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 (FcR), and toll like receptors (TLRs). Btk has become an attractive drug target as Btk inhibition may provide significant efficacy by blocking multiple disease mechanisms simultaneously. Consequently, a large number of Btk inhibitors have been developed. The compounds have diverse binding modes and both reversible and irreversible inhibitors have been developed. Reported herein, we have tested 9 Btk inhibitors and characterized on a molecular level how their interactions with Btk defines 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 found to be an important determinant of potency against FcϵR signaling as Y551 sequestering compounds were more potent at inhibiting basophils and mast cells. This was true for inhibition of Fcγ;R signaling as well. In contrast, Y551 sequestration was less of 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.
- Structure determinations
- Structure-activity relationships and modeling
- X-ray crystallography
- Leukocytes/Mast cells
- The American Society for Pharmacology and Experimental Therapeutics