Ability of Bruton’s Tyrosine Kinase Inhibitors to Sequester Y551 and Prevent Phosphorylation Determines Potency for Inhibition of Fc Receptor but not B-Cell Receptor Signaling

Abstract

Bruton's tyrosine kinase (Btk) is expressed in a variety of hematopoietic cells. Btk has been demonstrated to regulate signaling downstream of the B-cell receptor (BCR), Fc receptors (FcRs), and toll-like receptors. It has become an attractive drug target because its inhibition may provide significant efficacy by simultaneously blocking multiple disease mechanisms. Consequently, a large number of Btk inhibitors have been developed. These compounds have diverse binding modes, and both reversible and irreversible inhibitors have been developed. Reported herein, we have tested nine Btk inhibitors and characterized on a molecular level how their interactions with Btk define their ability to block different signaling pathways. By solving the crystal structures of Btk inhibitors bound to the enzyme, we discovered that the compounds can be classified by their ability to trigger sequestration of Btk residue Y551. In cells, we found that sequestration of Y551 renders it inaccessible for phosphorylation. The ability to sequester Y551 was an important determinant of potency against FcεR signaling as Y551 sequestering compounds were more potent for inhibiting basophils and mast cells. This result was true for the inhibition of FcγR signaling as well. In contrast, Y551 sequestration was less a factor in determining potency against BCR signaling. We also found that Btk activity is regulated differentially in basophils and B cells. These results elucidate important determinants for Btk inhibitor potency against different signaling pathways and provide insight for designing new compounds with a broader inhibitory profile that will likely result in greater efficacy.

Inhibition of Bruton’s tyrosine kinase interferes with pathogenic B-cell development in inflammatory CNS demyelinating disease

“…One promising approach is the inhibition of Bruton’s tyrosine kinase [ 20 ], an enzyme centrally involved in B-cell activation upon BCR antigen recognition [ 8 ]. Besides B-cell signaling, BTK is also crucial for Fc receptor (FcR) mediated activation of myeloid cells [ 4 , 8 ], an alternative pathway to facilitate antigen recognition via antibody-mediated opsonization. Based on this mechanism of action not relying on the depletion of immune cells, various BTK inhibitors are being developed for treatment of MS.…”

Diaminoimidazopyrimidines: Access via the Groebke–Blackburn–Bienaymé Reaction and Structural Data Mining

“…Type I‐active site kinase inhibitors bear the characteristic binding motif of a hydrogen‐bond donor in close proximity to a hydrogen‐bond acceptor. This common motif can be observed for instance in dabrafenib (a reversible ATP‐competitive kinase inhibitor targeting the BRAF, PDB ID 5CSW), lorlatinib (a macrocyclic kinase inhibitor targeting ALK, PDB ID 5A9U), an aminopyridine inhibitor targeting GLK kinase (PDB ID 5J5T) and ibrutinib (a covalent inhibitor of Bruton's tyrosine kinase, PDB ID 5P9J) . A similar motif of hydrogen‐bond donor in adjacent position to a hydrogen‐bond acceptor is also observed in ganciclovir (a guanine analogue targeting thymidine kinase, PDB ID 1KI2) and trimethoprim (an antifolate antibacterial inhibiting bacterial dihydrofolate reductase, PDB ID 1DG5) (Figure ).…”

Discovery and biological evaluation of N-(3-(7-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-4-methyl-2-oxo-2H-pyrimido[4,5-d][1,3]oxazin-1(4H)-yl)phenyl)acrylamide as potent Bruton’s tyrosine kinase inhibitors

“…We used covalent docking to evaluate the binding pattern of the compound through the software of Maestro 10.1. We chose the PDB code 5P9L from the Protein Date Bank, and this PDB includes the crystal structure of BTK in complex with a small molecule CC-2922 [ 24 ]. As we expected, the compound binds to the ATP-bingding pocket of BTK, and the acrylamide of this compound proceeds Michael addition reaction with the thiol of Cys481.…”