Targeting protein degradation

Abstract

This article provides a complex, method, and therapeutic use for inducing and / or enhancing the ubiquitination of proteins such as NRF2 via an E3 ubiquitin ligase (BTRC) containing β-transduction protein repeat sequences.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 545,875, filed October 26, 2023, the entire contents of which are incorporated herein by reference. Background Technology

[0003] Targeted protein degradation is an attractive area for developing therapeutics to treat diseases driven by the accumulation or inappropriate expression of proteins that reshape cellular physiology. The ubiquitin-proteasome system (UPS) is a major pathway regulating cellular protein abundance, involving the labeling of target proteins with ubiquitin for recognition and degradation by the proteasome. A key component of the UPS that provides specificity for this process is the recognition of sequences or structural motifs (degradation determinants) on target proteins by substrate recognition factors of E3 ubiquitin-binding enzymes. Post-translational modifications of degradation determinant motifs, such as phosphorylation of serine, threonine, or tyrosine residues, provide additional regulation of target protein recognition, increasing the affinity of substrate recognition factors. Each substrate recognition factor recognizes a specific combination of amino acid sequences and post-translational modifications; deviations from this sequence or suboptimal modifications result in inefficient target degradation, which may play a role in disease-promoting protein accumulation.

[0004] Synthetic small molecules that enhance the ubiquitination of target proteins by inducing and / or stabilizing the interaction between the target protein and the E3 ligase have been reported and are referred to as “molecular glues.” See, for example, Guoqiang Dong et al., J. Med. Chem. 2021, 64, 15, 10606–10620. Molecular glues differ from PROTACs in that PROTACs are bifunctional molecules, meaning the molecule has one part that selectively binds to the target protein and another part that recruits the E3 ubiquitin ligase, each part being molecularly separated from each other by a linker. Molecular glues, on the other hand, are small chemical entities composed of single molecules. While both PROTACs and molecular glues are effective, molecular glues are inherently more advantageous than PROTACs because they are analogous to traditional small molecule drug design principles. As smaller molecules, molecular glues inherently possess properties such as better solubility and membrane permeability based on fundamental druggability principles.

[0005] It is noteworthy that although over 600 types of E3 ligases exist in human cells, only a limited number have been targeted for PROTAC or molecular gel development. These E3 ligases include, for example, cereblon (CRBN), von Hippel–Lindau (VHL), inhibitor of apoptosis proteins (IAP), and mouse two-microsome 2 homolog (MDM2). Despite significant progress in developing drugs utilizing these ligases, the usefulness of other E3 ligases remains to be investigated. One such ligase is E3 ubiquitin protein ligase 1, which contains β-transduction protein repeat sequences.

[0006] E3 ubiquitin ligases 1 and 2 (β-TrCP1 and β-TrCP2, encoded by the BTRC and FBXW11 genes) containing β-transduction protein repeat sequences are substrate recognition factors with well-characterized degradation determinant motifs whose affinity is tightly regulated by serine phosphorylation. More than 800 human proteins contain β-TrCP degradation determinant motifs with varying degrees of identity to perfectly matching sequences, and many of these proteins have been identified as drivers of cancer and other diseases. Given these clinical implications and the abundance of β-TrCP degradation determinants in a wide range of proteins, identifying methods for “gluing” BTRC to target proteins with the necessary degradation determinant sequences could have a significant impact on the development of novel molecular glue therapeutics for a variety of diseases and conditions. Summary of the Invention

[0007] This article provides complexes, methods, and therapeutic uses related to or derived from our findings, which involve the induction or promotion of ubiquitination of proteins such as NRF2 using synthetic small molecules (i.e., molecular glues) via E3 ubiquitin ligases (BTRCs) containing β-transduction protein repeat sequences. In this regard, we have identified complexes with formula A containing certain degradation determinant recognition sites. 1 -A 2 -A 3 -φ-X (0-4) -A 4 Proteins with amino acid sequences that can enhance ubiquitination from BTRC are ideal targets.

[0008] On one hand, methods for designing, synthesizing, and using synthetic small molecules (i.e., molecular gels) that amplify the affinity between a target protein (POI) and an E3 ubiquitin protein ligase (BTRC) containing a β-transduction protein repeat sequence for targeted protein degradation are described, wherein the POI contains at least one degradation determinant moiety comprising an amino acid sequence of the following formula: A 1 -A 2 -A 3 -φ-X(0-4) -A 4 A 1 It is an aspartic acid (D) residue, a glutamic acid (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 2 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 3 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, an alanine (A) residue, a threonine (T) residue, an isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine (R) residue, or optionally a phosphorylated serine (S) residue; φ and X are each independently an amino acid residue; and A 4 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a threonine (T) residue, a leucine (L) residue, or optionally a phosphorylated serine (S) residue. Attached Figure Description

[0009] Figure 1 Data from TR-FRET proximity measurements are presented to investigate the affinity amplification induced by synthetic small molecules between the degradation determinant moiety of POI as defined herein and BTRC. Detailed Implementation

[0010] A complex is provided comprising a target protein (POI), an E3 ubiquitin ligase (BTRC) containing a β-transduction protein repeat sequence, and a synthetic small molecule, wherein the POI has at least one degradation determinant moiety comprising an amino acid sequence of the following formula: A 1 -A 2 -A 3 -φ-X (0-4) -A 4 A 1 It is an aspartic acid (D) residue, a glutamic acid (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 2 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 3 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, an alanine (A) residue, a threonine (T) residue, an isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine (R) residue, or optionally a phosphorylated serine (S) residue; φ and X are each independently an amino acid residue; and A 4It is a glutamate (E) residue, an aspartic acid (D) residue, a threonine (T) residue, a leucine (L) residue, or optionally a phosphorylated serine (S) residue, provided that the POI is not a β-catenin. Alternatively, A 1 It is an aspartic acid (D) residue or a glutamic acid (E) residue; A 2 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 3 It is a glycine residue (G), an alanine residue (A), a threonine residue (T), or optionally a phosphorylated serine residue (S); φ and X are each independently an amino acid residue; and A 4 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a threonine (T) residue, a leucine (L) residue, or optionally a phosphorylated serine (S) residue.

[0011] A complex is also provided comprising a target protein (POI), an E3 ubiquitin ligase (BTRC) containing a β-transduction protein repeat sequence, and a synthetic small molecule, wherein the POI has at least one degradation determinant moiety comprising an amino acid sequence of the following formula: A 1 -A 2 -A 3 -φ-X (0-4) -A 4 A 1 It is an aspartic acid (D) residue, a glutamic acid (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 2 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 3 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, an alanine (A) residue, a threonine (T) residue, an isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine (R) residue, or optionally a phosphorylated serine (S) residue; φ and X are each independently an amino acid residue; and A 4 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a threonine (T) residue, a leucine (L) residue, or optionally a phosphorylated serine (S) residue, provided that the synthetic small molecule is not selected from... (Compound A) (Compound B) (Compound C)

[0012] (Compound D) (Compound E) and (Compound F). Or, A 1 It is an aspartic acid (D) residue or a glutamic acid (E) residue; A 2 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 3 It is a glycine residue (G), an alanine residue (A), a threonine residue (T), or optionally a phosphorylated serine residue (S); φ and X are each independently an amino acid residue; and A 4 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a threonine (T) residue, a leucine (L) residue, or optionally a phosphorylated serine (S) residue.

[0013] A method for amplifying the affinity between POI and an E3 ubiquitin protein ligase (BTRC) containing a β-transduction protein repeat sequence is also provided, the method comprising contacting POI with a synthetic small molecule, wherein POI has at least one degradation determinant motif as described above and herein, and provided that POI is not a β-catenin.

[0014] A method for amplifying the affinity between a POI and an E3 ubiquitin protein ligase (BTRC) containing a β-transduction protein repeat sequence is also provided, the method comprising contacting the POI with a synthetic small molecule, wherein the POI has at least one degradation determinant moiety as described above and herein, and provided that the synthetic small molecule is not selected from compound A, compound B, compound C, compound D, compound E, or compound F.

[0015] It also provides the use of synthesizing small molecules to amplify the affinity between POI and E3 ubiquitin protein ligases (BTRC) containing β-transduction protein repeat sequences, wherein the POI has at least one degradation determinant motif as described above and herein, provided that the POI is not a β-catenin.

[0016] It also provides the use of synthesized small molecules to amplify the affinity between POI and E3 ubiquitin protein ligases (BTRC) containing β-transduction protein repeat sequences, wherein the POI has at least one degradation determinant motif as described above and herein, provided that the synthesized small molecule is not selected from compound A, compound B, compound C, compound D, compound E, or compound F.

[0017] Also provided is the use of synthesizing small molecules for the preparation of drugs for amplifying the affinity between POI and E3 ubiquitin protein ligases (BTRC) containing β-transduction protein repeat sequences, wherein the POI has at least one degradation determinant motif as described above and herein, provided that the POI is not a β-catenin.

[0018] Also provided is the use of synthetic small molecules for the preparation of drugs for amplifying the affinity between POI and E3 ubiquitin protein ligases (BTRC) containing β-transduction protein repeat sequences, wherein the POI has at least one degradation determinant moiety as described above and herein, provided that the synthetic small molecule is not selected from compound A, compound B, compound C, compound D, compound E, or compound F.

[0019] A method for inducing POI degradation caused by ubiquitination from BTRC is also provided, comprising contacting POI with a synthetic small molecule that enhances the affinity between BTRC and POI, wherein POI has at least one degradation determinant motif as described above and herein, provided that POI is not a β-catenin.

[0020] A method for inducing the degradation of POI caused by ubiquitination of POI from BTRC is also provided, comprising contacting POI with a synthetic small molecule that enhances the affinity between BTRC and POI, wherein POI has at least one degradation determinant moiety as described above and herein, provided that the synthetic small molecule is not selected from compound A, compound B, compound C, compound D, compound E or compound F.

[0021] Also provided is the use of synthetic small molecules for inducing POI degradation induced by ubiquitination of POI from BTRC, wherein the POI has at least one degradation determinant moiety as described above and herein, and wherein the synthetic small molecule enhances the affinity between BTRC and POI, provided that the POI is not a β-catenin.

[0022] Also provided is the use of synthetic small molecules for inducing POI degradation caused by ubiquitination from BTRC, wherein the POI has at least one degradation determinant moiety as described above and herein, and wherein the synthetic small molecule enhances the affinity between BTRC and POI, provided that the synthetic small molecule is not selected from compound A, compound B, compound C, compound D, compound E or compound F.

[0023] Also provided is the use of synthetic small molecules for the preparation of medicaments for inducing POI degradation caused by ubiquitination of POI from BTRC, wherein the POI has at least one degradation determinant moiety as described above and herein, and wherein said synthetic small molecule enhances the affinity between BTRC and POI, provided that the POI is not a β-catenin.

[0024] Also provided is the use of a synthetic small molecule for the preparation of a medicament for inducing POI degradation induced by ubiquitination of POI from BTRC, wherein the POI has at least one degradation determinant moiety as described above and herein, and wherein the synthetic small molecule enhances the affinity between BTRC and POI, provided that the synthetic small molecule is not selected from compound A, compound B, compound C, compound D, compound E, or compound F. Also provided is a method for treating a condition responsive to degradation of a target protein (POI) caused by an interaction between the POI and an E3 ubiquitin ligase (BTRC) containing a β-transduction protein repeat sequence, the method comprising administering an effective amount of the synthetic small molecule to a subject in need, wherein the POI has at least one degradation determinant moiety comprising an amino acid sequence of the following formula:

[0025] A 1 -A 2 -A 3 -φ-X (0-4) -A 4 A 1 It is an aspartic acid (D) residue, a glutamic acid (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 2 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 3 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, an alanine (A) residue, a threonine (T) residue, an isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine (R) residue, or optionally a phosphorylated serine (S) residue; φ and X are each independently an amino acid residue; and A 4 It is a glutamate (E) residue, an aspartic acid (D) residue, a threonine (T) residue, a leucine (L) residue, or optionally a phosphorylated serine (S) residue, provided that the POI is not a β-catenin; and wherein the synthesized small molecule promotes POI degradation by amplifying the affinity between the POI and BTRC, provided that the POI is not a β-catenin. Alternatively, A 1 It is an aspartic acid (D) residue or a glutamic acid (E) residue; A 2 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 3 It is a glycine residue (G), an alanine residue (A), a threonine residue (T), or optionally a phosphorylated serine residue (S); φ and X are each independently an amino acid residue; and A 4It is a glutamic acid (E) residue, an aspartic acid (D) residue, a threonine (T) residue, a leucine (L) residue, or optionally a phosphorylated serine (S) residue.

[0026] A method for treating a condition responsive to the degradation of a target protein (POI) caused by the interaction between the POI and an E3 ubiquitin ligase (BTRC) containing a β-transduction protein repeat sequence is also provided, the method comprising administering an effective amount of a synthetic small molecule to a subject in need, wherein the POI has at least one degradation determinant moiety comprising an amino acid sequence of the following formula:

[0027] A 1 -A 2 -A 3 -φ-X (0-4) -A 4 A 1 It is an aspartic acid (D) residue, a glutamic acid (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 2 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 3 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, an alanine (A) residue, a threonine (T) residue, an isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine (R) residue, or optionally a phosphorylated serine (S) residue; φ and X are each independently an amino acid residue; and A 4 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a threonine (T) residue, a leucine (L) residue, or optionally a phosphorylated serine (S) residue; and wherein the synthesized small molecule promotes POI degradation by amplifying the affinity between POI and BTRC, provided that the synthesized small molecule is not selected from compound A, compound B, compound C, compound D, compound E, or compound F. Or, A 1 It is an aspartic acid (D) residue or a glutamic acid (E) residue; A 2 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 3 It is a glycine residue (G), an alanine residue (A), a threonine residue (T), or optionally a phosphorylated serine residue (S); φ and X are each independently an amino acid residue; and A 4 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a threonine (T) residue, a leucine (L) residue, or optionally a phosphorylated serine (S) residue.

[0028] As used herein, a target protein (POI) is a naturally occurring protein that may or may not contain mutations. In some respects, a POI contains at least one mutation. In some respects, a POI is not a β-catenin.

[0029] The term "synthetic small molecule" refers to a synthetically derived low molecular weight (e.g., less than 1000 g / mol) organic compound. In some respects, synthetic small molecules have molecular weights of less than 900 g / mol, less than 800 g / mol, less than 700 g / mol, less than 600 g / mol, less than 500 g / mol, less than 400 g / mol, or less than 200 g / mol. In some respects, synthetic small molecules do not include peptides or small molecules derived from peptides. For the purposes of this document, the "synthetic small molecules" disclosed herein act as molecular glues because they promote, induce, or stabilize the interaction between POI and BTRC, or amplify the affinity between them.

[0030] In some respects, φ, as described herein, is a hydrophobic residue. As used herein, a “hydrophobic residue” refers to an amino acid residue that lacks an affinity for water and / or repels water rather than absorbs it. Examples of hydrophobic residues include, but are not limited to, glycine (G), alanine (A), leucine (L), isoleucine (I), valine (V), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), methionine (M), lysine (K), threonine (T), serine (S), and aspartic acid (D). In some respects, the hydrophobic residues of this disclosure are selected from leucine (L), isoleucine (I), valine (V), proline (P), phenylalanine (F), tyrosine (Y), lysine (K), threonine (T), serine (S), and aspartic acid (D).

[0031] In relation to the described complexes, methods, and uses, a portion of BTRC binds to the degradation determinant moiety of POI via one or more molecular interactions between BTRC and the degradation determinant moiety of POI. Molecular interactions include ionic bonds, covalent bonds, hydrogen bonds, electrostatic interactions, π-stacking, van der Waals interactions, and dipole-dipole interactions, as well as combinations thereof. In some respects, the molecular interactions mentioned herein include at least one covalent bond.

[0032] In terms of the described complexes, methods, and uses, the synthetic small molecules amplify the affinity between the degradation determinant moiety of POI and BTRC. In some aspects, the synthetic small molecules stabilize the molecular interaction between the degradation determinant moiety of POI and BTRC. In other aspects, the synthetic small molecules amplify the affinity between the degradation determinant moiety of POI and BTRC by stabilizing the molecular interaction between the degradation determinant moiety of POI and BTRC.

[0033] As used herein, "the molecular interaction between the degradation determinant moiety of the stable POI and the BTRC" refers to a protein-protein complex (i.e., POI and BTRC) with greater stability than in the absence of the described synthetic small molecule. In some respects, stabilization can be measured by one or more of time-resolved fluorescence resonance transfer, fluorescence polarization, fluorescence quenching, surface plasmon resonance, biolayer interferometry, natural mass spectrometry, nuclear magnetic resonance, thermal shift analysis, isothermal titration calorimetry, microthermophoresis, size exclusion chromatography, multi-angle light scattering, and enzyme complementation.

[0034] In relation to the described complex, method, and use, the amino acid sequence of the described degradation determinant is selected from DSGKS, DSGLS, DSGTS, DSAFQE, EEGFGS, DSAYGS, ESGSPS, DGSLPST, ESTDS, DDGFVD, DSGVHL, DSGLQPS, DSGIS, and DSAPGS. In relation to the described complex, method, and use, the amino acid sequence of the degradation determinant is selected from DSGIS and DSAPGS.

[0035] In respect of the described complexes, methods, and uses, the POIs described herein are selected from adenosine deaminase acting on RNA (ADAR), yes-associated protein (YAP), erythrocyte 2-associated factor 2 (NRF2), mouse two-microsome 2 homolog (MDM2), nuclear factor NF-κ-B (NFKB2), WEE1, MYC, myeloid leukemia-1 (MCL1), cell cycle 25A (CDC25A), echinoderm microtubule-associated protein-like 4 (EML4), and Werner syndrome (WRN). In other respects, the POIs are selected from nuclear factor erythrocyte 2-associated factor 2 (NRF2) and yes-associated protein 1 (YAP1). In other respects, the POI is NRF2.

[0036] Regarding the described complex, method, and use, the degradation determinant sequence is DSGIS or DSAPGS, and the POI is NRF2.

[0037] In relation to the described complexes, methods, and uses, when the POI is not a β-catenin, the synthetic small molecule is selected from synthetic small molecules that act as a molecular glue between the degradation determinant moiety of the β-catenin and the BTRC. Such compounds include, for example, compounds A, B, C, D, E, or F.

[0038] The terms "subject" and "patient" are used interchangeably and refer to mammals in need of treatment, such as companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cattle, pigs, horses, sheep, goats, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). Typically, a subject is a person in need of treatment.

[0039] As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of a disease or condition or one or more of its symptoms, or inhibiting its progression. In some respects, treatment may be administered after one or more symptoms have developed, i.e., therapeutic treatment. In other respects, treatment may be administered in the absence of symptoms. For example, treatment may be administered to susceptible individuals before the onset of symptoms (e.g., based on a history of symptoms and / or based on exposure to a specific organism or other susceptibility factor), i.e., preventative treatment. Treatment may also continue after symptoms have subsided, for example, to delay their recurrence.

[0040] Conditions that respond to POI degradation caused by the interaction between the target protein (POI) and the E3 ubiquitin protein ligase (BTRC) containing β-transferin repeat sequences include, but are not limited to, cancer, immune disorders, inflammatory diseases, genetic diseases, hypertension, heart disease, and cognitive disorders (Parkinson's disease, Alzheimer's disease).

[0041] The term “effective amount” or “therapeutic effective amount” refers to the amount of the compound described herein that is sufficient, under the conditions of administration, to achieve the desired therapeutic effect (such as treating the condition described herein).

[0042] example

[0043] In some embodiments, the affinity amplification between the degradation determinant moiety of the POI induced by the synthetic small molecule and BTRC is measured by TR-FRET (time-resolved fluorescence resonance energy transfer) proximity assay. In this assay, His-labeled BTRC-SKP1 is mixed with the donor fluorophore (LANCE anti-6xHis-EU-W1024, PerkinElmer) in or without the synthetic small molecule and diluted in buffer A. Alternatively, biotin-labeled synthetic peptides with either the NFE2L2 non-phosphorylated degradation determinant sequence (peptide 1) or the NFE2L2 diphosphorylated degradation determinant sequence (peptide 2) are mixed with the acceptor fluorophore (LANCE streptavidin-Ulight, PerkinElmer) and serially diluted 3-fold in buffer A to obtain eleven different concentrations. A background control solution is prepared by adding an equal volume of buffer, the peptide is dissolved in (buffer B) and mixed with the acceptor fluorophore, and serially diluted with the peptide as described. BTRC-SKP1 and serially diluted peptide solutions or background control solutions were aliquoted into ProxiPlate-384 F Plus black plates (PerkinElmer).

[0044] The total volume of each mixture was 10 μL, with a final concentration of 5 nanomoles for BTRC, 50 μmoles for the synthesized small molecule, 0.3 nanomoles for the donor fluorophore, a maximum concentration of 10 μmoles for the synthesized peptide, and a maximum concentration of 2 μmoles for the donor fluorophore. The mixtures were incubated at room temperature for 1 hour. The TR-FRET signals generated after 1 hour of incubation were read on a Pherastar FSX (BMG LABTECH) at excitation wavelengths of 337 nm and emission wavelengths of 665 nm and 620 nm. The values ​​were reported as the TR-FRET ratio, defined as the TR-FRET signal at 665 nm divided by the TR-FRET signal at 620 nm, and the quotient multiplied by 10,000. The background-subtracted TR-FRET ratio was generated by subtracting the TR-FRET ratio from the mixture containing the background control. Reagents: Buffer A (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM TCEP, 0.02 (v / v)% Tween-20, 0.1 mg / mL BSA), Buffer B (20 mM TRIS pH 8.0, 150 mM NaCl), synthetic small molecules in 100% DMSO, BTRC-SKP1 (internal), peptide 1 (internal), peptide 2 (internal).

[0045] Results: Representative data from TR-FRET proximity assays are shown in Figure 1. The data were fitted to a site-specific binding equation Y = (Bmax * X) / (Kd + X), where X is defined as the final concentration of the peptide, Y is the TR-FRET ratio minus the background, Bmax is the fitted maximum TR-FRET ratio signal, and Kd is the equilibrium dissociation constant.

[0046] All references cited throughout this application (including references, granted patents, published patent applications, and co-pending patent applications) are expressly incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly known to one of ordinary skill in the art.

Claims

1. A complex comprising a target protein (POI), an E3 ubiquitin ligase (BTRC) containing a β-transduction protein repeat sequence, and a synthetic small molecule, wherein the POI has at least one degradation determinant moiety comprising an amino acid sequence of the following formula: A 1 -A 2 -A 3 -φ-X (0-4) -A 4 , in: A 1 It is an aspartic acid (D) residue, a glutamic acid (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 2 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 3 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, an alanine (A) residue, a threonine (T) residue, an isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine (R) residue, or optionally a phosphorylated serine (S) residue. φ and X are each an amino acid residue; and A 4 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a threonine (T) residue, a leucine (L) residue, or optionally a phosphorylated serine (S) residue, provided that the POI is not a β-catenin.

2. The complex of claim 1, wherein the synthetic small molecule amplifies the affinity between the degradation determinant moiety of the POI and the BTRC.

3. A method for amplifying the affinity between a point of interest (POI) and an E3 ubiquitin protein ligase (BTRC) containing a β-transduction protein repeat sequence, the method comprising contacting the POI with a synthetic small molecule, wherein the POI has at least one degradation determinant moiety comprising an amino acid sequence of the following formula: A 1 -A 2 -A 3 -φ-X (0-4) -A 4 , in: A 1 It is an aspartic acid (D) residue, a glutamic acid (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 2 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 3 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, an alanine (A) residue, a threonine (T) residue, an isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine (R) residue, or optionally a phosphorylated serine (S) residue. φ and X are each an amino acid residue; and A 4 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a threonine (T) residue, a leucine (L) residue, or optionally a phosphorylated serine (S) residue, provided that the POI is not a β-catenin.

4. A method for inducing the degradation of a POI caused by ubiquitination from a BTRC, comprising contacting the POI with a synthetic small molecule that enhances the affinity between the BTRC and the POI, wherein the POI has at least one degradation determinant moiety comprising an amino acid sequence of the following formula: A 1 -A 2 -A 3 -φ-X (0-4) -A 4 , in: A 1 It is an aspartic acid (D) residue, a glutamic acid (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 2 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 3 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, an alanine (A) residue, a threonine (T) residue, an isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine (R) residue, or optionally a phosphorylated serine (S) residue. φ and X are each an amino acid residue; and A 4 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a threonine (T) residue, a leucine (L) residue, or optionally a phosphorylated serine (S) residue, provided that the POI is not a β-catenin.

5. A method for treating a condition responsive to degradation of a target protein (POI), said degradation being caused by an interaction between said POI and an E3 ubiquitin ligase (BTRC) containing a β-transduction protein repeat sequence, said method comprising administering an effective amount of a synthetic small molecule to a subject in need, said POI having at least one degradation determinant moiety comprising an amino acid sequence of the following formula: A 1 -A 2 -A 3 -φ-X (0-4) -A 4 , in: A 1 It is an aspartic acid (D) residue, a glutamic acid (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 2 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 3 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, an alanine (A) residue, a threonine (T) residue, an isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine (R) residue, or optionally a phosphorylated serine (S) residue. φ and X are each an amino acid residue; and A 4 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a threonine (T) residue, a leucine (L) residue, or optionally a phosphorylated serine (S) residue, provided that the POI is not a β-catenin.

6. The method of any one of claims 5, wherein the condition is selected from cancer, immune disorders, inflammatory diseases, genetic diseases, hypertension, heart disease, and cognitive disorders such as Parkinson's disease or Alzheimer's disease.

7. The complex or method of any one of claims 1 to 6, wherein a portion of the BTRC binds to the degradation determinant portion of the POI via one or more molecular interactions between the BTRC and the degradation determinant portion of the POI.

8. The complex or method of claim 7, wherein the one or more molecular interactions between BTRC and the degradation determinant moiety of the POI are stabilized by the synthetic small molecule.

9. The complex or method according to any one of claims 1 to 8, wherein A 1 It is an aspartic acid (D) residue or a glutamic acid (E) residue; A 2 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a glycine (G) residue, or optionally a phosphorylated serine (S) residue; A 3 It is a glycine residue (G), an alanine residue (A), a threonine residue (T), or optionally a phosphorylated serine residue (S); φ and X are each an amino acid residue; and A 4 It is a glutamic acid (E) residue, an aspartic acid (D) residue, a threonine (T) residue, a leucine (L) residue, or optionally a phosphorylated serine (S) residue.

10. The complex or method according to any one of claims 1 to 9, wherein X is 0, 1, 2 or 3.

11. The complex or method according to any one of claims 1 to 10, wherein X is 0.

12. The complex or method according to any one of claims 1 to 11, wherein φ is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), valine (V), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), methionine (M), lysine (K), threonine (T), serine (S) and aspartic acid (D).

13. The complex or method according to any one of claims 1 to 12, wherein φ is selected from leucine (L), isoleucine (I), valine (V), proline (P), phenylalanine (F), tyrosine (Y), lysine (K), threonine (T), serine (S) and aspartic acid (D).

14. The complex or method according to any one of claims 1 to 9, wherein the degradation determinant has a sequence selected from DSGKS, DSGLS, DSGTS, DSAFQE, EEGFGS, DSAYGS, ESGSPS, DGSLPST, ESTDS, DDGFVD, DSGVHL, DSGLQPS, DSGIS, and DSAPGS.

15. The complex or method according to any one of claims 1 to 9, wherein the degradation determinant has a sequence selected from DSGIS and DSAPGS.

16. The complex or method according to any one of claims 1 to 15, wherein the POI is selected from RNA-acting adenosine deaminase (ADAR), yes-associated protein (YAP), erythrocyte 2-associated factor 2 (NRF2), mouse two-microsome 2 homolog (MDM2), nuclear factor NF-κ-B (NFKB2), WEE1, MYC, myeloid leukemia-1 (MCL1), cell cycle 25A (CDC25A), echinoderm microtubule-associated protein-like 4 (EML4), and Werner syndrome (WRN).

17. The complex or method according to any one of claims 1 to 16, wherein the POI is selected from nuclear factor erythrocyte 2-associated factor 2 (NRF2) and yes-associated protein 1 (YAP1).

18. The complex or method according to any one of claims 1 to 17, wherein the POI is NRF2.

19. The complex or method of any one of claims 1 to 9, wherein the degradation determinant sequence is DSGIS or DSAPGS, and the POI is NRF2.

20. The complex or method according to any one of claims 1 to 19, wherein the POI has at least one mutation.

21. The complex or method according to any one of claims 1 to 20, wherein the synthetic small molecule is selected from... , , , , and .

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