BTK gatekeeper residue variation combined with cysteine 481 substitution causes super-resistance to irreversible inhibitors acalabrutinib, ibrutinib and zanubrutinib

Abstract

Irreversible inhibitors of Bruton tyrosine kinase (BTK), pioneered by ibrutinib, have become breakthrough drugs in the treatment of leukemias and lymphomas. Resistance variants (mutations) occur, but in contrast to those identified for many other tyrosine kinase inhibitors, they affect less frequently the “gatekeeper” residue in the catalytic domain. In this study we carried out variation scanning by creating 11 substitutions at the gatekeeper amino acid, threonine 474 (T474). These variants were subsequently combined with replacement of the cysteine 481 residue to which irreversible inhibitors, such as ibrutinib, acalabrutinib and zanubrutinib, bind. We found that certain double mutants, such as threonine 474 to isoleucine (T474I) or methionine (T474M) combined with catalytically active cysteine 481 to serine (C481S), are insensitive to ≥16-fold the pharmacological serum concentration, and therefore defined as super-resistant to irreversible inhibitors. Conversely, reversible inhibitors showed a variable pattern, from resistance to no resistance, collectively demonstrating the structural constraints for different classes of inhibitors, which may affect their clinical application.

Structure-Function Relationships of Covalent and Non-Covalent BTK Inhibitors

“…Fenebrutinib-BTK complex structure revealed specific interactions, which may explain its selectivity ( 25 ). This compound also showed retained activity in vitro towards BTK carrying the single and double variations C481S, T474A and T474S/C481S, respectively ( 32 ).…”

“…This distance allows for minor adjustments in the structures as well as interactions mediated by solvent (water) molecules. Crystallographic structures have been determined both for ibrutinib and zanubrutinib ( Figure 3 ), the corresponding complex for acalabrutinib has been modelled ( Figure 4 ) ( 32 ). Backbone amide groups of E475 and M477 form hydrogen bonds with the pyrazolopyrimidine core, which extends towards T474 and the area close to α-C-helix (5p9j and 5kup) ( 19 , 33 ).…”

“…Variants at amino acids T474 and C481 lead to tolerance of covalent inhibitors in CLL and other B-cell malignancies. Effects of several variants and their combinations were recently investigated and shown to impair inhibitor binding leading to drug resistance ( 32 ). These two positions are indicated in Figure 6B .…”

Resistance Mutations to BTK Inhibitors Originate From the NF-κB but Not From the PI3K-RAS-MAPK Arm of the B Cell Receptor Signaling Pathway

“…In a recent study, mutations affecting T474 and those yielding the C481S replacement were combined ( 18 ). Surprisingly, certain double mutants demonstrated super-resistance tolerating more than 16-fold the therapeutic BTKi concentration.…”

“…Many of these are investigated as treatment of autoimmune and inflammatory disorders ( 15 ). They also are explored as a treatment for patients with resistance mutations to irreversible inhibitors ( 17 , 18 ). The concept of irreversible kinase inhibitors was relatively novel around the time when BTKi were first generated ( 19 ).…”