# US 2017/0281784 A1 · Protein-protein interaction inducing technology

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- Number: US 2017/0281784 A1
- Document ID: US20170281784A1
- Status: Published application
- Kind: A1
- Application number: 15/477,966
- Filed: 2017-04-03
- Published: 2017-10-05
- Earliest claimed: 2016-04-05
- Applicants: Arvinas, Inc.
- Inventors: Jing Wang; Andrew P. Crew; Hanqing Dong; Taavi Neklesa; Brian Hamman
- Web page: https://patentgrep.com/patent/US20170281784A1

## CPC

- A61K 31/277: having a ring, e.g. verapamil
- A61K 31/4166: having oxo groups directly attached to the heterocyclic ring, e.g. phenytoin
- A61K 31/551: having two nitrogen atoms, e.g. dilazep
- A61K 38/05: Dipeptides
- A61K 47/55: the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug
- G01N 33/6845: {Methods of identifying protein-protein interactions in protein mixtures}
- G16B 15/30: Drug targeting using structural data; Docking or binding prediction
- G16B 20/00: ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B 20/30: Detection of binding sites or motifs
- G16C 20/50: Molecular design, e.g. of drugs
- G16B 15/00: ICT specially adapted for analysing two-dimensional [2D] or three-dimensional [3D] molecular structures, e.g. structural or functional relations or structure alignment
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16C: COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE

## Abstract

The present disclosure is based on the surprising and unexpected discovery that a ligand molecule with certain characteristics is able to bind to two protein molecules simultaneously and recruit them to form a transient or stable protein-protein interaction complex. The protein-protein interaction and other cross-domain interactions gained in this process contribute additional stabilization energy to the complex beyond the combination of the binary binding energies, and therefore, largely increase the binding potency of the ligand. Accordingly, the present disclosure provides a Protein-Protein Interaction Inducing Technology (PPIIT), which includes a method to design and identify the tripartite or bifunctional compounds and use such compounds to induce protein-protein interactions in various contexts. The present disclosure also provides a composition for the purpose of inducing protein-protein interactions.

## Claims

### Claim 1

A method of designing a bifunctional compound capable of effectuating protein-protein interactions between a first protein molecule (A) and a second protein molecule (B), the method comprising:
(a) providing a bifunctional ligand (L) of structure WA-Cn-WB, wherein WA is a warhead targeting the first protein A, WB is a warhead targeting the second protein B, and C is a connector with length or number of atoms n, covalently linked to WA and WB;
(b) measuring the ternary binding potency and the binary binding potencies of the ligand L with respect to the first protein A and the second protein B; and
(c) determining the capability of the ligand to induce an interaction between the first protein A and the second protein B.

### Claim 2 (depends on 1)

The method of claim 1, wherein at least one of the warhead WA, warhead WB, and connector C is a chemical group or moiety.

### Claim 3 (depends on 2)

The method of claim 2, wherein the warheads WA and WB are derived from compounds known to bind to proteins A and B, respectively.

### Claim 4 (depends on 1)

The method of claim 1, wherein the connector C is a linear chain of carbon atoms or a linear chain of alternating carbon atoms and heteroatoms.

### Claim 5 (depends on 4)

The method of claim 4, wherein any two heteroatoms are separated by at least two carbon atoms.

### Claim 6 (depends on 1)

The method of claim 1, wherein the method of designing the bifunctional ligand further comprises a step of modifying the chain length or the number of atoms of the connector C to determine the appropriate chain length or number of atoms for inducing protein-protein interactions.

### Claim 7 (depends on 6)

The method of claim 6, wherein the method includes the steps of determining comprising:
(a) synthesizing a set of compounds with the number of atoms in C varying n between 0 and 30 while keeping the warheads WA and WB constant;
(b) measuring the binding of each compound to determine which compounds have a superior ternary binding potency relative to a corresponding binary binding potency for proteins A and B; and
(c) determining the n values that give rise to the potencies indicative of the existence of protein-protein interactions.

### Claim 8 (depends on 7)

The method of claim 7, wherein determining the chain length or the number of atoms of the connector C (n) further comprises:
(d) changing the attachment points on WA and WB that are used to link the warheads to the connector C, and repeat steps (a) through (c) to find additional compounds with protein-protein interactions.

### Claim 9 (depends on 1)

The method of claim 1, wherein the connector C is a chain with branched groups and/or contains rings.

### Claim 10 (depends on 1)

The method of claim 1, wherein the measuring step further comprises:
measuring the influence of the first protein or the second protein on a binding constant of another protein toward the ligand to evaluate the capability of the ligand L to induce the protein-protein interaction.

### Claim 11 (depends on 10)

The method of claim 10, wherein the method comprises a step of:
performing molecular dynamics simulations to demonstrate protein-protein interactions and other cross-domain interactions in ternary systems composed of the first protein A, the second protein B, and the ligand L to evaluate the capability of the ligand L to induce the protein-protein interaction.

### Claim 12 (depends on 11)

The method of claim 11, wherein the first protein A and the second protein B are the same protein

### Claim 13 (depends on 11)

The method of claim 11, wherein the first protein A and the second protein B are different proteins.

### Claim 14 (depends on 1)

The method of claim 1, further comprising selecting a ligand with a ternary complex that results in surface area burial greater than the sum of the surface area burial of the corresponding warhead monomers with the first and second proteins.

### Claim 15 (depends on 1)

A compound resulting from the method of claim 1.

### Claim 16 (depends on 15)

A method of treating or preventing a disease or disorder, the method comprising: administering an effective amount of a compound of claim 15.

### Claim 17

A method of designing a tripartite or bifunctional ligand that induces protein-protein interaction(s) between a first protein molecule (A) and a second protein molecule (B), the method comprising:
designing, preparing, and/or synthesizing a plurality of tripartite and/or bifunctional compounds with the general structure WA-C-WB or WA-WB, wherein WA is a warhead that associates with the first protein, WB is a warhead that associates with the second protein, and C is a connector covalently linked or bound to WA and WB;
designing, preparing, and/or synthesizing control compounds;
quantifying induced protein-protein interactions with at least one of biochemical assays, cellular assays, and molecular dynamics simulations; and
selecting the tripartite or bifunctional compound/ligand that induces protein-protein interactions and/or other cross-domain interactions in the ternary complex.

### Claim 18 (depends on 17)

The method of claim 17, wherein designing, preparing, and/or synthesizing includes varying a length of the connector between 0 atoms to 30 atoms while maintaining the same warheads and connection points between the connector and the warheads.

### Claim 19 (depends on 18)

The method of claim 18, wherein the length of the connector is varied by an increment of 1 to 3 atoms.

### Claim 20 (depends on 17)

The method of claim 17, wherein the covalent link between the connector and WA and/or WB is at a solvent-exposed point.

### Claim 21 (depends on 17)

The method claim 17, wherein the plurality of tripartite and/or bifunctional compounds comprises subsets of compounds having a unique covalent link between warhead WA and warhead WB or a unique series of covalent links between warhead WA, the connector, and warhead WB, relative to the other subsets.

### Claim 22 (depends on 17)

The method of claim 17, wherein designing, preparing, and/or synthesizing control compounds comprises modifying either warhead WA or WB such that substantially all of its association/binding ability to protein A or protein B is removed.

### Claim 23 (depends on 17)

The method of claim 17, wherein quantifying protein-protein interactions using biochemical assays comprises determining whether (i) the tripartite or bifunctional compound binding/associating with protein A and protein B produce synergism, or (ii) the tripartite or bifunctional compound/ligand induces ternary binding potency.

### Claim 24 (depends on 17)

The method of claim 17, wherein selecting the tripartite or bifunctional compound/ligand that induces protein-protein interactions in the ternary complex comprises:
selecting at least one tripartite or bifunctional compound/ligands that have a ratio α that is greater than about 1, wherein the ratio α is IC50A over IC50A/B or IC50B over IC50B/A; and/or
selecting at least one tripartite or bifunctional compounds/ligands that have a ratio αT that is greater than about 1, wherein the ratio αT is a ratio of the lower of IC50A and IC50B over IC50T.

### Claim 25 (depends on 17)

The method of claim 17, wherein quantifying induced protein-protein interactions comprises performing molecular dynamics simulations on tripartite and/or bifunctional compounds that are determined to induce protein-protein interaction(s) by either biochemical assays or cellular assays.

### Claim 26 (depends on 17)

The method of claim 17, wherein the protein-protein interactions for a particular conformation determined by molecular dynamics simulations are examined by calculating at least one of atom distances, surface area burial, and interaction energies for the ternary complex formation and a binary complex formation.

### Claim 27 (depends on 17)

The method claim 17, wherein the protein-protein interactions for a particular conformation determined by molecular dynamics simulations are examined along the simulation trajectory and the critical distances related to the interactions and the intermolecular energies between critical groups can be calculated along the simulation time.