The cytoplasmic protein-tyrosine kinase BTK plays an essential role for differentiation and survival of B-lineage cells and, hence, represents a suitable drug target. The number of BTK inhibitors (BTKis) in the clinic has increased considerably and currently amounts to at least 22. First-in-class was ibrutinib, an irreversible binder forming a covalent bond to a cysteine in the catalytic region of the kinase, for which we have identified 228 active trials listed at ClinicalTrials.gov. Next-generation inhibitors, acalabrutinib and zanubrutinib, are approved both in the United States and in Europe, and zanubrutinib also in China, while tirabrutinib is currently only registered in Japan. In most cases, these compounds have been used for the treatment of B-lymphocyte tumors. However, an increasing number of trials instead addresses autoimmunity and inflammation in multiple sclerosis, rheumatoid arthritis, pemphigus and systemic lupus erythematosus with the use of either irreversibly binding inhibitors, e.g., evobrutinib and tolebrutinib, or reversibly binding inhibitors, like fenebrutinib. Adverse effects (AEs) have predominantly implicated inhibition of other kinases with a BTKi-binding cysteine in their catalytic domain. Analysis of the reported AEs suggests that ibrutinib-associated atrial fibrillation is caused by binding to ERBB2/HER2 and ERBB4/HER4. However, the binding pattern of BTKis to various additional kinases does not correlate with the common assumption that skin manifestations and diarrhoeas are off-target effects related to EGF receptor inhibition. Moreover, dermatological toxicities, diarrhoea, bleedings and invasive fungal infections often develop early after BTKi treatment initiation and subsequently subside. Conversely, cardiovascular AEs, like hypertension and various forms of heart disease, often persist.

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.