r-Haloacetophenone Derivatives As Photoreversible Covalent Inhibitors of Protein Tyrosine Phosphatases Gulnur Arabaci, † Xiao-Chuan Guo, † Kirk D. Beebe, ¶ K. Mark Coggeshall, § and Dehua Pei* ,†,¶ Departments of Chemistry and Microbiology Ohio State Biochemistry Program The Ohio State UniVersity 100 West 18th AVenue Columbus, Ohio 43210 ReceiVed March 2, 1999 Phosphorylation of proteins on tyrosine residues is one of the most important posttranslational modifications, playing central roles both in physiological processes such as transmembrane signaling and in pathological processes such as cancer and immune dysfunction. 1 The levels of tyrosine phosphorylation are regulated by the opposing actions of protein tyrosine kinases (PTKs), which catalyze the formation of phosphotyrosine (pY) in proteins, and protein tyrosine phosphatases (PTPs), which hydrolyze pY residues to give back tyrosine and inorganic phosphate. More than 100 PTPs have been identified to date, and it is estimated that the human genome contains as many as 500 PTP genes. 2 The precise functions of these PTPs in physiological and pathological states have remained largely unknown. Specific PTP inhibitors would provide valuable tools in studying the functions of these enzymes as well as potential therapeutic agents. It is with this premise that there has been a recently intensified interest in developing PTP inhibitors. 3 Here we report that R-halogenated acetophenones act as a novel class of potent, covalent PTP inhibitors, whose inhibitory effects can be conveniently reversed by photolysis at 350 nm. PTPs of all origins share a common catalytic domain of ∼250 amino acids, containing the unique “signature motif”, (I/V)- HC×AG××R(S/T). 2 PTP-catalyzed pY hydrolysis proceeds through a nucleophilic attack on the phosphate group by the side- chain thiol of the conserved cysteine in the signature motif, forming a covalent phosphocysteinyl enzyme intermediate, which is subsequently hydrolyzed by a water molecule (Scheme 1). 4 We envisioned that R-haloacetophenone 1 could bind to the PTP active site as a pY mimetic; its phenyl ring could engage in hydrophobic interactions with the protein as the phenyl ring of a substrate does, and the electron-rich halogen atom could mimic the negatively charged phosphate oxyanions. Binding of 1 to the PTP active site in such a manner would place the R-carbon, which is highly susceptible to nucleophilic attack, next to the catalytic cysteine. An S N 2 reaction between 1 and the cysteine thiol would result in the formation of a covalent enzyme-inhibitor adduct through a stable thioether linkage and loss of phosphatase activity. R-Bromo- and R-chloroacetophenone derivatives (1a-d) were prepared 5 and assayed against the prototypical phosphatase PTP1B, 6 a Src homology 2 (SH2) domain-containing phosphatase SHP-1, 7 and the catalytic domain of SHP-1, SHP-1(ΔSH2). 8 All four compounds resulted in time-dependent inhibition of the PTPs, and their inhibition kinetics can be described by equation where K I is the dissociation constant of the noncovalent complex, E•I, and k inact is the first-order rate constant for conversion of the E•I complex into a covalent complex, E-I. Inhibitor 1a binds to PTPs with the highest affinity, having K I values of 43 and 42 μM and k inact values of 0.40 and 0.57 min -1 for SHP-1(ΔSH2) and PTP1B, respectively (Table 1). Inhibitors 1b and 1c bind to SHP-1(ΔSH2) with 3-5-fold lower affinity but have 4-6-fold higher k inact , therefore having similar overall potency as 1a. The R-chloro derivative (1d) is 13-fold less potent than its R-bromo counterpart (1c), mainly because of lower binding affinity (higher K I ). This may be due to the smaller size of the chlorine atom, rendering R-chloroacetyl group a less effective mimetic of the phosphate group than the R-bromoacetyl group. Note that 1a has lower affinity to wild-type SHP-1 than its catalytic domain (K I ) 530 vs 43 μM), likely due to the fact that the SH2 domains can directly bind to the PTP active site and interfere with substrate/ inhibitor binding. 9 As controls, 1a was assayed against alkaline phosphatases, acid phosphatases, and a dual-specificity phos- phatase VHR. 10 Incubation with 10 mM 1a for 10 min resulted in no significant inhibition of any of the alkaline or acid * Corresponding author Telephone: (614) 688-4068. Fax: (614) 292-1532. E-mail: pei.3@osu.edu. † Department of Chemistry. § Department of Microbiology. ¶ Ohio State Biochemistry Program. (1) Hunter, T. Cell 1995, 80, 225-236. (2) Neel, B. G.; Tonk, N. K. Curr. Opin. Cell Biol. 1997, 9, 193-204. (3) For a recent review, see: Burke, T. R.; Zhang, Z.-Y. Biopolymers 1998, 47, 225-241. (4) Dixon, J. E.; Zhang, Z.-Y. AdV. Enzymol. Relat. Areas Mol. Biol. 1994, 68,1-36. (5) Compound 1a was synthesized as described: King, L. C.; Ostrum, G. K. J. Org. Chem. 1964, 29, 3459. Compound 1b is available from Fluka, whereas 1c and 1d were prepared as described under Supporting Information. (6) (a) Tonks, N. K.; Diltz, C. D.; Fischer, E. H. J. Biol. Chem. 1988, 263, 6722-6730. (b) Tonks, N. K.; Diltz, C. D.; Fischer, E. H. J. Biol. Chem. 1988, 263, 6731-6737. (7) Feng, G.-S.; Pawson, T. Trends Genet. 1994, 10, 54-58. (8) Pei, D.; Neel, B. G.; Walsh, C. T. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1092-1096. (9) (a) Pei, D.; Lorenz, U.; Klingmuller, U.; Neel, B. G.; Walsh, C. T. Biochemistry 1994, 33, 15483-15493. (b) Townley, R.; Shen, S.-H.; Banville, D.; Ramanchandran, C. Biochemistry 1993, 32, 13414-13418. Scheme 1. Mechanism of Catalysis and Inactivation by 1 Table 1. Kinetic Constant of PTP Inhibition by 1a-d a enzyme inhibitor KI (μM) kinact (min -1 ) SHP-1(ΔSH2) 1a 43 ( 10 0.40 ( 0.10 1b 128 ( 10 2.4 ( 0.2 1c 193 ( 38 1.8 ( 0.3 1d 2540 ( 610 1.8 ( 0.4 BrCH 2CO2H 77000 ( 14000 1.4 ( 0.2 SHP-1 1a 530 ( 120 2.6 ( 0.2 PTP1B 1a 42 ( 5 0.57 ( 0.05 VHR 1a 8900 ( 4500 3.4 ( 1.6 a Data reported are the mean (SD from three or more independent experiments carried out at pH 7.4 and at room temperature. E + I y \ z K I E•I 9 8 k inact E-I 5085 J. Am. Chem. Soc. 1999, 121, 5085-5086 10.1021/ja9906756 CCC: $18.00 © 1999 American Chemical Society Published on Web 05/12/1999