Kinase-Impaired BTK Mutations Are Susceptible to Clinical-Stage BTK and IKZF1/3 Degrader NX-2127

INTRODUCTION: Bruton’s tyrosine kinase (BTK) is a nonreceptor kinase in the B cell receptor (BCR) signaling cascade critical for B cell survival. As such, chronic lymphocytic leukemia (CLL) and other B cell cancers are sensitive to inhibition of BTK. Covalent and noncovalent inhibitors of BTK have revolutionized the treatment of these cancers. Therefore, understanding mechanisms by which acquired mutation in BTK confer drug resistance and developing new therapies to overcome resistance are critically important. RATIONALE: We recently discovered BTK mutations that confer resistance across covalent and noncovalent BTK inhibitors. In this study, we found that a group of these mutants impair BTK kinase activity despite still enabling downstream BCR signaling. We therefore set out to understand the nonenzymatic functions of BTK and explored targeted protein degradation to overcome the oncogenic scaffold function of mutant BTK. This effort included evaluation of BTK degradation in patients with CLL treated in a phase 1 clinical trial of NX-2127, a first-in-class BTK degrader (NCT04830137). RESULTS: BTK enzymatic activity assays revealed that drug resistance mutations in BTK fall into two distinct groups: kinase proficient and kinase impaired. Immunoprecipitation mass spectrometry of kinase-impaired BTK L528W (Leu528→Trp) revealed a scaffold function of BTK with downstream signaling and survival dependent on surrogate kinases that bind to kinase-impaired BTK proteoforms. To target the nonenzymatic functions of BTK, we developed NX-2127, a heterobifunctional molecule that engages the ubiquitin-proteasome system to simultaneously bind both BTK and the cereblon E3 ubiquitin ligase complex, inducing polyubiquitination and proteasome-dependent degradation of IKZF1/3 and all recurrent drug-resistant forms of mutant BTK. The activity of NX-2127 on BTK degradation was further demonstrated in patients with CLL treated in a phase 1 clinical trial of NX-2127, where \u3e80% BTK degradation was achieved and clinical responses were also seen in 79% of evaluable patients, independent of mutant BTK genotypes. CONCLUSION: We identified that BTK inhibitor resistance mutations fall into two distinct functional categories. Kinase-impaired BTK mutants disable BTK kinase activity while promoting physical interactions with other kinases to sustain downstream BCR signaling. This scaffold function of BTK was disrupted by NX-2127, a potent BTK degrader, which showed promising responses for patients with relapsed and refractory CLL, independently of mutant BTK functional category

Structure-function studies of M4 muscarinic acetylcholine receptor allosteric modulation

The M4 muscarinic acetylcholine receptor (mAChR) is implicated in many central nervous system disorders, however, due to a highly conserved acetylcholine (ACh) binding orthosteric site, there is a lack of highly selective ligands as therapeutics and experimental probes for this target. There are two classes of functionally selective M4 mAChR ligands, one being the allosteric modulators, typified by the small molecule LY2033298 (3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide) (Chan et al., 2008), and the other being the atypical agonists, exemplified by xanomeline and McN-A-343, whose mode of binding at the M4 mAChR is not clear. Challenges to understanding the activity of these ligands include the interplay of binding, efficacy and, when considering allosteric modulation, cooperativity. Thus, to investigate the molecular determinants of allosteric and atypical agonist activity, site-directed mutagenesis was utilised in conjunction with radioligand assays, to determine the role of specific amino acid residues on affinity or binding cooperativity, and M4 mAChR-mediated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, as a measure of efficacy or functional modulation by LY2033298. The endogenous agonist, ACh was used as a control agonist. Chapter 2 focused on four different regions of the M4 mAChR; extracellular loops (ECLs) 1, 2 and 3, and transmembrane domain (TM) 7. In the ECL1, we identified Ile93(2.65) and Lys95(2.67) as key residues that specifically governed the signalling efficacy of LY2033298 and its binding cooperativity with ACh, while Phe186(5.29) in the ECL2 was identified as a key contributor to the binding affinity of the modulator for the allosteric site. The highly conserved TM7 residues, Tyr439(7.39) and Tyr443(7.43), were important for both McN-A-343 and xanomeline affinity, while the ECL residues, Ile93(2.65), Phe186(5.29), Ser428(6.63) and Asp432(7.32) were detrimental to McN-A-343 affinity. Ser428(6.63) was exclusively involved in atypical agonist efficacy. In contrast, Tyr439(7.39) and Tyr443(7.43), were identified as contributing to a key activation switch utilized by all classes of agonists, except xanomeline. This initial study highlighted the general importance of aromatic residues for allosteric agonist activity, which led us to perform alanine scanning mutagenesis of selected aromatic residues in the top third the M4 mAChR. Additionally, due to the importance of Phe186(5.29) in allosteric agonist binding in the ECL2, residues lining the proximal and distal ends of ECL2 was also mutated. Results outlined in Chapter 3 showed that, Tyr89(2.61) and Trp435(7.35), on top of TM2 and TM7, respectively, were important for LY2033298 binding. Tyr89(2.61) was exclusively involved in LY2033298 efficacy compared to the other ligands, while other TM2/ECL1 residues also played a large role in LY2033298 efficacy. Multiple residues clustered between the putative allosteric and orthosteric sites, on TM2/ECL1 and TM7, appear to form the conformational link for transmitting ACh-LY2033298 cooperativity. Tyr89(2.61) was particularly important for the positive binding cooperativity between ACh and LY2033298. Only two residues (Tyr89(2.61) and Tyr439(7.39)) were identified to affect the functional modulation of ACh by LY2033298 in the current thesis. Orthosteric binding site residues, Trp164(4.57) and Trp413(6.48), were global activation switches for both allosteric and orthosteric agonists. The final study, outlined in Chapter 4, characterised the activity of the atypical agonists at the second set of mutant M4 mAChRs. It revealed that Trp413(6.48) is a critical contact residue for xanomeline, while playing a smaller role in ACh and McN-A-343 binding. In the distal ECL2, Ile187(5.30), may play a role in both xanomeline and McN-A-343 binding, while Ile187(5.30), Gln188(5.31) and Phe189(5.32) played a large role in McN-A-343 efficacy. Trp164(4.57) and Trp413(6.48), like Tyr439(7.39) and Tyr443(7.43), were global activation switches for all three classes of agonists. These results provide new insights into the existence of multiple binding pockets and activation switches in G protein-coupled receptors (GPCRs), some of which can be selectively exploited by allosteric and atypical agonists for future development of selective M4 mAChR ligands, whereas others represent global activation mechanisms for all classes of ligand

Structure-function studies of M4 muscarinic acetylcholine receptor allosteric modulation

The M4 muscarinic acetylcholine receptor (mAChR) is implicated in many central nervous system disorders, however, due to a highly conserved acetylcholine (ACh) binding orthosteric site, there is a lack of highly selective ligands as therapeutics and experimental probes for this target. There are two classes of functionally selective M4 mAChR ligands, one being the allosteric modulators, typified by the small molecule LY2033298 (3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide) (Chan et al., 2008), and the other being the atypical agonists, exemplified by xanomeline and McN-A-343, whose mode of binding at the M4 mAChR is not clear. Challenges to understanding the activity of these ligands include the interplay of binding, efficacy and, when considering allosteric modulation, cooperativity. Thus, to investigate the molecular determinants of allosteric and atypical agonist activity, site-directed mutagenesis was utilised in conjunction with radioligand assays, to determine the role of specific amino acid residues on affinity or binding cooperativity, and M4 mAChR-mediated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, as a measure of efficacy or functional modulation by LY2033298. The endogenous agonist, ACh was used as a control agonist. Chapter 2 focused on four different regions of the M4 mAChR; extracellular loops (ECLs) 1, 2 and 3, and transmembrane domain (TM) 7. In the ECL1, we identified Ile93(2.65) and Lys95(2.67) as key residues that specifically governed the signalling efficacy of LY2033298 and its binding cooperativity with ACh, while Phe186(5.29) in the ECL2 was identified as a key contributor to the binding affinity of the modulator for the allosteric site. The highly conserved TM7 residues, Tyr439(7.39) and Tyr443(7.43), were important for both McN-A-343 and xanomeline affinity, while the ECL residues, Ile93(2.65), Phe186(5.29), Ser428(6.63) and Asp432(7.32) were detrimental to McN-A-343 affinity. Ser428(6.63) was exclusively involved in atypical agonist efficacy. In contrast, Tyr439(7.39) and Tyr443(7.43), were identified as contributing to a key activation switch utilized by all classes of agonists, except xanomeline. This initial study highlighted the general importance of aromatic residues for allosteric agonist activity, which led us to perform alanine scanning mutagenesis of selected aromatic residues in the top third the M4 mAChR. Additionally, due to the importance of Phe186(5.29) in allosteric agonist binding in the ECL2, residues lining the proximal and distal ends of ECL2 was also mutated. Results outlined in Chapter 3 showed that, Tyr89(2.61) and Trp435(7.35), on top of TM2 and TM7, respectively, were important for LY2033298 binding. Tyr89(2.61) was exclusively involved in LY2033298 efficacy compared to the other ligands, while other TM2/ECL1 residues also played a large role in LY2033298 efficacy. Multiple residues clustered between the putative allosteric and orthosteric sites, on TM2/ECL1 and TM7, appear to form the conformational link for transmitting ACh-LY2033298 cooperativity. Tyr89(2.61) was particularly important for the positive binding cooperativity between ACh and LY2033298. Only two residues (Tyr89(2.61) and Tyr439(7.39)) were identified to affect the functional modulation of ACh by LY2033298 in the current thesis. Orthosteric binding site residues, Trp164(4.57) and Trp413(6.48), were global activation switches for both allosteric and orthosteric agonists. The final study, outlined in Chapter 4, characterised the activity of the atypical agonists at the second set of mutant M4 mAChRs. It revealed that Trp413(6.48) is a critical contact residue for xanomeline, while playing a smaller role in ACh and McN-A-343 binding. In the distal ECL2, Ile187(5.30), may play a role in both xanomeline and McN-A-343 binding, while Ile187(5.30), Gln188(5.31) and Phe189(5.32) played a large role in McN-A-343 efficacy. Trp164(4.57) and Trp413(6.48), like Tyr439(7.39) and Tyr443(7.43), were global activation switches for all three classes of agonists. These results provide new insights into the existence of multiple binding pockets and activation switches in G protein-coupled receptors (GPCRs), some of which can be selectively exploited by allosteric and atypical agonists for future development of selective M4 mAChR ligands, whereas others represent global activation mechanisms for all classes of ligand

Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics

Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular-level understanding of the general principles that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. In this study, we rigorously quantified the affinity, efficacy, and magnitude of modulation of two different positive allosteric modulators, LY2033298 (LY298) and VU0467154 (VU154), combined with the endogenous agonist acetylcholine (ACh) or the high-affinity agonist iperoxo (Ipx), at the human M4 mAChR. By determining the cryo-electron microscopy structures of the M4 mAChR, bound to a cognate Gi1 protein and in complex with ACh, Ipx, LY298-Ipx, and VU154-Ipx, and applying molecular dynamics simulations, we determine key molecular mechanisms underlying allosteric pharmacology. In addition to delineating the contribution of spatially distinct binding sites on observed pharmacology, our findings also revealed a vital role for orthosteric and allosteric ligand–receptor–transducer complex stability, mediated by conformational dynamics between these sites, in the ultimate determination of affinity, efficacy, cooperativity, probe dependence, and species variability. There results provide a holistic framework for further GPCR mechanistic studies and can aid in the discovery and design of future allosteric drugs

Kinase-impaired BTK mutations are susceptible to clinical-stage BTK and IKZF1/3 degrader NX-2127

Increasing use of covalent and noncovalent inhibitors of Bruton's tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients