example 2 Development of cell-based reporter assays using generated/ engineered high affinity variants
  • the initial design of the assay system is based on the dual-luciferase reporter (DLR) technology (Promega), the theoretical model of which is presented in Figure 1A.
  • DLR dual-luciferase reporter
  • the invention takes advantage of high affinity interaction between the target protein (e.g. UBAN nem0 , SEQ ID NO: 2) and a variant (e.g. Ubv-A, SEQ ID NO: 6), coupling the expression of Firefly luciferase to the occurrence of a given interaction.
  • UBAN ne 0 (SEQ ID NO: 2) is expressed as a fusion to the Gal-4 DNA binding domain (DBD, SEQ ID NO: 10) that binds to the Gal4 promoter (SEQ ID NO: 11 ) upstream of the FIREFLY gene.
  • the second component of the DLR system is the fusion of 2xUbv-A to a transcriptional activation module (ACT).
  • ACT transcriptional activation module
  • the ACT fusion vector also contains the RENILLA luciferase gene that is constitutively expressed irrespective of the interaction between the fusion partners.
  • Firefly and Renilla luciferases are evolutionary distinct and therefore have dissimilar enzyme structures and substrate requirements. Therefore, activity determinations of both luciferases using subsequent addition of substrate reagents enable assay normalization to correct for variability associated with transient transfection.
  • HEK293 cells were co-transfected with modified DLR vectors, incubated for 20 hours, lysed and then assayed for Firefly and Renilla luciferase activity by subsequent addition of assay reagents according to manufacturer’s manual. Quantification of relative light units generated by Firefly and Renilla luciferases was recorded and Firefly/Renilla signal ratio served for normalization between samples. Since no activation of luciferase should occur in the absence of high affinity variant of the PPI under study, any signal indicative of activity of this reporter is considered to represent“background” for the DLR system.
  • the background level of luciferase is measured in the presence of DBD and ACT empty vector controls.
  • Co-transfection of cells with DLR assay vectors expressing the DBD-UBAN NEMO first fusion protein and the ACT-2xUbv-A second fusion protein enabled the detection of the specific DLR assay activation.
  • the F312A UBAN NEMO mutant SEQ ID NO: 19
  • Ubv-A SEQ ID NO: 6
  • Figure 4A did not produce any signal above the background activation
  • the inventors present herein the first example of an approach for generation of cell-based assays that utilize high affinity variants to given PPI.
  • the inventors utilized the DLR system and exchanged the NEMO/Ubv-A binding pair with other engineered PPI pairs.
  • the inventors tested a recently reported SUM02 variant (SEQ ID NO: 7) that was engineered to bind UBC9 enzyme (SEQ ID NO: 3) with high affinity and block its enzymatic activity 3 .
  • the DLR assay vectors were modified to express ACT-UBC9 and fusions of the DBD domain to either control SUM02 or high affinity SUM02 variant (SEQ ID NO: 7).
  • the inventors use a GATE16 high affinity variant (SEQ ID NO: 8) developed to the ATG3 enzyme (SEQ ID NO: 4), which plays a key role in autophagy.
  • the amino acid sequence of this engineered variant is given in SEQ ID NO: 8.
  • the inventors of the present invention modified DLR assay vectors to express DBD-ATG3 and fusions of ACT to either control GATE16 or GATE16 variant (SEQ ID NO: 8).
  • control GATE16 or GATE16 variant SEQ ID NO: 8
  • high affinity GATE16 variant SEQ ID NO: 8
  • FIG. 4C the examples presented in Figure 4 illustrate that specific high affinity variants significantly improve performance of the reporter assays for PPIs.
  • the NanoBiT assay system was then utilised as it is more advanced and enables HTS in live cells 1 .
  • the NanoBiT assay activation is independent of protein expression, which provides notable advantages in chemical screening as it limits the identification of compounds that affect protein expression machinery and therefore reduce false discovery rate (Figure 1 B).
  • the invention takes advantage of high affinity PPI between UBAN NEMO (SEQ ID NO: 2) and Ubv-A (SEQ ID NO: 6) that are fused to the small bit (SmBiT) and the large bit (LgBiT) of split nanoluciferase, respectively.
  • the assay background is determined by transfecting the LgBiT assay fusion together with a fusion of an unrelated protein (HaloTag) to SmBiT.
  • the assay specificity is determined by the F312A point mutation in UBAN NEMO (SEQ ID NO: 19) that abolishes the interaction between UBAN NEMO (SEQ ID NO: 2) and high affinity Ubv-A (SEQ ID NO: 6) ( Figure 5A).
  • NanoBiT technology was then used for other target PPIs to demonstrate the flexibility of this novel approach. It was demonstrated that the current invention also enables targeting of a key selective autophagy receptor Optineurin (OPTN) with its partner protein LC3B. This PPI has a broad therapeutic application and a robust reporter assay for detecting this interaction is presented in Figure 5B. Taken together, the proof-of-concept experiments of the inventors demonstrate that application of phage display improved high affinity variants that target a specific PPI in assay development results in the generation of robust cell-based assays.
  • OPTN autophagy receptor Optineurin
  • Example 3 Method validation in high-throughput screening
  • NanoBiT NEMO assay was miniaturized and optimized for screening in 384-well plate format.
  • a panel of available cell lines was first tested to identify a suitable cell line demonstrating a robust assay response.
  • HEK293, CHO or A549 cells were co-transfected with constructs expressing LgBiT-Ubv-A in combination with either SmBiT-wt UBAN NEMO or SmBiT-HaloTag fusion proteins and dispensed into 384-well plates. Following 20-hour incubation nanoluciferase activity was quantified as relative light units and Z’ score for each cell line was determined. As shown in Figure 6A, co- transfection of A549 cells results in Z’ score > 0.5 that is indicative of high assay quality.
  • A549 cells were co-transfected with a combination of LgBiT-Ubv-A and SmBiT-wt UBAN NEMO constructs, dispensed into 384- well plates and following 20-hour incubation the indicated concentrations of Aloe Emodin were dispensed into the wells and allowed to incubate for additional two hours.
  • Figure 6B shows a concentration-dependent response of the assay to Aloe Emodin, validating its use as a reference inhibitor in HTS.
  • NanoBiT NEMO assay As a pharmacological screening platform, the inventors used a compound library comprised of 774 FDA approved drugs. A549 cells were reverse transfected as before with a combination of LgBiT-Ubv-A and SmBiT-wt UBAN MEMO constructs and dispensed into 384-well plates. Following 20-hour incubation, test compounds were dispensed at 10 mM concentration and plates were further incubated for additional two hours. Assay response to test compounds was quantified relative to the maximum inhibition by Aloe Emodin reference inhibitor.
  • An FDA approved drug library can also be used for drug repurposing screening.
  • FDA approved medicines the detailed understanding of their molecular mechanism of action is currently unknown. Therefore, providing novel insights about drug mechanism of action can facilitate drug repurposing.
  • Method for detecting a modulator compound of a protein-protein interaction between a first and a second protein comprising the steps of:
  • step (b) comparing the measurements made in step (b), wherein an alteration of the signal of said reporter polypeptide in step a) ii) to the signal of said reporter polypeptide in step a) i) indicates the presence of a modulator compound of said protein-protein interaction, and
  • step c) quantifying the extent of modulation of the protein-protein interaction by the test modulator compound by step c).
  • step a) is within a cell, cell lysate, within a reaction mixture or outside a cell, preferably within a cell.
  • the modulator compound is a compound being capable of inhibiting said protein-protein interaction, preferably a protein- protein interaction of the nuclear factor kappa B signaling pathway, more preferably the prate in- prate in interaction between NEMO and linear ubiquitin chains and most preferably the interaction between the UBAN domain of NEMO (SEQ ID NO: 2) and an Ubiquitin variant (SEQ ID NO: 6).
  • Method according to any one of the preceding items wherein fusing the first protein of said prate in- prate in interaction with the reporter polypeptide or part thereof is carried out by recruiting the first protein via an interaction between a DNA binding domain and an upstream activator sequence of the reporter polypeptide or part thereof to the reporter polypeptide or part thereof.
  • Method according to item 6 wherein, in the absence of said test modulator compound according to step a) ii), the binding of the DNA binding domain to the upstream activator sequence and the binding of the transcription activation domain to a reporter sequence of the reporter polypeptide causes expression of the reporter polypeptide.
  • the protein-protein interaction is the interaction between a protein of interest or a part thereof and an engineered high affinity variant, preferably the interaction between an UBAN domain of NEMO (SEQ ID NO: 2) and an Ub variant (SEQ ID NO: 6), the interaction between UBC9 (SEQ ID NO: 3) and a small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), the interaction between the ATG3 enzyme (SEQ ID NO: 4) and a GATE16 variant (SEQ ID NO: 8), or the interaction between OPTN (SEQ ID NO: 5) and a LC3B variant (SEQ ID NO: 9).
  • the first protein of the protein- protein interaction is selected from the group consisting of UBAN domain of NEMO (SEQ ID NO: 2), UBC9 enzyme (SEQ ID NO: 3), ATG3 enzyme (SEQ ID NO: 4) and OPTN (SEQ ID NO: 5), preferably the UBAN domain of NEMO (SEQ ID NO: 2)
  • the second protein of the protein-protein interaction is selected from the group consisting of an Ub variant (SEQ ID NO: 6), a small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), a GATE16 variant (SEQ ID NO: 8) and a LC3B variant (SEQ ID NO: 9), preferably an Ub variant (SEQ ID NO: 6).
  • the reporter polypeptide is selected from the group consisting of a split ubiquitin, a fluorescent protein, preferably the green, red or yellow fluorescent protein, more preferably a split green fluorescent protein, beta-lactamase, beta-galactosidase, chloramphenicol acetyltransferase (CAT) and a luciferase, preferably Firefly luciferase, Renilla luciferase or split nanoluciferase.
  • a split ubiquitin a fluorescent protein, preferably the green, red or yellow fluorescent protein, more preferably a split green fluorescent protein, beta-lactamase, beta-galactosidase, chloramphenicol acetyltransferase (CAT) and a luciferase, preferably Firefly luciferase, Renilla luciferase or split nanoluciferase.
  • the method is a high- throughput screening assay, preferably a cell-based high-throughput screening assay, for detecting the modulator compound of the prate in -prate in interaction.
  • Engineered high affinity variant selected from the group consisting of an Ub variant (SEQ ID NO: 6), a small ubiquitin-like modifier 2 (SUM02) variant (SEQ ID NO: 7), a GATE16 variant (SEQ ID NO: 8) and a LC3B variant (SEQ ID NO: 9), preferably an Ub variant (SEQ ID NO: 6).
  • a kit-of-parts comprising an engineered high affinity variant according to item 13 and a test system that is capable of detecting the extent of modulation of the modulator compound on a protein-protein interaction between a first protein and a second protein, wherein the second protein is the engineered high affinity variant.
  • Kit-of-parts according to item 14, wherein the test system further comprises a reporter polypeptide, wherein the reporter polypeptide is selected from the group consisting of a split ubiquitin, a fluorescent protein, preferably the green, red or yellow fluorescent protein, more preferably a split green fluorescent protein, beta-lactamase, beta-galactosidase, chloramphenicol acetyltransferase (CAT) and a luciferase, preferably Firefly luciferase or Renilla luciferase or split nanoluciferase.
  • a reporter polypeptide is selected from the group consisting of a split ubiquitin, a fluorescent protein, preferably the green, red or yellow fluorescent protein, more preferably a split green fluorescent protein, beta-lactamase, beta-galactosidase, chloramphenicol acetyltransferase (CAT) and a luciferase, preferably Firefly luciferase

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Abstract

La présente invention concerne un procédé de détection d'un composé modulateur d'une interaction protéine-protéine entre une première et une seconde protéine, un composé modulateur détecté par l'un quelconque des procédés selon la présente invention destiné à être utilisé dans la prophylaxie et/ou le traitement d'une maladie, un variant modifié d'affinité élevée utilisé dans le procédé selon la présente invention et un kit de composants comprenant ledit variant modifié d'affinité élevée selon la présente invention et un système de test qui permet de détecter l'étendue de la modulation du composé modulateur sur ladite interaction protéine-protéine entre la première et la seconde protéine de l'interaction protéine-protéine. De plus, la présente invention concerne l'utilisation desdits procédés et également des compositions comprenant ledit variant modifié d'affinité élevée.
PCT/EP2019/073058 2018-08-30 2019-08-29 Procédé de détection d'un composé modulateur pour le ciblage chimique d'interactions protéine-protéine Ceased WO2020043812A1 (fr)

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CN114164255A (zh) * 2021-12-10 2022-03-11 南通大学 一种筛选d2r与nr2b相互作用阻断剂的方法
CN116068198A (zh) * 2022-11-30 2023-05-05 深圳湾实验室 Ppi原位检测方法及其载体、诊断试剂、试剂盒和应用
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112694535A (zh) * 2021-01-05 2021-04-23 重庆医科大学 用于抗体检测的多功能蛋白分子开关
CN112694535B (zh) * 2021-01-05 2023-03-31 重庆医科大学 用于抗体检测的多功能蛋白分子开关
CN114164255A (zh) * 2021-12-10 2022-03-11 南通大学 一种筛选d2r与nr2b相互作用阻断剂的方法
CN116068198A (zh) * 2022-11-30 2023-05-05 深圳湾实验室 Ppi原位检测方法及其载体、诊断试剂、试剂盒和应用
CN116068198B (zh) * 2022-11-30 2024-01-09 深圳湾实验室 Ppi原位检测方法及其载体、诊断试剂、试剂盒和应用
WO2024113229A1 (fr) * 2022-11-30 2024-06-06 深圳湾实验室 Procédé de détection in situ de ppi, vecteur, réactif de diagnostic, kit et utilisation
WO2025160115A1 (fr) * 2024-01-22 2025-07-31 Integrated Dna Technologies, Inc. Inhibition de nhej, mmej et 53bp1 favorisant des niveaux élevés de hdr

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