Crystal Structures of IL‐2‐inducible T cell Kinase Complexed with Inhibitors: Insights into Rational Drug Design and Activity Regulation

Abstract

IL-2-inducible T cell kinase plays an essential role in T cell receptor signaling and is considered a drug target for the treatment of Th2-mediated inflammatory diseases. By applying high-throughput protein engineering and crystallization, we have determined the X-ray crystal structures of IL-2-inducible T cell kinase in complex with its selective inhibitor BMS-509744 and the broad-spectrum kinase inhibitors sunitinib and RO5191614. Sunitinib uniquely stabilizes IL-2-inducible T cell kinase in the helix C-in conformation by inducing side chain conformational changes in the ATP-binding site. This preference of sunitinib to bind to an active kinase conformation is reflective of its broad-spectrum kinase activity. BMS-509744 uniquely stabilizes the activation loop in a substrate-blocking inactive conformation, indicating that structural changes described for Src family kinases are also involved in the regulation of IL-2-inducible T cell kinase activity. The observed BMS-509744 binding mode allows rationalization of structure-activity relationships reported for this inhibitor class and facilitates further structurebased drug design. Sequence-based analysis of this binding mode provides guidance for the rational design of inhibitor selectivity.Key words: BMS-509744, conformational changes, high-throughput protein engineering, IL-2-inducible T cell kinase, kinase inhibitor, sunitinib, X-ray protein crystallography Abbreviations: ITK, IL-2-inducible T cell kinase; SAR, structureactivity relationship; SPR, surface plasmon resonance; TEV, tobacco etch virus.Received 5 March 2010, revised 13 April 2010 and accepted for publication 18 April 2010 IL-2-inducible T cell kinase (ITK) is involved in the regulation of actin reorganization, PLCc activation, calcium mobilization, and NFAT activation in T cells. IL-2-inducible T cell kinase mediates the secretion of Th2 cytokines and the development of effective Th2 response during allergic asthma or infection by parasitic worms. Consequently, ITK is considered a promising drug target for the treatment of Th2-mediated inflammatory diseases like asthma, rhinitis, allergies, and atopic dermatitis (1,2). Recently, ITK inhibition has also been shown to block HIV infection by affecting multiple steps of HIV replication (3).ITK is a member of the TEC family of non-receptor tyrosine kinases. It consists of an N-terminal Pleckstrin homology domain that targets ITK reversibly to the membrane, followed by a TEC-homology, SH3, SH2, and kinase domain (2). The involvement of the SH3 and SH2 domains in the regulation of ITK activity has been studied in detail (4-6), and the solution structure of the binary SH3 ⁄ SH2 complex has been determined by NMR (PDB accession numbers 2K7A and 2K79) (7). Phosphorylation of Y512 within the activation loop of the kinase domain by the Src family kinase Lck is critical for ITK activity (8). The crystal structure of the ITK kinase domain in complex with the pan-kinome inhibitor staurosporine has been solved for non-phosphorylated (PDB accession number 1SNU) and ...

Insights into the conformational flexibility of Bruton's tyrosine kinase from multiple ligand complex structures

“…Compound 1, a reported inhibitor of the protein kinases Aurora‐A,10 BTK (half maximal inhibitory concentration (IC 50 ) = 100 n M ) and SYK,11 forms three hydrogen bonds via its amino‐pyrazole moiety to the BTK hinge region [Fig. 3(A)], similar as observed for an analogous compound bound to the Tec kinase family member ITK 12. The phthalazinone core of compound 1 is stacked between V427 in the N‐terminal BTK lobe and G480 in the C‐terminal lobe.…”

Substrate and Inhibitor Specificity of the Type II p21-Activated Kinase, PAK6

“…There are currently 6 co-crystal structures of protein kinases in complex with sunitinib deposited in the PDB. These kinases are of VEGFR (4AGD) [41], KIT (3G0E) [42], KIT-D816H mutation (3G0F) [42], ITK (3MIY) [43], CDK2 (3TI1) [44] and phosphorylase kinase γ2 (2Y7J) (unpublished). In each of these structures the dihydrooxaindole and pyrrole rings of sunitinib make hydrogen-bonds to the kinase linker region, a pattern that is replicated in the PAK6 co-crystal structure.…”

Emergence of Polyclonal FLT3 Tyrosine Kinase Domain Mutations during Sequential Therapy with Sorafenib and Sunitinib in FLT3-ITD–Positive Acute Myeloid Leukemia

“…Our data are consistent with a previous investigation showing that sunitinib inhibits FLT3 phosphorylation in cells harboring a single D835H mutation with an IC50 < 10 nM (4, 5). Sunitinib has also been shown to exhibit type I inhibitor properties, which may explain its activity against some FLT3 activation loop mutations (38, 39). Homology modeling predicts that histidine substitution at D835 may retain partial ability to make a hydrogen bond with S838 to stabilize the inactive kinase conformation; whereas, a tyrosine exchange would entirely disrupt hydrogen bond formation.…”