Targeted protein degradation

Abstract

Provided herein are complexes, methods, and therapeutic uses for inducing and / or enhancing the ubiquitination of proteins such as NRF2 by beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC).

Description

TARGETED PROTEIN DEGRADATIONRELATED APPLICATIONS

[0001] This application claims the benefit of 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

[0002] Targeted protein degradation is an attractive area for the development of therapeutics to treat diseases driven by the accumulation or inappropriate expression of proteins that remodel cellular physiology. The ubiquitin-proteasome system (UPS) is a major pathway for regulating cellular protein abundance that involves tagging target proteins with ubiquitin for recognition and degradation by the proteasome. A key component of the UPS that lends specificity to the process is the recognition of sequence or structural motifs (degrons) on the target protein by a substrate recognition factor of E3 ubiquitin conjugating enzymes. Additional regulation of target protein recognition is provided by post-translational modification (such as phosphorylation of serine, threonine, or tyrosine residues) of the degron motif that increases the affinity of the substrate recognition factor. Each substrate recognition factor recognizes a defined combination of amino acid sequence and post-translational modifications; deviations from this sequence or suboptimal modification result in inefficient target degradation which may play a role in the accumulation of disease-promoting proteins.

[0003] Synthetic small molecules which enhance the ubiquitination of a target protein by inducing and / or stabilizing the interaction between the target protein and an E3 ligase have been reported and are referred to as “molecular glues”. See e.g., Guoqiang Dong et al., J.Med. Chem. 2021, 64, 15, 10606-10620. Molecular glues are distinct from PROTACS in that PROTACS are bifunctional molecules, i.e., the molecules have one moiety that selectively binds a target protein and another moiety that recruits an E3 ubiquitin ligase, each moiety being separated from each other on the molecule by a linker. Molecular glues, on the other hand, are small chemical entities made up of a single molecule. While both PROTACS and molecular glues are effective, molecular glues are inherently more advantageous than PROTACs because they resemble traditional small molecule drug design principles. Being a smaller molecule, a molecular glue intrinsically poses qualities such as better solubility and membrane permeability, based on basic druggability principles.

[0004] Strikingly, even though there are over 600 types of E3 ligases in human cells, only a limited number have been targeted for PROTAC or molecule glue exploitation. These E3 ligase include, e.g., cereblon (CRBN), von Hippel-Lindau (VHL), inhibitors of apoptosis protein (IAP), and mouse double minute 2 homolog (MDM2). Although great strides have been made towards the development of drugs that take advantage of these ligases, the need to investigate the usefulness of other E3 ligases remains. One such ligase is Beta-Transducin Repeat Containing E3 Ubiquitin Protein Ligase 1.

[0005] The proteins Beta-Transducin Repeat Containing E3 Ubiquitin Protein Ligase 1 and 2 (P-TrCPl and P-TrCP2, encoded by the BTRC and FBXW 11 genes) are substrate recognition factors with a well-characterized degron motif whose affinity is tightly regulated by serine phosphorylation. Over 800 human proteins contain the P-TrCP degron motif with varying degrees of identity to the perfectly matched sequence, and many of these proteins have been implicated as drivers of cancer and other diseases. Given these clinical implications, and the abundance of P-TrCP degron across a variety of proteins, identifying means for “gluing” BTRC to target proteins having the requisite degron sequence can have meaningful implications in developing new molecular glue therapeutics to treat a wide variety of diseases and disorders.SUMMARY

[0006] Provided herein are complexes, methods, and therapeutic uses relating to, or arising from, our discoveries for using synthetic small molecule (i.e., molecular glues) to induce or facilitate the ubiquitination of proteins such as NRF2 by beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC). In this regard, we have identified that proteins comprising certain degron recognition sites having an amino acid sequence of the formula A1-A2-A3-cp-X(o-4)-A4are ideal targets in which ubiquitination from BTRC can be enhanced.

[0007] In one aspect, described are methods for designing, synthesizing, and using synthetic small molecules (i.e., molecule glues) which amplify the affinity between a protein of interest (POI) and beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC), for targeted protein degradation, wherein the POI comprises at least one degron moiety comprising an amino acid sequence of the formula: A1-A2-A3-cp-X(o-4)-A4, wherein A1is an aspartate (D) residue, a glutamate (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue; A2is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue; A3is a glutamate (E) residue, an aspartate (D) residue, a glycine (G)residue, an alanine (A) residue, a threonine (T) residue, a isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine (R) residue, or an optionally phosphorylated serine(S) residue; cp and X are each independently an amino acid residue; and A4is a glutamate (E) residue, an aspartate (D) residue, a threonine (T) residue, a leucine (L) residue, or an optionally phosphorylated serine (S) residue.BRIEF DESCRIPTION OF THE FIGURES

[0008] FIG. 1 shows data from a TR-FRET proximity assay to study the amplification in affinity induced by a synthetic small molecule between the degron moiety of the POI and BTRC as defined herein.DETAILED DESCRIPTION

[0009] Provided is a complex comprising a protein of interest (POI), beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC), and a synthetic small molecule, wherein the POI has at least one degron moiety comprising an amino acid sequence of the formula: A1-A2-A3-cp-X(o-4)-A4, wherein A1is an aspartate (D) residue, a glutamate (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue; A2is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue; A3is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, an alanine (A) residue, a threonine(T) residue, a isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine(R) residue, or an optionally phosphorylated serine (S) residue; cp and X are each independently an amino acid residue; and A4is a glutamate (E) residue, an aspartate (D) residue, a threonine (T) residue, a leucine (L) residue, or an optionally phosphorylated serine(S) residue, provided the POI is not P-Catenin. Alternatively, A1is an aspartate (D) residue or a glutamate (E) residue; A2is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue; A3is a glycine residue (G), an alanine (A) residue, a threonine (T) residue, or an optionally phosphorylated serine (S) residue; cp and X are each independently an amino acid residue; and A4is a glutamate (E) residue, an aspartate (D) residue, a threonine (T) residue, a leucine (L) residue, or an optionally phosphorylated serine (S) residue.

[0010] Also provided is a complex comprising a protein of interest (POI), beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC), and a synthetic small molecule, wherein the POI has at least one degron moiety comprising an amino acid sequence of theformula: A1-A2-A3-cp-X(o-4)-A4, wherein A1is an aspartate (D) residue, a glutamate (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue; A2is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue; A3is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, an alanine (A) residue, a threonine (T) residue, a isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine(R) residue, or an optionally phosphorylated serine (S) residue; cp and X are each independently an amino acid residue; and A4is a glutamate (E) residue, an aspartate (D) residue, a threonine (T) residue, a leucine (L) residue, or an optionally phosphorylated serine(S) residue, provided the synthetic small molecule is not selected fromresidue or a glutamate (E) residue; A2is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue; A3is a glycine residue (G), an alanine (A) residue, a threonine (T) residue, or an optionally phosphorylated serine (S) residue; cp and X are each independently an amino acid residue; and A4is a glutamate (E) residue, an aspartate (D) residue, a threonine (T) residue, a leucine (L) residue, or an optionally phosphorylated serine (S) residue.

[0011] Also provided is a method of amplifying the affinity between a POI and beta- transducin repeat containing E3 ubiquitin protein ligase (BTRC), said method comprising contacting the POI with a synthetic small molecule, wherein the POI has at least one degron moiety as described above and herein, and provided the POI is not P-Catenin.

[0012] Also provided is a method of amplifying the affinity between a POI and beta- transducin repeat containing E3 ubiquitin protein ligase (BTRC), said method comprising contacting the POI with a synthetic small molecule, wherein the POI has at least one degron moiety as described above and herein, and provided the synthetic small molecule is not selected from Compound A, Compound B, Compound C, Compound D, Compound E, or Compound F.

[0013] Also provided is the use of a synthetic small molecule for amplifying the affinity between a POI and beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC), wherein the POI has at least one degron moiety as described above and herein, provided the POI is not P-Catenin.

[0014] Also provided is the use of a synthetic small molecule for amplifying the affinity between a POI and beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC), wherein the POI has at least one degron moiety as described above and herein, provided the synthetic small molecule is not selected from Compound A, Compound B, Compound C, Compound D, Compound E, or Compound F.

[0015] Also provided is the use of a synthetic small molecule for use in the manufacture of a medicament for amplifying the affinity between a POI and beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC), wherein the POI has at least one degron moiety as described above and herein, provided the POI is not P-Catenin.

[0016] Also provided is the use of a synthetic small molecule for use in the manufacture of a medicament for amplifying the affinity between a POI and beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC), wherein the POI has at least one degron moiety as described above and herein, provided the synthetic small molecule is not selectedfrom Compound A, Compound B, Compound C, Compound D, Compound E, or Compound F.

[0017] Also provided is a method of inducing degradation of a POI arising from the ubiquitination of the POI from BTRC, comprising contacting the POI with a synthetic small molecule that enhances the affinity between BTRC and the POI, wherein the POI has at least one degron moiety as described above and herein, provided the POI is not P-Catenin.

[0018] Also provided is a method of inducing degradation of a POI arising from the ubiquitination of the POI from BTRC, comprising contacting the POI with a synthetic small molecule that enhances the affinity between BTRC and the POI, wherein the POI has at least one degron moiety as described above and herein, provided the synthetic small molecule is not selected from Compound A, Compound B, Compound C, Compound D, Compound E, or Compound F.

[0019] Also provided is the use of a synthetic small molecule for inducing degradation of a POI arising from the ubiquitination of the POI from BTRC, wherein the POI has at least one degron moiety as described above and herein, and wherein said synthetic small molecule enhances the affinity between BTRC and the POI, provided the POI is not P-Catenin.

[0020] Also provided is the use of a synthetic small molecule for inducing degradation of a POI arising from the ubiquitination of the POI from BTRC, wherein the POI has at least one degron moiety as described above and herein, and wherein said synthetic small molecule enhances the affinity between BTRC and the POI, provided 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 a synthetic small molecule for the manufacture of a medicament for inducing degradation of a POI arising from the ubiquitination of the POI from BTRC, wherein the POI has at least one degron moiety as described above and herein, and wherein said synthetic small molecule enhances the affinity between BTRC and the POI, provided the POI is not P-Catenin.

[0022] Also provided is the use of a synthetic small molecule for the manufacture of a medicament for inducing degradation of a POI arising from the ubiquitination of the POI from BTRC, wherein the POI has at least one degron moiety as described above and herein, and wherein said synthetic small molecule enhances the affinity between BTRC and the POI, provided 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 of treating a condition responsive to the degradation of a protein of interest (POI) resulting fromthe interaction between the POI and beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC), said method comprising administering to a subject in need thereof an effective amount of a synthetic small molecule, wherein the POI has at least one degron moiety comprising an amino acid sequence of the formula:A1-A2-A3-cp-X(o-4)-A4, wherein A1is an aspartate (D) residue, a glutamate (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue; A2is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue; A3is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, an alanine (A) residue, a threonine (T) residue, a isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine(R) residue, or an optionally phosphorylated serine (S) residue; cp and X are each independently an amino acid residue; and A4is a glutamate (E) residue, an aspartate (D) residue, a threonine (T) residue, a leucine (L) residue, or an optionally phosphorylated serine(S) residue; and wherein the synthetic small molecule facilitates degradation of the POI by amplifying the affinity between the POI and BTRC, provided the POI is not P-Catenin. Alternatively, A1is an aspartate (D) residue or a glutamate (E) residue; A2is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue; A3is a glycine residue (G), an alanine (A) residue, a threonine (T) residue, or an optionally phosphorylated serine (S) residue; cp and X are each independently an amino acid residue; and A4is a glutamate (E) residue, an aspartate (D) residue, a threonine (T) residue, a leucine (L) residue, or an optionally phosphorylated serine (S) residue.

[0023] Also provided is a method of treating a condition responsive to the degradation of a protein of interest (POI) resulting from the interaction between the POI and beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC), said method comprising administering to a subject in need thereof an effective amount of a synthetic small molecule, wherein the POI has at least one degron moiety comprising an amino acid sequence of the formula: A1-A2-A3-cp-X(o-4)-A4, wherein A1is an aspartate (D) residue, a glutamate (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue; A2is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue; A3is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, an alanine (A) residue, a threonine(T) residue, a isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine (R) residue, or an optionally phosphorylated serine (S) residue; cp and X are each independently an amino acid residue; and A4is a glutamate (E) residue, an aspartate (D)residue, a threonine (T) residue, a leucine (L) residue, or an optionally phosphorylated serine (S) residue; and wherein the synthetic small molecule facilitates degradation of the POI by amplifying the affinity between the POI and BTRC, provided the synthetic small molecule is not selected from Compound A, Compound B, Compound C, Compound D, Compound E, or Compound F. Alternatively, A1is an aspartate (D) residue or a glutamate (E) residue; A2is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue; A3is a glycine residue (G), an alanine (A) residue, a threonine (T) residue, or an optionally phosphorylated serine (S) residue; cp and X are each independently an amino acid residue; and A4is a glutamate (E) residue, an aspartate (D) residue, a threonine (T) residue, a leucine (L) residue, or an optionally phosphorylated serine (S) residue.

[0024] As used herein, a protein of interest (POI) refers to a naturally occurring protein which may or may not contain mutations. In certain aspects, the POI comprises at least one mutation. In certain aspects, the POI is not P-Catenin.

[0025] The term “synthetic small molecule” refers to a low molecular weight (e.g., less than 1000 g / mol) organic compound that is synthetically derived. In certain aspects, the synthetic small molecule has a molecular weight 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 aspects, synthetic small molecules exclude peptides or small molecules derived from peptides. For the purposes herein, “synthetic small molecules” of the present disclosure act as molecule glues in that they encourage, induce, or stabilize the interaction, or amplify the affinity, between a POI and BTRC.

[0026] In some aspect, cp as described herein is a hydrophobic residue. As used herein, a “hydrophobic residue” refers to an amino acid residue which lacks affinity for water and / or repels water rather than absorbing 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 aspartate (D). In some aspects, the hydrophobic residue of the disclosure is selected from leucine (L), isoleucine (I), valine (V), proline (P), phenylalanine (F), tyrosine (Y), lysine (K), threonine (T), serine (S), and aspartate (D).

[0027] In aspects of the described complexes, methods, and uses, a portion of BTRC is bound to the degron moiety of the POI through one or more molecular interactions between BTRC and the degron moiety of the POI. Molecular interactions include ionic bonds, covalent bonds, hydrogen bonds, electrostatic interactions, pi stacking, van der Waalsinteractions, and dipole-dipole interactions, and combinations thereof. In certain aspects, the molecular interactions referred to herein include at least one covalent bond.

[0028] In aspects of the described complexes, methods, and uses, the synthetic small molecule amplifies the affinity between the degron moiety of the POI and BTRC. In certain aspects, the synthetic small molecule stabilizes the molecular interactions between the degron moiety of the POI and BTRC. In certain aspects, the synthetic small molecule amplifies the affinity between the degron moiety of the POI and BTRC by stabilizing the molecular interactions between the degron moiety of the POI and BTRC.

[0029] As used herein “stabilizing the molecular interactions between the degron moiety of the POI and BTRC” means that the stability of the protein-protein complex, i.e., the POI and BTRC is greater than in the absence of the described synthetic small molecule. In certain aspects, stabilization can be measured by one for more of time-resolved fluorescence resonance transfer, fluorescence polarization, fluorescence quenching, surface plasmon resonance, biolayer interferometry, native mass spectrometry, nuclear magnetic resonance, thermal shift analysis, isothermal titration calorimetry, microscale thermophoresis, size exclusion chromatography, multi-angle light scattering, and enzyme complementation.

[0030] In aspects of the described complexes, methods, and uses, the amino acid sequence of the described degron is selected from DSGKS, DSGLS, DSGTS, DSAFQE, EEGFGS, DSAYGS, ESGSPS, DGSLPST, ESTDS, DDGFVD, DSGVHL, DSGLQPS, DSGIS, and DSAPGS. In aspects of the described complexes, methods, and uses, amino acid sequence of the degron is selected from DSGIS and DSAPGS.

[0031] In aspects of the described complexes, methods, and uses, the POI described herein is selected from adenosine deaminases acting on RNA (ADAR), yes-associated protein (YAP), erythroid 2-related factor 2 (NRF2), mouse double minute 2 homolog (MDM2), nuclear factor NF-kappa-B (NFKB2), WEE1, MYC, myeloid cell leukemia-1 (MCL1), cell division cycle 25A (CDC25A), echinoderm microtubule-associated protein-like 4 (EML4), and Werner syndrome (WRN). In other aspects, the POI is selected from nuclear factor erythroid 2-related factor 2 (NRF2), yes-associated protein 1 (YAP1). In other aspects, the POI is NRF2.

[0032] In aspects of the described complexes, methods, and uses, the degron sequence is DSGIS or DSAPGS and the POI is NRF2

[0033] In aspects of the described complexes, methods, and uses, when the POI is not beta-catenin, the synthetic small molecule is selected from one that serves as a molecular gluebetween the degron moiety of beta-catenin and BTRC. Such compounds include e.g., Compound A, Compound B, Compound C, Compound D, Compound E, or Compound F.

[0034] The terms “subject” and “patient” may be used interchangeably, and means a mammal in need of treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horses, sheep, goats and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like). Typically, the subject is a human in need of treatment.

[0035] As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof. In some aspects, treatment may be administered after one or more symptoms have developed, i.e., therapeutic treatment. In other aspects, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of exposure to a particular organism, or other susceptibility factors), i.e., prophylactic treatment. Treatment may also be continued after symptoms have resolved, for example to delay their recurrence.

[0036] Conditions responsive to the degradation of a protein of interest (POI) resulting from the interaction between the POI and beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC)include, but are not limited to, cancer, an immune disorder, an inflammatory disease, a hereditary disease, hypertension, heart disease, a cognitive disease (Parkinson’s, Alzheimer’s).

[0037] The term “effective amount” or “therapeutically effective amount” refers to an amount of a compound described herein that is sufficient to achieve the desired therapeutic effect (such as treatment of a condition recited herein) under the conditions of administration.EXEMPLIFICATION

[0038] In some embodiments the amplification in affinity induced by a synthetic small molecule between the degron moiety of the POI and BTRC is measured by a TR-FRET (time resolved-fluorescence resonance energy transfer) proximity assay. In this assay a His-tagged BTRC-SKP1 is mixed with the donor fluorophore (LANCE anti-6xHis-EU-W1024, PerkinElmer) with or without a synthetic small molecule present and diluted in buffer A. Additionally, a biotin-labeled synthetic peptide of the NFE2L2 non-phosphorylated degron sequence (Peptide 1) or NFE2L2 di-phosphorylated degron sequence (Peptide 2) is mixed with the acceptor fluorophore (LANCE streptavidin-Ulight, PerkinElmer) and serially diluted 3-fold in buffer A for a total of eleven different concentrations. Background control solutionsare generated by adding an equivalent volume of the buffer peptides are dissolved in (Buffer B) and mixed with the acceptor fluorophore and serially diluted as described with peptides. BTRC-SKP1 and serially diluted peptide solutions or background control solutions are dispensed in equal parts into a ProxiPlate-384 F Plus black plate (PerkinElmer).

[0039] The total volume of each mixture is 10 microliters, with a final concentration of BTRC at 5 nanomolar, 50 micromolar synthetic small molecule, 0.3 nanomolar donor fluorophore, top concentration of 10 micromolar synthetic peptide, and top concentration of 2 micromolar donor fluorophore. The mixture is incubated for 1-hour at room temperature.The generated TR-FRET signal after the 1-hour incubation is read on a PHERAstar FSX (BMG LABTECH) with excitation: 337 nm and emissions: 665 nm and 620 nm. Values are reported as the TR-FRET ratio which is defined as the TR-FRET signal at 665 nm divided by the TR-FRET signal at 620 nm, the quotient is multiplied by 10,000. The background subtracted TR-FRET ratio is generated by subtracting out the TR-FRET ratio from mixtures 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 (in-house), Peptide 1 (in-house), Peptide 2 (in-house).

[0040] Results: Representative data from the TR-FRET proximity assay is shown in FIG. 1. Data is fit to a one site — specific binding equation Y=(Bmax*X) / (Kd + X), where X is defined as the final concentration of peptide, Y as the background subtracted TR-FRET ratio, Bmax as the fitted maximum TR-FRET ratio signal, and Kd as the equilibrium dissociation constant.

[0041] The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated herein in their entireties by reference. Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly known to one with ordinary skill in the art.

Claims

Listing of Claims:

1. A complex comprising a protein of interest (POI), beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC), and a synthetic small molecule, wherein the POI has at least one degron moiety comprising an amino acid sequence of the formula:A1-A2-A3-(p-X(0-4)-A4, wherein:A1is an aspartate (D) residue, a glutamate (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue;A2is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue;A3is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, an alanine (A) residue, a threonine (T) residue, a isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine (R) residue or an optionally phosphorylated serine (S) residue;(p and X are each independently an amino acid residue; andA4is a glutamate (E) residue, an aspartate (D) residue, a threonine (T) residue, a leucine (L) residue, or an optionally phosphorylated serine (S) residue, provided the POI is not P-Catenin.

2. The complex of Claim 1, wherein the synthetic small molecule amplifies the affinity between the degron moiety of the POI and BTRC.

3. A method of amplifying the affinity between a POI and beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC), said method comprising contacting the POI with a synthetic small molecule, wherein the POI has at least one degron moiety comprising an amino acid sequence of the formula:AJ-A2-A3-(p-X(o-4)-A4, wherein:A1is an aspartate (D) residue, a glutamate (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue;A2is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue;A3is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, an alanine (A) residue, a threonine (T) residue, a isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine (R) residue or an optionally phosphorylated serine (S) residue;(p and X are each independently an amino acid residue; andA4is a glutamate (E) residue, an aspartate (D) residue, a threonine (T) residue, a leucine (L) residue, or an optionally phosphorylated serine (S) residue, provided the POI is not P-Catenin.

4. A method of inducing degradation of a POI arising from the ubiquitination of the POI from BTRC, comprising contacting the POI with a synthetic small molecule that enhances the affinity between BTRC and the POI, wherein the POI has at least one degron moiety comprising an amino acid sequence of the formula:AJ-A2-A3-(p-X(o-4)-A4, wherein:A1is an aspartate (D) residue, a glutamate (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue;A2is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue;A3is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, an alanine (A) residue, a threonine (T) residue, a isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine (R) residue or an optionally phosphorylated serine (S) residue; cp and X are each independently an amino acid residue; andA4is a glutamate (E) residue, an aspartate (D) residue, a threonine (T) residue, a leucine (L) residue, or an optionally phosphorylated serine (S) residue, provided the POI is not P-Catenin.

5. A method of treating a condition responsive to the degradation of a protein of interest (POI) resulting from the interaction between the POI and beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC), said method comprising administering to a subject in need thereof an effective amount of a synthetic small molecule, wherein the POI has at least one degron moiety comprising an amino acid sequence of the formula:AJ-A2-A3-(p-X(o-4)-A4, wherein:A1is an aspartate (D) residue, a glutamate (E) residue, a threonine (T) residue, a leucine (L) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue;A2is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue;A3is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, an alanine (A) residue, a threonine (T) residue, a isoleucine (I) residue, a leucine (L) residue, a valine (V) residue, an arginine (R) residue or an optionally phosphorylated serine (S) residue;(p and X are each independently an amino acid residue; andA4is a glutamate (E) residue, an aspartate (D) residue, a threonine (T) residue, a leucine (L) residue, or an optionally phosphorylated serine (S) residue, provided the POI is not P-Catenin.

6. The method of any one of Claim 5, wherein the condition is selected from a cancer, an immune disorder, an inflammatory disease, a hereditary disease, hypertension, heart disease, and a cognitive disease such as Parkinson’s or Alzheimer’s.

7. The complex or method of any one of Claims 1 to 6, wherein a portion of BTRC is bound to the degron moiety of the POI through one or more molecular interactions between BTRC and the degron moiety of the POI.

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

9. The complex or method of any one of Claims 1 to 8, whereinA1is an aspartate (D) residue or a glutamate (E) residue;A2is a glutamate (E) residue, an aspartate (D) residue, a glycine (G) residue, or an optionally phosphorylated serine (S) residue;A3is a glycine residue (G), an alanine (A) residue, a threonine (T) residue, or an optionally phosphorylated serine (S) residue;(p and X are each independently an amino acid residue; andA4is a glutamate (E) residue, an aspartate (D) residue, a threonine (T) residue, a leucine (L) residue, or an optionally phosphorylated serine (S) residue.

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

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

12. The complex or method of any one of Claims 1 to 11, wherein cp 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 aspartate (D).

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

14. The complex or method of any one of Claims 1 to 9, wherein the degron 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 of any one of Claims 1 to 9, wherein the degron has a sequence selected from DSGIS and DSAPGS.

16. The complex or method of any one of Claims 1 to 15, wherein the POI is selected from adenosine deaminases acting on RNA (ADAR), yes-associated protein (YAP), erythroid 2-related factor 2 (NRF2), mouse double minute 2 homolog (MDM2), nuclear factor NF- kappa-B (NFKB2), WEE1, MYC, myeloid cell leukemia-1 (MCL1), cell division cycle 25A (CDC25A), echinoderm microtubule-associated protein-like 4 (EML4), and Werner syndrome (WRN).

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

18. The complex or method of 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 degron sequence is DSGIS or DSAPGS and the POI is NRF2.

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

21. The complex or method of any one of Claims 1 to 20, wherein the synthetic small

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