Prospective discovery of small molecule enhancers of an E3 ligase-substrate interaction

Protein–protein interactions (PPIs) governing the recognition of substrates by E3 ubiquitin ligases are critical to cellular function. There is significant therapeutic potential in the development of small molecules that modulate these interactions; however, rational design of small molecule enhancers of PPIs remains elusive. Herein, we report the prospective identification and rational design of potent small molecules that enhance the interaction between an oncogenic transcription factor, β-Catenin, and its cognate E3 ligase, SCFβ-TrCP. These enhancers potentiate the ubiquitylation of mutant β-Catenin by β-TrCP in vitro and induce the degradation of an engineered mutant β-Catenin in a cellular system. Distinct from PROTACs, these drug-like small molecules insert into a naturally occurring PPI interface, with contacts optimized for both the substrate and ligase within the same small molecule entity. The prospective discovery of ‘molecular glue’ presented here provides a paradigm for the development of small molecule degraders targeting hard-to-drug proteins.

P rotein-protein interactions (PPIs) are central to most signaling pathways in cells and represent important nodes for therapeutic intervention. Although most small molecule PPI modulators are inhibitors, a small subset of PPI modulators are known to work by stabilizing PPIs 1,2 . These include immunosuppressants cyclosporine, FK506 and rapamycin as well as anti-cancer agents lenalidomide, pomalidomide, and thalidomide (collectively referred as immunomodulatory drugs or IMiDs). These IMiD molecules work by binding to the CRL4 E3 ligase substrate receptor protein, Cereblon (CRBN) and promoting interaction with the non-native lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IFZF3), as well as Casein kinase 1α (CK1α) leading to their ubiquitylation by RING E3 ligase CRL4 CRBN and subsequent degradation by the 26S proteasome [3][4][5][6] . This degradation contributes to the clinical efficacy of IMiDs. Although thalidomide was discovered over 70 years ago, the molecular mechanism of IMiDs as PPI enhancers were discovered only recently following careful characterization of their pharmacologically relevant targets 7,8 . In addition to the IMiDs, other ligase-binding molecules are known in plant physiology. In particular, the small molecule hormone auxin, also known as indole-3-acetic acid (IAA), regulates plant growth and development by modulating the recognition of Aux/IAA transcriptional repressors by the SCF TIR1 E3 ligase 9 . Auxin binds to the substrate receptor protein TIR1 and promotes the interaction between TIR1 and the Aux/IAA substrate 10 . Importantly, auxin enhances the substrate interaction with TIR1 by extending the protein interaction interface for substrate binding, acting as a molecular glue by forming a continuous hydrophobic core for the TIR1 and substrate interaction 10 .
The use of PPI enhancer molecules as 'molecular glue' to potentiate substrate:ligase interactions could be a therapeutically useful modality to employ against drug targets previously considered undruggable. However, rational design of such small drug-like molecules to degrade proteins that are prospectively identified remains elusive. In recent years, significant progress has been made to target protein degradation by employing heterobifunctional molecules (also referred as PROTACs or PROteolysis-TArgeting Chimeras) [11][12][13][14] . These bifunctional molecules consist of two small molecules connected by a linker. One small molecule binds a protein of interest (substrate) and the other binds an E3 ubiquitin ligase, thereby bringing the substrate in close proximity with the ligase, promoting its ubiquitylation and degradation. Although PROTACs represent important new chemotypes to induce substrate degradation, the molecular weight, physiochemical, and pharmaceutical properties fall outside of the typical range for small molecule drugs potentially leading to challenges in drug development. These challenges might be circumvented by discovering and rationally designing smaller molecular glue-like molecules that bind both the substrate (either native-substrate or neo-substrate) and the ligase without the need for a linker to induce substrate degradation.
Transcription factors remain extremely challenging proteins to target, despite being implicated in multiple diseases 15 . One such example is the Wnt signaling effector protein, β-catenin that is often dysregulated and stabilized in cancer 16,17 . In normal resting cells, the cytoplasmic pool of β-catenin is maintained at low levels through a process involving phosphorylation, ubiquitylation, and subsequent degradation. β-catenin is phosphorylated by the cytoplasmic destruction complex (DC) consisting of the tumor suppressor proteins, Axin and Adenomatous Polyposis Coli (APC), and two serine-threonine kinases, glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). β-catenin is initially phosphorylated at Ser45 by CK1, followed by GSK3 phosphorylation at Thr41, Ser37, and Ser33 [18][19][20][21] . However, the sequential nature of the β-catenin phosphorylation cascade is not essential in certain colorectal cancer cells where phosphorylation at Ser45 is not required for phosphorylation at residues Ser33, Ser37, and Thr41 22 . The phosphorylated Ser33 and Ser37 in β-catenin are part of the phosphodegron sequence (DpSGφXpS) that binds to the F-box-containing E3 ubiquitin ligase protein β-TrCP, leading to β-catenin ubiquitylation by the SCF β-TrCP cullin-RING ligase and subsequent degradation by the 26S proteasome 23 . In the majority of colorectal cancers, β-catenin is stabilized either by inactivating mutations to APC or Axin, which prevent proper βcatenin phosphorylation or by mutations within the β-catenin phosphodegron sequence itself 24,25 . While disruption of βcatenin phosphorylation frequently accounts for elevated protein levels, mutations in β-TrCP are rarely observed 26 . These βcatenin phosphodegron mutations including Ser33 and Ser37, impair the ability of β-catenin to effectively bind to β-TrCP leading to its stabilization and thereby facilitating an enhanced oncogenic transcriptional program. Despite being an important biological target, no small molecule inhibitors of β-catenin are available as therapeutic agents 16,27 . Therefore, using the concept of molecular glue, we sought to prospectively discover small molecule β-catenin degraders that work by restoring mutant βcatenin binding to β-TrCP. This molecular glue strategy represents a unique approach to target tumors with elevated levels of β-catenin. More broadly, it represents a therapeutic strategy to target proteins defective in ligase binding, ubiquitylation and degradation that have been implicated in various diseases 28 .
In this study, we report the prospective identification and rational structure-guided optimization of potent molecular gluelike small molecules intentionally designed to enhance the PPI between mutant β-catenin and β-TrCP that potentiate cellular degradation of an engineered mutant β-catenin in a cellular system. This work establishes a paradigm for rational design of small molecule enhancers to target ubiquitylation and degradation of proteins, including oncogenic transcription factors, by harnessing the ubiquitin proteasome system.
Structural characterization of the β-catenin phosphodegron lacking Ser37 phosphorylation reveals that the monophosphorylated degron binds to β-TrCP in a similar conformation as the WT pSer33/pSer37 degron 30 (Supplementary Fig. 3a). Removal of the phosphate from Ser37 eliminates several key electrostatic and hydrogen bonding interactions between the β-catenin peptide and β-TrCP, leading to reduced binding affinity, but also reveals a small hydrophobic pocket between the β-catenin:β-TrCP interface ( Supplementary Fig. 3b). We performed a high-throughput screen (HTS) on a 350,000 compound library utilizing the FP assay configured at 20% saturation of β-TrCP binding to the monophosphorylated pSer33/Ser37 β-catenin peptide. This HTS identified four structurally related compounds including NRX-1532 (1) that contains a 6-trifluoromethylpyridone core linked to a biaryl group via a secondary amide linkage (Fig. 1b, c). Further characterization of NRX-1532 confirmed that it enhances the pSer33/Ser37 β-catenin:β-TrCP interaction in a dose-dependent manner in the FP assay with a potency (EC 50 ) of 206 ± 54 μM, as well as in orthogonal TR-FRET and SPR binding assays with similar EC 50 values of 246 ± 17 μM and 129 ± 33 μM, respectively (Fig. 1d).
To elucidate the binding mode of the enhancer molecule, we solved the crystal structure of the ternary complex of a related but more soluble tetrazole analog, NRX-1933 (2), bound to the βcatenin:β-TrCP complex (Fig. 2a). The structure reveals that NRX-1933 binds at the β-catenin:β-TrCP interface with the trifluoromethylpyridone occupying the small pocket that is revealed by the absence of Ser37 phosphorylation (Fig. 2b). Enhancer binding to the complex has minimal impact on the conformation of either β-catenin or β-TrCP. The trifluoromethyl substituent fills the small hydrophobic pocket formed by the Leu31 and Ile35 residues of the β-catenin peptide and by the Ala434 and Leu472 residues of β-TrCP, forming important hydrophobic interactions with both β-catenin and β-TrCP (Fig. 2c). The trifluoromethylpyridone is anionic (pK a of NRX-1532 is 5.6) and functions as a phosphate mimetic that occupies the β-catenin Ser37 phosphate binding site and mimics the phosphate by forming a hydrogen bond with the backbone N-H of Gly432 on β-TrCP. Lastly, the phenyltetrazole tail group extends away from the β-catenin:β-TrCP interface, with the phenyl ring stacking against Arg431 of β-TrCP and the anionic tetrazole residing in close proximity to the cationic Arg410 and Arg431 residues of β-TrCP ( Fig. 2d and Supplementary Fig. 3c).
To determine the degree to which NRX-1532 enhances the binding affinity of β-TrCP for the monophosphorylated degron peptide (referred to as the cooperativity of compound binding), we monitored the binding affinity of β-catenin to the β-TrCP at increasing concentrations of compound (Fig. 2e). In a TR-FRETbased binding assay, NRX-1532 enhances the binding of pSer33/ Ser37 β-catenin to β-TrCP with 10-fold cooperativity, increasing the binding affinity from 689 nM in the absence of compound to 68 nM at 500 µM compound. The increase in binding affinity plateaus at high concentrations, indicating saturation of the NRX-1532:β-catenin:β-TrCP ternary complex formation. Importantly, Identification of small molecule enhancers of β-catenin:β-TrCP interaction. a β-TrCP complex was titrated against 5 nM BODIPY-TMR-labeled βcatenin peptide (residues 17-48) of various phosphorylation status and binding was monitored by fluorescence polarization. Binding affinities were estimated by curve fitting to a one-site binding model. Circled on the pSer33/Ser37 β-catenin binding curve is the β-TrCP concentration that results in 20% binding saturation, which was used in the HTS. b Binding enhancement for all compounds screened in the HTS using the FP assay. Each dot represents an individual compound, with NRX-1532 highlighted. c Chemical structure of the enhancers, NRX-1532 and NRX-1933. d Enhancement of pSer33/Ser37 βcatenin peptide binding to β-TrCP with increasing concentrations of NRX-1532. In each assay, the concentration of either β-TrCP or β-catenin was adjusted to obtain 20% binding saturation in the absence of compound. Shown is a representative experiment, values reported are averages the binding affinity of doubly-phosphorylated pSer33/pSer37 βcatenin to the β-TrCP was unaffected by the compound, implying that these enhancers are specific for β-catenin defective in Ser37 phosphorylation. Similar enhancement of binding affinity was observed in an orthogonal FP binding assay ( Supplementary  Fig. 4a). Additionally, these enhancer molecules are specific for βcatenin and do not potentiate the binding of other β-TrCP substrates, such as Emi1, IκBα, and Wee1 ( Supplementary  Fig. 4b).
Optimized enhancers have improved potency and cooperativity. The small molecule enhancers NRX-1532 and NRX-1933 have reasonable molecular weight and drug-like physiochemical properties to serve as starting points for a structure-enabled design campaign to improve both potency and cooperativity (Supplementary Table 2). Optimizing this system is challenging because of the small volume of the enclosed binding site 31 , the cationic nature of the phosphate binding site, and the increased entropic penalty of forming a ternary complex. However, analysis of the bound NRX-1933 crystal structure b-factors indicates that the N-terminus of the β-catenin peptide has the potential for movement. Therefore, we initially focused on strategies to induce movement in this region of β-catenin to induce a larger, more druggable binding pocket. This strategy was realized early in the design campaign, with analogues such as NRX-2663 (3), where phenoxy substitution at the 4-position of the trifluoromethyl pyridone core induces rearrangement of β-catenin Leu31 to create a larger binding pocket (Fig. 3a, b and Supplementary Fig. 5a).  Table 1). b Unbiased Fo-Fc electron density for NRX-1933 at 3.0 standard deviations above the mean is shown (green). The compound is shown as sticks (yellow), bound between the surface of the β-TrCP β-propeller (gray) and the monophosphorylated β-catenin degron peptide, shown as sticks (magenta). c Cutaway view of the hydrophobic pocket formed by residues Leu472 and Ala434 from β-TrCP and Ile35 and Leu31 from β-catenin. d Side view of the same interaction with the β-TrCP surface removed, highlighting the β-catenin and β-TrCP residues that interact with NRX-1933. e The binding affinities of the pSer33/Ser37 and pSer33/pSer37 β-catenin peptides for β-TrCP were measured in the TR-FRET assay by titrating BODIPY-FL-labeled β-catenin peptides (residues 17-48) against 300 pM β-TrCP at varying concentrations of NRX-1532. The binding curves for the pSer33/pSer37 β-catenin peptide titrations were fitted to one-site binding model. For the pSer33/Ser37 β-catenin peptide titrations, which do not reach saturation owing to limitations in the maximum amount of fluorophore compatible with this assay. Curves were fitted to the same one-site binding model, with the upper plateau constrained to that of the pSer33/pSer37 β-catenin peptide binding curve. The K D s are reported for both pSer33/Ser37 and pSer33/pSer37 β-catenin peptides NRX-2663 displays both improved potency in the binding assay with pSer33/Ser37 β-catenin (EC 50 = 80 ± 4 µM) and cooperativity of binding (13-fold, Fig. 3c). NRX-2663 also markedly enhances the ubiquitylation of the pSer33/Ser37 β-catenin peptide (Fig. 3d), with ubiquitin chain formation at ligand concentrations of 16 μM and above resembling ubiquitin chain formation on WT pSer33/pSer37 β-catenin peptide. Further optimization of the phenoxy substituent gave diarylthioether derivatives NRX-103094 (4) and NRX-103095 (5) with significant improvements in potency (EC 50 = 457 ± 23 nM for NRX-103094) with pSer33/ Ser37 β-catenin peptide ( Fig. 3b and Supplementary Fig. 5b) Table 1), whereas the NRX-252114 pose was determined by computational docking. c Binding affinities of the βcatenin peptides (as indicated) for β-TrCP were measured in the TR-FRET assay in presence of enhancers at concentration indicated. For NRX-252114, the binding assay is bottoming out due to 300 pM β-TrCP concentration, with the curve fit yielding a higher than expected hill slope of 1.8. Therefore, the K D reported for NRX-252114 represents an upper estimate. For reference, the binding curve for pSer33/pSer37 β-catenin peptide in the same TR-FRET assay is represented in dotted gray line with a K D of 2 nM.  Table 2). Since these enhancers show the best activity against the β-catenin S37A mutant, and because of the ready access to the homozygous S37A mutant line TOV-112D, we prioritized our efforts on this isoform. With pSer33/S37A β-catenin, NRX-103094 gives an EC 50 of 62 ± 3 nM and displays a 1000-fold cooperativity of peptide binding to β-TrCP (Fig. 3c). Importantly, NRX-103094 potentiates the ubiquitylation of pSer33/S37A β-catenin peptide to levels greater than WT pSer33/pSer37 levels at 250 nM (Fig. 3d). Introduction of the cyanoisoindoline in NRX-252114 (6) resulted in another significant gain in potency (EC 50 = 6.5 ± 0.3 nM).
Computational docking of NRX-252114 bound to the β-catenin: β-TrCP complex suggested that the cyanoisoindoline projects out of the plane from the trifluoromethylpyridone and makes additional hydrophobic contacts with β-TrCP. NRX-252114 enhances the binding of pSer33/S37A β-catenin peptide for β-TrCP with >1500-fold cooperativity, surpassing the affinity of WT doublyphosphorylated peptide (Fig. 3c). However, the full extent of cooperativity could not be measured in this assay since the K D of the peptide in the presence of compound is lower than the concentration of β-TrCP in the assay, resulting in stoichiometric binding and a curve hill slope > 1. Thus, the K D represents only a lower limit of the binding affinity. Corresponding to improvement in binding affinity, NRX-252114 further enhances the ubiquitylation of pSer33/S37A β-catenin peptide to form long ubiquitin chains (Fig. 3d). Finally, another isoindoline analog, NRX-252262 (7) with dimethoxy substitution displays slightly improved potency (EC 50 = 3.8 ± 0.2 nM). Compared to the original HTS hit NRX-1532, with an EC 50 > 200 μM and only eightfold cooperativity, NRX-252114 and NRX-252262 represent significant improvements in both measures. Importantly, improvements in both potency and cooperativity translated into enhanced ubiquitylation of mutant β-catenin peptide, with enhancer molecules potentiating ubiquitin chain formation to levels greater than WT β-catenin peptide.
Enhancers potentiate unphosphorylated β-catenin ubiquitylation. The optimized enhancers NRX-252114 and NRX-252262 had suitable drug-like properties for cellular evaluation (Supplementary Table 2); however, they did not induce degradation of endogenous S37A mutant β-catenin in TOV-112D cells. We reasoned that the lack of degradation could be due to insufficient Ser33 phosphorylation in S37A mutant β-catenin ( Supplementary  Fig. 6a), which is consistent with the sequential nature of βcatenin phosphorylation 18,21 . Since the non-phosphorylated Ser33/S37A β-catenin could potentially be the prevalent βcatenin species in the TOV-112D cells, we evaluated the magnitude of the defect in non-phosphorylated Ser33/S37A β-catenin binding to β-TrCP and determined how effectively these enhancers potentiate this interaction. The non-phosphorylated Ser33/ Ser37 peptide binds β-TrCP with very weak affinity (>100 μM); however, in the presence of 20 μM NRX-252114 that binding affinity is enhanced to 180 nM (Fig. 4a). This represents a >500fold enhancement in PPI binding affinity, although this is still 90fold lower than WT pSer33/pSer37 β-catenin peptide binding to β-TrCP. Consistent with binding enhancement, NRX-252114 also potentiates the ubiquitylation of unphosphorylated Ser33/S37A βcatenin peptides by the SCF β-TrCP complex (Fig. 4b). Interestingly, in the presence of NRX-252114, unphosphorylated βcatenin peptides are ubiquitylated with chain lengths similar to WT doubly-phosphorylated peptide; however, only a small percentage of unphosphorylated β-catenin is ubiquitylated. Consistent with peptide ubiquitylation, these enhancers also potentiate the ubiquitylation activity of full-length mutant Ser33/ S37A β-catenin protein with the more potent NRX-252114 displaying a higher degree of ubiquitylation than NRX-103094 ( Fig. 4c and Supplementary Fig. 6b, c). In the presence of 40 μM NRX-252114, the level of ubiquitylation activity on Ser33/S37A mutant β-catenin approaches that of WT full-length protein (Fig. 4c). The robust binding and ubiquitylation enhancement of unphosphorylated Ser33/S37A β-catenin with NRX-252114 in biochemical assays suggests that unphosphorylated β-catenin might be degraded in cellular system with further improvement of these enhancer molecules. Induced cellular degradation of phosphomimetic β-catenin. The lack of cellular activity in TOV-112D cells suggested that non-phosphorylated Ser33/S37A might be the prevalent β-catenin species in the TOV-112D cells. To overcome this lack of Ser33 phosphorylation, we developed a S33E phosphomimetic system to evaluate the cellular activity of these enhancers. In this system, the Ser33 status of β-catenin is not impacted by the sequential nature of the phosphorylation cascade (Fig. 5a). Replacement of Ser33 with glutamic acid (S33E/S37A β-catenin) results in a >10fold decrease in the binding affinity for β-  pSer33/S37A peptide (we could not estimate the binding affinities due to partial binding curves). In this system, NRX-103094 enhances the S33E/S37A phosphomimetic peptide binding affinity for β-TrCP from >5 μM in the absence of compound to 22 nM in the presence of 40 μM compound (Fig. 5b), which is similar to the binding enhancement observed for pSer33/S37A peptide. Consistent with the binding affinities, S33E/S37A βcatenin peptide had limited ubiquitylation by the SCF β-TrCP complex in the absence of compound. However, in the presence of 20 μM NRX-103094, robust time-dependent ubiquitylation of S33E/S37A β-catenin peptide is observed (Fig. 5c). Likewise, the full-length S33E/S37A β-catenin protein is strongly ubiquitylated in the presence of 20 μM NRX-103094, with the ubiquitylation levels approaching that of WT full-length β-catenin protein (Fig. 5d). The potency trends observed in the enhancer compounds for pSer33/S37A β-catenin peptide binding to β-TrCP translated proportionately for the S33E/S37A phosphomimetic peptide binding. However, the potencies were consistently diminished with the S33E/S37A peptide (Fig. 5e). Twentymicromolar NRX-252114 enhanced the binding affinity of the S33E/S37A β-catenin peptide for β-TrCP to 6 nM, which is within three-fold of that of WT doubly-phosphorylated peptide (Fig. 5f). Importantly, NRX-252114 also markedly enhances the ubiquitylation of S33E/S37A β-catenin protein (Fig. 5g), with the ubiquitin chain formation at ligand concentrations of 10 μM and above resembling that of WT β-catenin protein.
These experiments confirmed that NRX-252114 is competent to form a ternary complex with the full-length S33E/S37A βcatenin and β-TrCP in cells. We then examined whether these enhancers can potentiate the degradation of S33E/S37A mutant β-catenin in cells. To test this, we established an engineered HEK293T cell line stably expressing the S33E/S37A phosphomimetic mutant β-catenin. As expected, the mutant S33E/S37A βcatenin was stable relative to WT β-catenin ( Supplementary  Fig. 7a). In agreement with the biochemical results, NRX-252114 treatment of phosphomimetic expressing cells resulted in a dosedependent degradation of S33E/S37A mutant β-catenin starting at~50 μM (Fig. 6c). Importantly, the degradation of S33E/S37A mutant β-catenin can be completely blocked by either inactivation of the ubiquitylation pathway using the Nedd8 E1 inhibitor, MLN4924, or by inactivation of the degradation pathway using the proteasome inhibitor, MG132 (Fig. 6c). Furthermore, this cellular degradation of β-catenin is dependent on β-TrCP, as siRNA knockdown of β-TrCP eliminates the degradation of S33E/S37A mutant β-catenin ( Supplementary Fig. 7b). The protein levels of other β-TrCP substrates including WT βcatenin and IκBα remained unchanged. The more potent NRX-252262 also induces S33E/S37A mutant β-catenin degradation at an even lower concentration of~35 μM (Fig. 6d). Collectively, these results demonstrate that these enhancers act as molecular glue in cells to promote the ubiquitylation and subsequent degradation of mutant β-catenin by the 26S proteasome. This provides an enticing path towards targeting β-catenin overexpressing cancer cells.

Discussion
Here we report the prospective discovery and rational design of small molecule molecular glues that enhance a substrate:ligase interaction to induce substrate degradation. These molecules potentiate the binding of S37A mutant β-catenin to β-TrCP. The enhanced PPI affinity results in increased K48-linked ubiquitylation of mutant β-catenin by its natural ubiquitin ligase SCF β-TrCP , thereby promoting its proteasomal degradation. Since S37A mutant β-catenin is defective in β-TrCP binding, improving the cooperativity was essential to overcome the binding defect and restore functional ubiquitylation in the mutant system. Strategically, we prioritized molecular modifications that were contained at the β-catenin:β-TrCP interface rather than growing the enhancer molecule away from the protein-protein interface. This strategy allowed concomitant improvements in both the cooperativity (>1500-fold) and potency (>10,000-fold) of the enhancers. This required improving affinity by increasing ligand efficiency, rather than improving affinity by increasing ligand molecular weight and growing away from the PPI interface. This allowed the enhancers to retain drug-like physiochemical properties throughout the optimization process even though they evolved from a weak screening hit.
While a few retrospectively characterized enhancers of substrate:ligase interactions have been reported 3,4,6-8,32-34 this work represents a prospective discovery of such molecules for a specific ligase-substrate pair. Compared to the better characterized enhancers, auxin and the IMiDs that directly bind to their respective ligases to create surfaces for substrate binding 7,8,10 , these enhancers do not exhibit appreciable binding directly to β-TrCP alone and only bind in the context of the ternary complex. The enhancers herein also differ from the other known substrate: ligase enhancers by promoting the interaction of β-TrCP with its native substrate, β-catenin, that has otherwise lost affinity because of its mutational status. The enhancer molecules provide the essential binding surface between β-catenin and β-TrCP that is lost as a result of the S37A β-catenin mutation. In contrast, the IMiD enhancers promote the binding of neo-substrates IKZF1, IKZF3, CK1α, and GSPT1 to the ubiquitin ligase CRBN [3][4][5][6][7][8]32 , while another enhancer, indisulam, promotes the binding of neosubstrate Rbm39 to the ubiquitin ligase DCAF15 33,34 . These previously reported substrates have no endogenous connection to the ligase and display no binding affinity in the absence of enhancers. Compared to hetero-bifunctional molecules, these enhancers have an acceptable molecular weight for small molecules with desirable drug-like properties. Rather than degrading substrates with bifunctional molecules by hijacking an unrelated ligase where tissue-specific expression of both proteins could be a challenge to drive ternary complex formation for efficient ubiquitylation, these enhancers co-opt the native ligase to degrade substrate proteins that has otherwise lost binding due to mutations. Additionally, utilizing a native substrate-ligase pair that enhances PPI through the naturally occurring interface ensures both cooperative interaction as well as provide accessible substrate lysines for efficient ubiquitylation.
Developing small molecule therapeutics targeting the Wnt/βcatenin signaling pathway remains a challenge despite extensive efforts to target various components of the pathway 27 . Our work provides an approach to target the Wnt signaling pathway by promoting degradation of mutant β-catenin by SCF β-TrCP . Since these enhancer ligands bind at the phosphoserine binding site on β-TrCP, binding of the doubly-phosphorylated WT β-catenin is not enhanced by these ligands. This ensures the specificity of the enhancers for the mutant form of β-catenin without impacting the function of WT β-catenin in normal tissues. Due to the uncertainty of β-catenin Ser33 phosphorylation status in the mutant TOV-112D cells, we utilized an engineered Ser33 phosphomimetic β-catenin model to demonstrate cellular mechanistic proof-of-concept for mutant β-catenin degradation. Additionally, the small molecule NRX-252114 enhanced the affinity of unphosphorylated Ser33/S37A β-catenin for β-TrCP from >10,000-fold to within 90-fold of WT doubly-phosphorylated βcatenin affinity. Importantly, these enhancers potentiated unphosphorylated β-catenin ubiquitylation with chain lengths similar to that of doubly-phosphorylated WT β-catenin. This tantalizing result suggests that further potency improvements to the small molecule could drive unphosphorylated β-catenin ternary complex formation in cellular and in vivo contexts, and enable degradation of unphosphorylated β-catenin in β-catenin dependent tumors typically observed in cancers with APC, Axin or β-catenin mutations. This work shows that a prospective discovery approach can be rationally applied to develop small molecule β-catenin degraders beyond Ser37 mutations.
Transcription factors such as β-catenin are implicated in the development of many diseases, but with few exceptions, effectively remain as an undruggable target class. It is now evident that post-translational modification via the ubiquitin proteasome system is a common mechanism by which the levels of transcription factors are regulated in cells 15,28 . Dysregulation often results from mutations leading to compromised binding of transcription factors to their cognate ligases. This work provides a rational framework to prospectively employ small molecules that target such oncogenic transcription factors by enhancing substrate:ligase interactions to promote their degradation. Ultimately, this strategy could be useful to target therapeutically important classes of proteins that were previously deemed undruggable and might find further application to other PPIs of different classes as well.
The unbound fraction was further buffer exchanged into low salt, low pH buffer (50 mM MES pH 6.0, 50 mM NaCl, 2 mM DTT), bound to a 5 mL HiTrap SP column (GE) and eluted by a linear salt gradient. Fractions containing β-TrCP/ Skp1 complex were concentrated and further purified by size exclusion chromatography (Superdex200 HiLoad 16/600, GE) ( Supplementary Fig. 8). β-TrCP/Skp1 used in biochemical assays was buffer exchanged into 50 mM NaH 2 PO 4 , 200 mM NaCl, and 2 mM DTT during this final SEC step, concentrated to 100 µM, aliquoted, frozen in liquid nitrogen and stored at −80°C. β-TrCP/Skp1 used in crystallization was buffer exchanged into 10 mM BTP pH 6.8, 200 mM NaCl, and 5 mM DTT during the final size exclusion step.
Fluorescence polarization (FP). Compounds were incubated in a 20 μl reaction volume with β-TrCP/Skp1 (116 nM in binding enhancement assays and a concentration dilution series in peptide binding and cooperativity assays) and 5 nM BODIPY-TMR labeled β-catenin peptides (see figure legends for peptide details) in a black 384-well microplate (Perkin Elmer ProxiPlate), in a PBS buffer containing 0.01% Triton X-100 and 2 mM DTT with 2% final DMSO. Following 1-h incubation at room temperature, plates were read on an Envision plate reader (Perkin Elmer) for FP signal using a P-pol and S-pol 595/60 filter and a D555 single mirror. Binding and enhancement data were fit to a standard Hill equation (Y = B min + (B max − B min )(X^h)/(X^h + EC 50^h )) in Prism (GraphPad). Errors are standard error of the fit.
Time-resolved fluorescence resonance energy transfer (TR-FRET). Compounds were incubated in a 20 μl reaction volume with 300 pM biotinylated β-TrCP/Skp1 preincubated with 1.2 nM terbium-coupled streptavidin (Cisbio) and BODIPY-FL labeled β-catenin peptides (see figure legends for peptide details) in a white 384-well microplate (Perkin Elmer ProxiPlate), using a PBS buffer containing 0.01% Triton X-100 and 2 mM DTT with 2% final DMSO. In binding enhancement assays, the peptide concentrations were held constant at 107, 85, 10, 5, and 85 nM for the pSer33/Ser37, pS33/S37A, pS33/S37C, pS33/S37F, and S33E/S37A peptides, respectively. In peptide binding and cooperativity assays, the peptide concentrations were varied across the concentration range displayed on the x-axis. Following 1-h incubation, plates were read for TR-FRET signal after excitation at 320 nm and read at 520 and 620 nm using an EnVision plate reader (Perkin Elmer). TR-FRET signal was calculated using a 520/620 nm ratio. Binding and enhancement data were fit to a standard Hill equation (Y = B min + (B max − B min ) (X^h)/(X^h + EC 50^h )) in Prism (GraphPad). Errors are standard error of the fit.
High-throughput screen. High-throughput screen activity was assessed with FP (as described above) and calculated using the percent change from the null control and standard deviation (σ) from the plate median. Hit thresholds were defined by two standard deviations from the plate median or 25% enhancement from the null control.
Surface plasmon resonance (SPR). SPR binding assays were performed on a GE Healthcare Biacore T200 using a Series B Biacore Sensor Chip SA. Biotinylated βcatenin pSer33/Ser37 peptide was immobilized at 27.6 RU. 333 nM β-TrCP/Skp1 and titrated compound were preincubated and flowed at a rate of 30 μl/min in 10 mM HEPES, 150 mM NaCl, 0.05% Surfactant P20, 1 mM TCEP, and 2% DMSO at pH 7.4 and 20°C. Engagement of the ternary complex was assessed with a contact and dissociation time of 120 s. Equilibrium binding levels were assessed using Biacore T200 Evaluation Software (GE) and these data were fit to a standard Hill equation (Y = B min + (B max − B min )(X^h)/(X^h + EC 50^h )) in Prism (GraphPad). Errors are standard error of the fit.
Data for the pSer33/Ser37, NRX-2663, NRX-1933, and NRX-103094 containing crystals were collected at the Canadian lightsource on beamline 08ID at 100 K using and a wavelength of 0.9795 Å. Data for the NRX-2776 containing crystal were collected at the Advanced Photon Source on beamline 22ID at 100 K using and a wavelength of 1 Å. Data were processed using XDS and scaled in XSCALE, using CC1/2 as the resolution cutoff criteria 37 . For the highest resolution NRX-2776 containing structure, the coordinates of β-TrCP and Skp1 from PDB ID 1P22 30 were used as search ensembles in PHASER 38 to obtain an initial solution for this structure by molecular replacement. The final model was built through iterative rounds of refinement in Phenix 39,40 and Coot 41 . Ligand restraints were generated in Maestro and Phenix Elbow 42,43 . The resulting model for the NRX-2776 containing complex was used as the starting point and refinement constraints for refinement of the other structures ( Supplementary Fig. 9). Model quality was determined by Molprobity 44  Ligand docking. The X-ray crystal structure of NRX-103094 was used in the docking of the various analogs. The structure was prepared with the protein preparation procedure in Maestro. All hydrogens were added, and charge states defined using PROTKA procedure, follow by h-bond optimization and constraint relaxation to 0.2 rmsd tolerance. The resulting structure was used to generate docking grids and perform docking studies using GLIDE. The GLIDE extra precision scoring function (XP) was used to obtain suitable docking poses, which were subsequently refined with Macromodel embrace procedure using default options. All the modeling was done with Maestro 10.3 Release 2015-2 (Schrodinger LLC).
Pull down from cells treated with compound: HEK293T cells were plated in 10 cm dishes at a density of 1.2 × 10 6 cells per plate. Cells were transfected 24 h later with 2.5 µg of a WT or S33E/S37A Myc/FLAG-tagged β-catenin expression construct using the transfection reagent FuGeneHD. Forty-eight hours post transfection, cells were treated with a combination of 20 µM MG132 and NRX-252114 at the described concentrations. Cells were then liberated from 10 cm dishes using a cell scraper, pelleted, washed with PBS, resuspended in an equal volume of hypotonic lysis buffer (20 mM HEPES 7.5, 5 mM KCl, 1.5 mM MgCl 2 , 2 mM DTT, 0.01% TX-100, protease and phosphatase inhibitors, 1% DMSO), and sheered by eight passages through 21 G and 25 G needles. Samples were then supplemented up to 150 mM NaCl and clarified by centrifugation. Thirty microliters of each lysate was incubated over 10 µL of anti-c-myc magnetic beads (Pierce, Catalog #PI88842) and incubated at room temp with rotation for 30 min. Beads were pelleted, washed twice with lysis buffer, and resuspended in SDS loading buffer. Samples were analyzed by western blot.
S33E/S37A β-catenin stable cell line generation. HEK293T cells were plated in a 10 cm dish in regular growth media (DMEM + 10% FBS) and allowed to adhere to the plate overnight. The following day, cells were transfected with S33E/S37 βcatenin using FuGeneHD transfection reagent. Transfected cells were then selected with media containing 700 μg/ml Geneticin. A pooled population of selected cells were used for all described experiments.
β-catenin degradation with compound treatment. HEK293T cells expressing S33E/S37A β-catenin were plated in six-well plates at a density of 9 × 10 5 cells/well. The following day, cells were incubated with compounds diluted in regular growth media to the appropriate concentration. The final concentration of DMSO for all cell treatments was kept at 0.5% or less. Cell treatments with the Nedd8 inhibitor (MLN4924) and the proteasome inhibitor (MG132) were performed at a final concentration of 5 μM and 10 μM respectively. Cycloheximide treatment was performed at a final concentration of 100 μg/ml. β-TrCP knockdown in S33E/S37A β-catenin expressing cell line. HEK293T cells expressing S33E/S37A β-catenin were plated in six-well plates at a density of 5 × 10 5 cells/well. The following day, cells were transfected with a nontargeting siRNA or a combination of Dharmacon SMARTPool siRNAs targeting β-TrCP1 & β-TrCP2 using Dharmafect1 lipid reagent (Dharmacon/Horizon Discovery). Transfected cells were grown for 48 h before being re-plated in six-well plates at a density of 9 × 10 5 per well. The following day (72-h post siRNA transfection), cells were treated with either DMSO or NRX-252114 for 6 h. Harvested lysate was assessed for levels of FLAG-tagged S33E/S37A β-catenin by Western analysis.