Structural and biophysical insights into the mode of covalent binding of rationally designed potent BMX inhibitors

Abstract

We identified potent, functionalisable BMX inhibitors and revealed their covalent mode of binding to BMX by X-ray crystallography.

Selective Inhibition of Bruton’s Tyrosine Kinase by a Designed Covalent Ligand Leads to Potent Therapeutic Efficacy in Blood Cancers Relative to Clinically Used Inhibitors

“…The rate of inactivation ( k inact / K I ) is a second-order event, which describes the efficacy of the covalent bond binding event. To characterize the covalent interactions of JS25 with BTK, evaluation of the irreversible binding efficacy was performed as previously described . Additionally, we included ibrutinib, acalabrutinib, and BMX-IN-1 (Figure a).…”

“…Additionally, we had previously shown that JS25 did not react with other Scr kinases. 23 On comparing the JS25 selectivity profile with other BTKi (Supporting Table S3), we find that JS25 is less reactive than ibrutinib for TEC, TXK, ITK, EGFR, JAK3, BLK, and Her2; less reactive for TEC and TXK than acalabrutinib; less reactive toward EGFR, JAK3, and Her2 than zanubrutinib; and less reactive toward TEC, TXK, and BLK than tirabrutinib, although slightly more reactive against ITK. The BTKi acalabrutinib, zanubrutinib, and tirabrutinib are secondgeneration inhibitors, and relative to ibrutinib, these molecules presented fewer "off-target" effects in early clinical trials.…”

“…JS25 is also a dual inhibitor of BMX and BTK and presents lower reactivity for TEC, ITK, and TXK and nonreactivity toward EGFR, BLK, JAK3, and Her2. Additionally, we had previously shown that JS25 did not react with other Scr kinases . On comparing the JS25 selectivity profile with other BTKi (Supporting Table S3), we find that JS25 is less reactive than ibrutinib for TEC, TXK, ITK, EGFR, JAK3, BLK, and Her2; less reactive for TEC and TXK than acalabrutinib; less reactive toward EGFR, JAK3, and Her2 than zanubrutinib; and less reactive toward TEC, TXK, and BLK than tirabrutinib, although slightly more reactive against ITK.…”

“…In this study, we investigate the therapeutic potential of a small covalent molecule (JS25) with nanomolar potency against BTK (5.8 nM). JS25 was obtained from the scaffold of BMX-IN-1, a recently discovered molecule that has been shown to also inhibit BTK, as part of our efforts to identify regions of the molecule that could be modulated for improved efficacy and selectivity. , Initially, we had explored the JS25 potential for treating prostate cancer, but later experiments revealed that JS25 was highly selective for BTK, and therefore, it could have therapeutic importance in blood malignancies that derive from BTK’s abnormal expression. Following the preliminary data, we sought to characterize the binding mode of JS25 to BTK and asserted its selectivity against a panel of eight kinases related to BTK’s signaling pathway or with an equally placed cysteine as to the Cys481 of BTK.…”

The role of reversible and irreversible covalent chemistry in targeted protein degradation

“…Nevertheless, recently there have been successful examples of PROTACs using covalent warheads that have degraded a range of targets, including ERK1/2 (Lebraud et al, 2016), Bruton tyrosine kinase (BTK) (Xue et al, 2020), and KRAS G12C (Bond et al, 2020). There have also been examples of TCIs that degrade a target POI themselves, such as the BMX inhibitors BMX-IN-1 and highly potent JS25 (Seixas et al, 2020), but this is beyond the scope of this review.…”