Differences in JAK Isoform Selectivity Among Different Types of JAK Inhibitors Evaluated for Rheumatic Diseases Through In Vitro Profiling

Abstract

ObjectiveThe selectivity of JAK inhibitors (Jakinibs) forms the basis for understanding their clinical characteristics; however, evaluation of selectivity is hampered by the lack of comprehensive head‐to‐head studies. Our objective was to profile in parallel Jakinibs indicated or evaluated for rheumatic diseases for their JAK and cytokine selectivity in vitro.MethodsWe analyzed 10 Jakinibs for JAK isoform selectivity by assaying their inhibition of JAK kinase activity, binding to kinase and pseudokinase domains, and inhibition of cytokine signaling using blood samples from healthy volunteers and using isolated peripheral blood mononuclear cells (PBMCs) from patients with rheumatoid arthritis and from healthy donors.ResultsPan‐Jakinibs effectively suppressed kinase activity of 2 to 3 JAK family members, whereas isoform‐targeted Jakinibs possessed varying degrees of selectivity for 1 or 2 JAK family members. In human leukocytes, Jakinibs predominantly inhibited the JAK1‐dependent cytokines interleukin‐2 (IL‐2), IL‐6, and interferons (IFNs). In PBMCs from patients with rheumatoid arthritis compared with healthy controls, inhibition of these cytokines was more pronounced, and some cell‐type and STAT isoform differences were observed. Novel Jakinibs demonstrated high selectivity: the covalent Jakinib ritlecitinib showed 900‐ to 2,500‐fold selectivity for JAK3 over other JAKs and specific suppression of IL‐2‐signaling, whereas the allosteric TYK2 inhibitor deucravacitinib inhibited IFNα signaling with high specificity. Interestingly, deucravacitinib targeted the regulatory pseudokinase domain and did not affect JAK in vitro kinase activity.ConclusionInhibition of JAK kinase activity did not directly translate into cellular inhibition of JAK/STAT signaling. Despite differences in JAK selectivity, the cytokine inhibition profiles of currently approved Jakinibs were highly similar, with preference for JAK1‐mediated cytokines. Novel types of Jakinibs showed narrow cytokine inhibition profile specific for JAK3‐ or TYK2‐mediated signaling.image