In-Silico Based Screening System To Develop Allosteric Moducators Of Bruton'S Tyrosine Kinase

Abstract Ibrutinib is a covalent inhibitor that binds to the Bruton’s tyrosine kinase (BTK) ATP-dependent active site and it is currently used for treating a variety of B cell tumors. A high rate of drug resistance develops in Ibrutinib-treated patients through mutations at the BTK kinase domain, resulting in more aggressive diseases. Thus, there is significant interest in identifying modulators that inhibit BTK in an active site-independent manner, specifically via allosteric modulation. We used an in-silico system to identify small molecules that bind outside of the BTK active site. Candidate compounds were then tested for direct binding to full length BTK protein using Saturated Transfer Difference Nuclear Magnetic Resonance (STD-NMR) and were subsequently used in primary mouse B cell-based assays to determine their functional relevance. Ex vivo B cells were triggered via the BCR in the presence of compounds and signaling events downstream of the BCR, including Nur77 upregulation and calcium flux, were measured by flow cytometry. A series of candidate allosteric BTK inhibitors were then confirmed to retain suppressive activity in an ABC-DLBC lymphoma line that carries the Ibrutinib resistant mutation. Additionally, primary human CLL, but not healthy B cells, were susceptible to candidate inhibitor-mediated death. These results indicate that a screening approach integrating in silico docking, NMR measurements, and cellular assays with primary B cells can be used to identify BTK inhibitors that act outside of the kinase domain. Allosteric BTK inhibitors will broaden the range of treatment options for B cell cancers, possibly overcoming or preventing the Ibrutinib-induced resistance when used as a combination therapy.