Differential impact of BTK active site inhibitors on the conformational state of full-length BTK

Abstract

Bruton's tyrosine kinase (BTK) is targeted in the treatment of B-cell disorders including leukemias and lymphomas. Currently approved BTK inhibitors, including Ibrutinib, a first-in-class covalent inhibitor of BTK, bind directly to the kinase active site. While effective at blocking the catalytic activity of BTK, consequences of drug binding on the global conformation of full-length BTK are unknown. Here we uncover a range of conformational effects in full-length BTK induced by a panel of active site inhibitors, including large-scale shifts in the conformational equilibria of the regulatory domains. Additionally, we find that a remote Ibrutinib resistance mutation, T316A in the BTK SH2 domain, drives spurious BTK activity by destabilizing the compact autoinhibitory conformation of full-length BTK, shifting the conformational ensemble away from the autoinhibited form. Future development of BTK inhibitors will need to consider long-range allosteric consequences of inhibitor binding, including the emerging application of these BTK inhibitors in treating COVID-19.

A Combined Approach Reveals a Regulatory Mechanism Coupling Src’s Kinase Activity, Localization, and Phosphotransferase-Independent Functions

“…In addition to Src, several other tyrosine kinase N termini add a layer of regulation to a core SH3-SH2-linker-CD architecture (Devkota et al, 2017;Joseph et al, 2017;Wang et al, 2015). For example, Abl's N-terminal myristate binds to its CD, reducing phosphotransferase activity (Hantschel et al, 2003).…”

Effects of the Btk-Inhibitors Remibrutinib (LOU064) and Rilzabrutinib (PRN1008) With Varying Btk Selectivity Over Tec on Platelet Aggregation and in vitro Bleeding Time

“…They could be related to effects on the Btk protein itself. Recently it was found that binding of certain irreversible BTKi (except fenebrutinib) to the kinase domain had long-range allosteric effects on the SH2-and SH3-regulatory domains changing their conformation toward an activated state of the protein (53).…”

Reining in BTK: Interdomain Interactions and Their Importance in the Regulatory Control of BTK

“…The C481S resistance mutation was first identified in the samples of five out of six relapsed CLL patients ( Woyach et al, 2014 ) and has surfaced in other B-cell lymphomas such as WM and MCL supporting the notion that this mutation is a primary acquired resistance mutation that is a consequence of treatment with Ibrutinib ( Chiron et al, 2014 ; Xu et al, 2017 ). The BTK C481S mutation does not affect the activity of BTK ( Joseph et al, 2020 ) but mutation of this residue renders BTK insensitive to Ibrutinib due to the loss of the covalent bond to Ibrutinib ( Cheng et al, 2015 ; Figure 2 ). The reversible binding of Ibrutinib by the BTK C481S mutant together with the rapid clearance of Ibrutinib from the plasma (mean half-life of 2–3 h, Advani et al, 2013 ; Davids and Brown, 2014 ), would significantly reduce the occupancy of BTK by Ibrutinib, leaving BTK uninhibited and signaling competent.…”

“…Understanding how these point mutations contribute to Ibrutinib resistance at the molecular level is important as this information can aid inhibitor design to provide treatments for patients who acquire resistance. Recent solution nuclear magnetic resonance (NMR), hydrogen/deuterium exchange mass spectrometry (HDX-MS), and biochemical studies have revealed that the T316A mutation disrupts the autoinhibitory conformation of BTK (described in detail below) thereby increasing the active population of BTK, evading Ibrutinib inhibition ( Joseph et al, 2020 ). There are similar reports of active site inhibitor resistance mutations disrupting autoinhibitory contacts in ABL, another multi-domain kinase ( Saleh et al, 2017 ; Xie et al, 2020 ) suggesting that this mode of resistance may be a shared mechanism to bypass inhibition.…”

“…An increasing volume of work is being published on understanding the molecular level influence of small molecule inhibitors on full-length kinases ( Skora et al, 2013 ; Tong et al, 2017 ; Chakraborty et al, 2019 ; Fang et al, 2020 ). Detailed evaluation of a panel of BTK inhibitors has demonstrated that different active site inhibitors exert a range of distinct dynamic and conformational effects on the remote non-catalytic regulatory domains ( Joseph et al, 2020 ). Ibrutinib proves the most interesting case as covalent binding of Ibrutinib to the BTK active site promotes release of both the SH3 and SH2 domains, as well as the SH2-kinase linker, from their autoinhibitory poses.…”