Tofacitinib

There is convincing evidence demonstrating direct target engagement of JAK1, JAK2, JAK3 and TYK2 by tofacitinib in vitro using X-ray crystallography (i.e., PMIDs 20478313, 19361440). Based on the reported IC₅₀ values in cell-free systems, tofacitinib will most likely inhibit JAK1, JAK2 and JAK3 to varying degrees at the recommended concentrations (1-120 nM); therefore, users should be cautious about assuming isoform selectivity in their particular system. The original report (PMID 14593182) does not demonstrate direct target engagement in cells, but rather uses indirect measurements of JAK and STAT phosphorylation, as well as inflammatory/disease endpoints in animal models. The relative engagement of each JAK isoform by tofacitinib is unclear in these systems (i.e., IC₅₀ values may or may not reflect the cellular inhibition by tofacitinib). Users should consider assaying each JAK isoform (e.g., phosopho-JAK1/2/3) in their cell-based and in vivo experiments to define the specific contributions of each JAK-dependent pathway, especially if isoform-specific effects are being studied.

In human primary cell types, tofacitinib is active and non-cytotoxic from 14 nM to 90 uM. Between 14-120 nM, it is active in human primary cell-based assays that are JAK3/JAK1 dependent. At 370 nM and above, it is active in assays that are JAK1/JAK2 dependent (DOI:10.1039/9781782620136-00088).

This probe will always inhibit both JAK1 and JAK3 at any in vitro and in vivo dose explored. At higher doses, it will inhibit JAK2 , and at even higher doses, it inhibits TYK2. Care in data interpretation and target association is, therefore, warranted within the JAK family of kinases.