Tunable Methacrylamides for Covalent Ligand Directed Release Chemistry.

Rambabu N Reddi,Haim Barr,Kim Goldenberg,Adi Rogel,Boddu Venkateswara Rao,Ziv Shulman,Neta Gurwicz,Daniel Zaidman,Nir London,Efrat Resnick,Ronen Gabizon,Alexander Plotnikov

doi:10.1021/jacs.0c10644

Abstract

Targeted covalent inhibitors are an important class of drugs and chemical probes. However, relatively few electrophiles meet the criteria for successful covalent inhibitor design. Here we describe α-substituted methacrylamides as a new class of electrophiles suitable for targeted covalent inhibitors. While typically α-substitutions inactivate acrylamides, we show that hetero α-substituted methacrylamides have higher thiol reactivity and undergo a conjugated addition–elimination reaction ultimately releasing the substituent. Their reactivity toward thiols is tunable and correlates with the pKa/pKb of the leaving group. In the context of the BTK inhibitor ibrutinib, these electrophiles showed lower intrinsic thiol reactivity than the unsubstituted ibrutinib acrylamide. This translated to comparable potency in protein labeling, in vitro kinase assays, and functional cellular assays, with improved selectivity. The conjugate addition–elimination reaction upon covalent binding to their target cysteine allows functionalizing α-substituted methacrylamides as turn-on probes. To demonstrate this, we prepared covalent ligand directed release (CoLDR) turn-on fluorescent probes for BTK, EGFR, and K-RasG12C. We further demonstrate a BTK CoLDR chemiluminescent probe that enabled a high-throughput screen for BTK inhibitors. Altogether we show that α-substituted methacrylamides represent a new and versatile addition to the toolbox of targeted covalent inhibitor design.

Highlights

Acrylamides have been widely used as electrophiles for irreversible covalent inhibitors for many proteins bearing noncatalytic cysteines.[1−5] For example, afatinib, ibrutinib, and AMG-510 are acrylamide-based inhibitors of EGFR, Bruton’s tyrosine kinase (BTK), and K-RasG12C, respectively (Figure S1)
Such irreversible inhibitors have the advantages of nonequilibrium kinetics, full target occupancy, and flexibility to modify the structure for ADME issues without sacrificing potency and selectivity.[6−8] The efficiency of a covalent inhibitor depends upon initial reversible binding with the protein and the rate of subsequent covalent bond formation with the target nucleophile.[9,10]
■ RESULTS Reactivity and Substitution Propensity of α-Methacrylamides Can Be Tuned by Different Substitutions

Summary

■ INTRODUCTION

Acrylamides have been widely used as electrophiles for irreversible covalent inhibitors for many proteins bearing noncatalytic cysteines.[1−5] For example, afatinib, ibrutinib, and AMG-510 are acrylamide-based inhibitors of EGFR, BTK, and K-RasG12C, respectively (Figure S1). Ibrutinib is an irreversible inhibitor of Bruton’s tyrosine kinase (BTK) and is FDA approved for several B cell oncogenic malignancies.[27] Starting from the parent ibrutinib, we used the Morita−Baylis−Hillmann reaction to functionalize the acrylamide (Figure S9) and synthesized various ibrutinib-based methacrylamide derivatives with different leaving groups including phenols, acids, carbonates, amines, and quaternary ammonium salts (3a−3k; Figure 4A). All of these compounds exhibited covalent binding of the recombinant BTK kinase domain as assessed by intact protein LC/MS (Figure 4B; Figure S10; Table S1). Pluripotin, a reported ERK1 and RasGAP inhibitor, fully inhibited pBTK at all tested concentrations (down to 64 nM; Figure 7G, Figure S24)

■ DISCUSSION

■ ACKNOWLEDGMENTS

■ REFERENCES