Abstract
Ibrutinib is the first covalent inhibitor of Bruton's tyrosine kinase (BTK) to be used in the treatment of B-cell cancers. Understanding the mechanism of covalent inhibition will aid in the design of safer and more selective covalent inhibitors that target BTK. The mechanism of covalent inhibition in BTK has been uncertain because there is no appropriate residue nearby that can act as a base to deprotonate the cysteine thiol prior to covalent bond formation. We investigate several mechanisms of covalent modification of C481 in BTK by ibrutinib using combined quantum mechanics/molecular mechanics (QM/MM) molecular dynamics reaction simulations. The lowest energy pathway involves direct proton transfer from C481 to the acrylamide warhead in ibrutinib, followed by covalent bond formation to form an enol intermediate. There is a subsequent rate-limiting keto–enol tautomerisation step (ΔG‡ = 10.5 kcal mol−1) to reach the inactivated BTK/ibrutinib complex. Our results represent the first mechanistic study of BTK inactivation by ibrutinib to consider multiple mechanistic pathways. These findings should aid in the design of covalent drugs that target BTK and other similar targets.
Highlights
quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics (MD) simulations indicate that the lowest energy pathway of C481 modi cation by ibrutinib proceeds by a direct proton transfer (PT) from the C481 thiol group to the carbonyl oxygen atom of ibrutinib, resulting in a Cys-SÀ/C]OH+ ion pair (E-I1, Fig. 2)
An equivalent solvent-assisted PT and enol formation has been modelled for the modi cation of cysteine residues by microcystins, where a high barrier of 21.9 kcal molÀ1 was calculated for the reaction pathway involving water.[26]
The 1,2-addition pathway, consisting of a direct PT from the thiol to the a-carbon of the acrylamide warhead and simultaneous S–C formation has been reported as a high energy pathway by Rowley et al who calculated the barrier of the 1,2-s addition of methylvinyl ketone and methyl thiolate to be 65.2 kcal molÀ1 at the CCSD(T)//uB97X-D level.[27]
Summary
Covalent inhibitor drug discovery has re-emerged because of advantages compared with conventional non-covalent reversible binding that can include complete target blockage, increased selectivity and longer duration of action.[1,2,3] Recent years have seen the approval of several new marketed covalent drugs targeting protein kinases.[4,5] In particular, inhibition of Bruton's tyrosine kinase (BTK) is an attractive target for blood cancers and autoimmune diseases, due to its function in signal transduction in the B-cell antigen receptor (BCR) pathway.[6,7] BTK inhibitors have been explored as possible inhibitors against the SARS-CoV-2 coronavirus in drug repurposing studies.[8]. Utilising warheads of this type to target poorly conserved cysteine residues is a common technique to developing covalent inhibitors in drug discovery.[10]
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