title: Selectively targeting an inactive conformation of interleukin-2-inducible T-cell kinase by allosteric inhibitors.
abstract: ITK (interleukin-2-inducible T-cell kinase) is a critical component of signal transduction in T-cells and has a well-validated role in their proliferation, cytokine release and chemotaxis. ITK is an attractive target for the treatment of T-cell-mediated inflammatory diseases. In the present study we describe the discovery of kinase inhibitors that preferentially bind to an allosteric pocket of ITK. The novel ITK allosteric site was characterized by NMR, surface plasmon resonance, isothermal titration calorimetry, enzymology and X-ray crystallography. Initial screening hits bound to both the allosteric pocket and the ATP site. Successful lead optimization was achieved by improving the contribution of the allosteric component to the overall inhibition. NMR competition experiments demonstrated that the dual-site binders showed higher affinity for the allosteric site compared with the ATP site. Moreover, an optimized inhibitor displayed non-competitive inhibition with respect to ATP as shown by steady-state enzyme kinetics. The activity of the isolated kinase domain and auto-activation of the full-length enzyme were inhibited with similar potency. However, inhibition of the activated full-length enzyme was weaker, presumably because the allosteric site is altered when ITK becomes activated. An optimized lead showed exquisite kinome selectivity and is efficacious in human whole blood and proximal cell-based assays.
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Seungil
authors.initials: S
authors.lastname: Han
authors.affiliation: †Pfizer Worldwide Research, 200 Cambridge Park Drive, Cambridge, MA 02140, U.S.A.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Czerwinski
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Nicole L
authors.initials: NL
authors.lastname: Caspers
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: David C
authors.initials: DC
authors.lastname: Limburg
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: WeiDong
authors.initials: W
authors.lastname: Ding
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Jeffrey F
authors.initials: JF
authors.lastname: Ohren
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Francis
authors.initials: F
authors.lastname: Rajamohan
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: McLellan
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Ray
authors.initials: R
authors.lastname: Unwalla
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Chulho
authors.initials: C
authors.lastname: Choi
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Mihir D
authors.initials: MD
authors.lastname: Parikh
authors.affiliation: †Pfizer Worldwide Research, 200 Cambridge Park Drive, Cambridge, MA 02140, U.S.A.
authors.forename: Nilufer
authors.initials: N
authors.lastname: Seth
authors.affiliation: †Pfizer Worldwide Research, 200 Cambridge Park Drive, Cambridge, MA 02140, U.S.A.
authors.forename: Jason
authors.initials: J
authors.lastname: Edmonds
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Chris
authors.initials: C
authors.lastname: Phillips
authors.affiliation: †Pfizer Worldwide Research, 200 Cambridge Park Drive, Cambridge, MA 02140, U.S.A.
authors.forename: Subarna
authors.initials: S
authors.lastname: Shakya
authors.affiliation: †Pfizer Worldwide Research, 200 Cambridge Park Drive, Cambridge, MA 02140, U.S.A.
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.affiliation: †Pfizer Worldwide Research, 200 Cambridge Park Drive, Cambridge, MA 02140, U.S.A.
authors.forename: Vikki
authors.initials: V
authors.lastname: Spaulding
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Samantha
authors.initials: S
authors.lastname: Hughes
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Andrew
authors.initials: A
authors.lastname: Cook
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Colin
authors.initials: C
authors.lastname: Robinson
authors.affiliation: †Pfizer Worldwide Research, 200 Cambridge Park Drive, Cambridge, MA 02140, U.S.A.
authors.forename: John P
authors.initials: JP
authors.lastname: Mathias
authors.forename: Iva
authors.initials: I
authors.lastname: Navratilova
authors.affiliation: †Pfizer Worldwide Research, 200 Cambridge Park Drive, Cambridge, MA 02140, U.S.A.
authors.forename: Quintus G
authors.initials: QG
authors.lastname: Medley
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: David R
authors.initials: DR
authors.lastname: Anderson
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Ravi G
authors.initials: RG
authors.lastname: Kurumbail
authors.affiliation: *Pfizer Worldwide Research, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Ann
authors.initials: A
authors.lastname: Aulabaugh
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D000495:Allosteric Site; D003460:Crystallization; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D020349:Surface Plasmon Resonance
chemical_list: D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; C080813:emt protein-tyrosine kinase