1521-0111/85/4/553–563$25.00 http://dx.doi.org/10.1124/mol.113.089847 MOLECULAR PHARMACOLOGY Mol Pharmacol 85:553–563, April 2014 Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics Allosteric Noncompetitive Small Molecule Selective Inhibitors of CD45 Tyrosine Phosphatase Suppress T-Cell Receptor Signals and Inflammation In Vivo s Michael D. Perron, Shafinaz Chowdhury, Isabelle Aubry, Enrico Purisima, Michel L. Tremblay, and H. Uri Saragovi Lady Davis Institute-Jewish General Hospital (M.P., S.C., H.U.S.), Departments of Pharmacology and Therapeutics (M.P., H.U.S.), Biochemistry (I.A., E.P., M.L.T.), and Oncology (H.U.S.), Goodman Cancer Research Center (M.L.T.), and Segal Cancer Center, McGill University, Montreal, Quebec, Canada (H.U.S.); and Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada (E.P.) Received September 24, 2013; accepted January 14, 2014 ABSTRACT CD45 is a receptor-like member of the protein tyrosine phospha- tase (PTP) family. We screened in silico for small molecules binding at a predicted allosteric pocket unique to the CD45 intracellular domain, and validated inhibitors by in vitro phosphatase assays. Compound 211 exhibited a CD45 IC 50 value of 200 nM and had .100-fold selectivity over six related PTPs. The relevance of the allosteric pocket was verified through site-directed mutagenesis. Compound 211 has a noncompetitive mechanism of action, and it is extremely effective at preventing dephosphorylation of substrate Lck phosphotyrosine (pY)-505 versus preventing dephosphoryla- tion of Lck pY-393. In cultured primary T cells, compound 211 prevents T-cell receptor–mediated activation of Lck, Zap-70, and mitogen-activated protein kinase, and interleukin-2 production. In a delayed-type hypersensitivity reaction in vivo, compound 211 abolished inflammation. This work demonstrates a novel approach to develop effective allosteric inhibitors that can be expanded to target the corresponding allosteric domains of other receptor PTPs. Introduction CD45 is a transmembrane receptor–like protein tyrosine phosphatase (PTP), expressed on cells of hematopoietic origin. The function of CD45 is to regulate signal transduction and T-cell activation in response to antigen stimulation at the T-cell receptor (TCR) (Justement, 1997). CD45 exists as a variably glycosylated extracellular domain with no known physiologic ligand (Streuli et al., 1987), and a cytoplasmic portion with tandem D1 and D2 domains (Glover and Tracey, 2000). D1 is the catalytically active domain, because it harbors the cysteine residue involved in the enzymatic reaction that hydrolyzes phosphate from tyrosine residues. D2 is catalytically inactive (Streuli et al., 1990). CD45 regulates the phosphorylation of the Src-family kinases such as Lck and Fyn, as well as z-immunoreceptor tyrosine-based activation motif of the CD3 component of the TCR. In T cells, proper activation of Lck causes rapid Ca 21 fluxes and the phosphorylation of Zap-70 and Erk, which then stimulate the production of interleukin-2 (IL-2). These are standard readouts for T-cell activation and CD45 activity (Thomas and Brown, 1999; Alexander, 2000). CD45-deficient T cells have diminished proliferation and cytokine production in response to TCR stimulation (Pingel and Thomas, 1989). Furthermore, mutations in the human CD45 gene have been associated with autoimmune diseases, such as severe combined immunodeficiency and multiple sclerosis (Jacobsen et al., 2000; Kung et al., 2000). All of these events implicate CD45 as playing a key role in lymphocyte development and activation (Trowbridge and Thomas, 1994). Full activation of Lck requires the dephosphorylation of an inhibitory phosphotyrosine (pY)-505. Dephosphorylation of Lck pY-505 by CD45 facilitates Lck autophosphorylation at an activating tyrosine, Lck phosphotyrosine 393 (pY-393). The activating Lck pY-393 is also under CD45 regulation. Thus, CD45 essentially functions as a rheostat to keep Lck in a “primed” state, ready for activation (Hermiston et al., 2003) but avoiding sustained hyperactivation (D’Oro et al., 1996). Drug inhibitors of CD45 would disrupt the rheostat and could cause immunosuppression. Competitive CD45 inhibitors that block substrates from docking (Beers et al., 1997) and substrate analogs that poison the catalytic pocket have been This research was supported by the Canadian Institutes of Health Research [Proof-of-Principle Grant (to H.U.S. and M.L.T.)]. dx.doi.org/10.1124/mol.113.089847. s This article has supplemental material available at molpharm.aspetjournals. org. ABBREVIATIONS: 1,2-NQ, 1,2-napthoquinone; DiFMUP, 6,8-difluoro-4-methylumbelliferyl phosphate; DTH, delayed-type hypersensitivity; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; GST, glutathione S-transferase; IL-2, interleukin-2; MAPK, mitogen-activated protein kinase; MTT, 4-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; NCI, National Cancer Institute; OVA, ovalbumin; PTP, protein tyrosine phosphatase; pY, phosphotyrosine; RWJ-60475, [[2-(4-bromophenoxy)-5-nitrophenyl]-hydroxymethyl]phosphonic acid; SH, sulfhydryl; TCR, T-cell receptor. 553 http://molpharm.aspetjournals.org/content/suppl/2014/01/28/mol.113.089847.DC1 Supplemental material to this article can be found at: at ASPET Journals on June 21, 2017 molpharm.aspetjournals.org Downloaded from at ASPET Journals on June 21, 2017 molpharm.aspetjournals.org Downloaded from at ASPET Journals on June 21, 2017 molpharm.aspetjournals.org Downloaded from at ASPET Journals on June 21, 2017 molpharm.aspetjournals.org Downloaded from at ASPET Journals on June 21, 2017 molpharm.aspetjournals.org Downloaded from at ASPET Journals on June 21, 2017 molpharm.aspetjournals.org Downloaded from at ASPET Journals on June 21, 2017 molpharm.aspetjournals.org Downloaded from at ASPET Journals on June 21, 2017 molpharm.aspetjournals.org Downloaded from at ASPET Journals on June 21, 2017 molpharm.aspetjournals.org Downloaded from at ASPET Journals on June 21, 2017 molpharm.aspetjournals.org Downloaded from at ASPET Journals on June 21, 2017 molpharm.aspetjournals.org Downloaded from