title: Insights into kinetic mechanism of Janus kinase 3 and its inhibition by tofacitinib.
abstract: JAK3 kinase plays a critical role in several cytokine signaling pathways involved in immune cell development and function. The studies presented in this report were undertaken to elucidate the kinetic mechanism of the JAK3 kinase domain, investigate the role of activation loop phosphorylation in regulating its catalytic activity, and examine its inhibition by the anti-rheumatoid arthritis drug, tofacitinib. Phosphorylation of two Tyr residues in JAK3's activation loop has been reported to impact its kinase activity. The recombinant JAK3 kinase domain used in our studies was heterogeneous in its activation loop phosphorylation, with the non-phosphorylated protein being the dominant species. Kinetic analysis revealed similar kinetic parameters for the heterogeneously phosphorylated JAK3, JAK3 mono-phosphorylated on Tyr 980, and the activation loop mutant YY980/981FF. Bisubstrate and product inhibition kinetic results were consistent with both sequential random and sequential ordered kinetic mechanisms. Solvent viscosometric experiments showed perturbation of kcat, suggesting the phosphoryl transfer step is not likely rate limiting. This was supported by results from quench-flow experiments, where a rapid burst of product formation was observed. Kinetic analysis of JAK3 inhibition by tofacitinib indicated inhibition is time dependent, characterized by on- and off-rate constants of 1.4 ± 0.1 μM-1s-1and 0.0016 ± 0.0005 s-1, respectively.
authors.affiliation: Department of Biochemical Pharmacology, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA. Electronic address: mohammad.hekmat-nejad@novartis.com.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Hekmatnejad
authors.affiliation: Department of Biochemical Pharmacology, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA. Electronic address: sara.conwell@gmail.com.
authors.forename: Sara
authors.initials: S
authors.lastname: Conwell
authors.affiliation: Department of Molecular and Protein Sciences, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA. Electronic address: stephenjlok@gmail.com.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Lok
authors.affiliation: Department of Molecular and Protein Sciences, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA. Electronic address: akutach@hotmail.com.
authors.forename: Alan
authors.initials: A
authors.lastname: Kutach
authors.affiliation: Department of Molecular and Protein Sciences, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA. Electronic address: shaw.david@gmail.com.
authors.forename: David
authors.initials: D
authors.lastname: Shaw
authors.affiliation: Protein Sciences, Novartis Institutes for Biomedical Research, 4560 Horton St., Emeryville, CA 94608, USA. Electronic address: eric.fang@novartis.com.
authors.forename: Eric
authors.initials: E
authors.lastname: Fang
authors.affiliation: Department of Biochemical Pharmacology, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA. Electronic address: david.swinney@ymail.com.
authors.forename: David C
authors.initials: DC
authors.lastname: Swinney
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D000818:Animals; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020128:Inhibitory Concentration 50; D007313:Insecta; D053616:Janus Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D010766:Phosphorylation; D010880:Piperidines / Q000737:chemistry*; D047428:Protein Kinase Inhibitors / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry*; D011758:Pyrroles / Q000737:chemistry*; D061987:Sf9 Cells; D015398:Signal Transduction; D012997:Solvents; D014783:Viscosity
chemical_list: D010880:Piperidines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D011758:Pyrroles; D012997:Solvents; C479163:tofacitinib; C507933:JAK3 protein, human; D053616:Janus Kinase 3; D000251:Adenosine Triphosphatases
keywords: JAK3 activation mechanism; Kinetic mechanism; Sequential random; Time dependent inhibition; Tofacitinib