Cell-Based HTS Campaign to Identify Negative Allosteric Modulators of the Leukemia-associated Tyrosine Kinase, BTK”.

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

Investigators

Abstract

Abstract. The non-receptor tyrosine kinase Bruton’s tyrosine kinase (BTK) is essential for the development and function of B cells. Small molecule inhibitors of BTK (BTKi’s) like Ibrutinib (IMBRUVICA®) have revolutionized the treatment of B cell malignancies including chronic lymphocytic leukemia (CLL), the most common form of leukemia in adults. Ibrutinib, and related next-generation FDA-approved BTKi’s, covalently and irreversibly bind to cysteine 481 (C481) in the ATP binding pocket of the kinase domain. Unfortunately, intrinsic and acquired resistance to ibrutinib develops in the majority of CLL patients, often due to mutations in the BTK active site cysteine essential for drug action. While the C481S point mutation is most common, double mutations involving the BTK gatekeeper residue (T474) in addition to C481 produce super resistance to ibrutinib, acalabrutinib, and zanubrutinib. Durable responses to orthosteric ATP-site BTKi’s in the cancer setting are therefore limited by these drug resistance mechanisms, resulting in the evolution of inhibitor-resistant tumor cell clones. In addition, toxicity and adverse events cause discontinuation of BTKi therapy in up to 26% of patients. In this proposal, which is responsive to PAR-23-264: Assay development and screening for discovery of chemical probes, drugs or immunomodulators, we will capitalize on our advanced knowledge of BTK structure and how the non-catalytic regulatory domains of BTK control kinase function. We have developed and optimized a suite of cell-based high-throughput and high content screening (HTS & HCS) assays to identify allosteric (non-ATP site) inhibitors of BTK dimerization, a prerequisite for BTK autophosphorylation and catalytic activation. Our team of investigators combines expertise in Tec family tyrosine kinases including BTK biochemistry and structural biology (Andreotti), broad experience (in both the pharmaceutical industry and academia) in HTS/HCS drug discovery (Johnston), expertise in pharmacology and molecular oncology (Smithgall) and medicinal chemistry (Iyer). In Aim 1 we will conduct an HTS campaign using a statistically validated BTK dimerization NanoBiT assay (based on NanoLuc complementation) to identify compounds that block BTK dimerization and subsequent activation in cells. Aim 2 will confirm primary HTS actives in counter screens and orthogonal assays, and prioritize hits based on potency and chemical tractability. We will determine whether BTK dimerization inhibitors are cytotoxic to B- cell cancers and explore synergy with approved BTKi’s and other cancer drugs to enhance killing. Aim 3 makes use of our established biochemical, cellular, and structural approaches to fully characterize hit compounds that block BTK dimerization, kinase activity, and signal transduction. Successful completion of the proposed HTS campaign will identify novel small molecule allosteric inhibitors that disrupt BTK signaling. Drugs developed from these compounds are anticipated to suppress emergence of acquired resistance mutations that abolish the efficacy of ibrutinib and related drugs that target the BTK active site.