Characterization of Neuropathy Target Esterase Using Trifluoromethyl Ketones
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Biochemml Pharmocolog,v, Vol.40. No. 12. pp. 2587-2596. 1990. lloO6-2952/90$3.00+ 0.00 PrintedinGreat Britain. Q 1990Pergamon Press plc CHARACTERIZATION OF NEUROPATHY TARGET ESTERASE USING TRIFLUOROMETHYL KETONES THOMAS C.THOMAS,* ANDRASSZ~KACS,~SSCOTTKOJAS,* BRUCED.HAMMOCK,~~ BARRY W. WILSON/]and MARK G.McNAMEE*~ Departments of *Biochemistry and Biophysics, //Avian Sciences, fEntomology, and liEnvironmenta1 Toxicology, University of California, Davis, CA 95616, U.S.A. (Receioed 8 Janunry 1990; accepted 8 June 1990) Abstract-Neuropathy target esterase (NTE) is a membrane-bound carboxylesterase activity which is proposed as the target site in nerve tissue for initiation of organophosphate-induced delayed neuropathy. This activity is identified as phenyl valerate hydrolysis which is resistant to treatment with paraoxon and sensitive to co-incubation with paraoxon and mipafox. NTE preparations were obtained, which did not contain paraoxon-sensitive or mipafox-resistant hydrolases, by selective reconstitution of detergent-solubilized NTE from chick embryo brain into asolectin vesicles during gel filtration. The topography of the catalytic site of NTE was then examined by investigating the inhibition of NTE by a series of 3-alkylthio- and 3-arylthio-l,l,l-trifluoro-propan-2-ones. These trifluoromethyl ketones were found to be rapidly reversible, competitive inhibitors of NTE with I,, values from 1.3 X 1Om4 M to 4.9 x lo-” M. Correlation of Is<, values with octanol/water partition coefficients (Pi. in the range of log P = 1.5 to 5.9, indicated that the optimal lipophilicity for NTE substrates and inhibitors is in the range of log P = 3.0 to 3.4. Electrophilic substitution at the meta position of aromatic rings increased the inhibitory capacity of these inhibitors, whereas substitution at the orrho position reduced inhibitory capacity. These results indicate both that a large hydrophobic pocket is closely associated with the catalytic residue of NTE, and that affinity for the active site is affected by steric and electronic parameters. Neuropathy target esterase (NTE,** also known as inhibitors has been examined in two previous studies neurotoxic &erase) is an integral membrane protein [ll, 12]. In excess of 150 different phosphates, found predominantly in central and peripheral nerve phosphonates, phosphinates, carbamates, and sul- tissue [l-3]. NTE is proposed as the target site at fates were examined. Analysis of the results involved which certain organophosphates (OPs) such as the examination of a homologous series of diisopropyl phosphoro~uoridate (DFP) and di- compounds, but attempts were not made to isopropylphosphorodiamidic Auoride (mipafox) act quantitatively relate observed trends to the physico- to initiate organophosphate-induced delayed neur- chemical parameters of the compounds. Although a opathy (OPIDN) [4-71. The symptoms of this strong correlation between structure and activity was potentially irreversible neuropathy appear 2-3 weeks not identified, the results indicated that both after a single acute exposure to OPs and are hydrophobic and steric parameters were important. accompanied by a distal degeneration of the long, It has been reported previously that trifluoromethyl large diameter peripheral nerves and the distal ends ketones are potent inhibitors of a large number of of the ascending and descending tracts of the spinal serine active site hydrolases 113-151, including human cord [&lo]. Determination of the role of NTE in brain NTE (Talcott, Ketterman and Lotti, personal the proper function of the nerve axon will require communication to B.D.H.). The substitution of the both the purification of NTE and the characterization hydrogen atoms LYto the carbonyl group with of its physical and catalytic properties. fluorines enhances the reactivity of the carbonyl with The relationship between the structures and the serine in the catalytic site [ 131. and it is postulated inhibitory potencies of a wide range of NTE that these compounds behave as transition state analogs [16]. In the present study we investigated the interaction of NTE with a series of twenty-two $ On leave from the Plant Protection Institute of the aliphatic and aromatic trifluoromethyl ketone Hungarian Academy of Sciences, H-1525 Budapest POB sulfides. The Iso values for inhibition of NTE by 102, Hungary. each of these compounds were determined, and an 1 Author to whom all correspondence should be addres- analysis of quantitative structure-activity relation- sed. ships (QSAR) was performed based on the ** Abbreviations: NTE, neuropathy target esterase; physicochemical properties of each inhibitor. The OPIDN, organophosphate-induced delayed neuropathy; purpose of this analysis was 3-fold: (1) to characterize OTFP, 3-octyIthio-l,l,l-t~fluoropropan-2-one; TFK; tri- fluoromethyl ketone; mipafox, N,N’-diisopropylphospho- the active site of NTE, (2) to provide information rodiamidic fluoride; DFP, diisopropyl phosphorofluori- for the synthesis of more specific substrates and date; paraoxon, phosphoric acid diethyl 4nitrophenyl inhibitors, and (3) to identify appropriate ligands ester; P, octanol/water partition coefficient; and EGTA, for affinity chromatography of NTE. ethyleneglycoibis(aminoethylether)tetra-acetate. In addition, we report a method for obtaining an 2587 2588 ‘I-. c. ‘rHOMAS et al NTE preparation which is free from contamination the following protocol. Samples (lC-50 pL) were by non-NT&type phenyl valerate hydrolases. incubated for 20 min at 37” in an appropriate volume Operationally, NTE is identified as that portion of of 50 mM Tris-HCl, 0.2 mM EDTA, pH 8.0, and an total phenyi valerate hydrolyzing activity which is inhibitor solution such that the finai volume was resistant to pretreatment with non-neuropathic 0.74mL. The inhibitor solution was one of the paraoxon (40 kcM), but sensitive to pretreatment following: (a) 5OpL of above buffer; (b) 5OpL of with paraoxon and neuropathic mipafox (50(*M) 0.6 mM paraoxon in 50 mM Tris-citrate, pH 6.0; (c) [17]. Therefore, to determine NTE activity, pairs of 50 PL of 0.75 mM mipafox in 50 mM Tris-citrate, differentially inhibited samples must be assayed. pH 6.0; or (d) 50 PL each of the above paraoxon Protocols to characterize the interaction of NTE and mipafox solutions. Ten microliters of phenyl with other inhibitory compounds must confront two valerate (12.5 mg PV/mL in dimethylformamide) potential problems. First, the catalytic sites of NTE was added and tubes were incubated for 30min at may be less than 50% saturated with substrate during 37”. Enzymatic hydrolysis was stopped by adding assay [18, 191, in which case, nonbound paraoxon 0.5 mL of 1% (w/v) SDS, 0.25% (w/v) 4- and mipafox may compete with the substrate or the aminoantipyrine, 50 mM Tris-HCl, 0.2 mM EDTA, inhibitor of interest for interaction with hydrolytic pH 8.0. Then the colored complex was developed sites. Second, if nonbound paraoxon and mipafox by adding 0.25 mL of 0.4% (w/v) K3Fe(CN)h. The are removed prior to addition of test compounds absorbance of each assay tube was determined in and substrate, then reversibly inhibited hydrolases triplicate using a Bio-Tek 96-well plate reader may be reactivated. Evidence identifying the (Winooski, VT) by addition of 3OOpL from each existence of such a hydrolase in chick embryo brain tube to each of three wells of a Falcon 96-well plate membrane fractions is presented. The problems (Becton-Dickinson, Oxnard, CA). Absorbance was associated with these methods were avoided in the determined at 490 nm and the concentration of present study by screening inhibitors against an phenol was calculated using an absorption coefficient NTE-type phenyl valerate hydrolase preparation equal to l6,812M-‘cm-‘. Units of activity are from which all paraoxon-sensitive and mipafox- reported as micromoles of phenol produced per resistant activities had been removed. minute (I.U.). Protein determinations. Protein was quantitated using the method of Lowry et al. [25] with bovine MATERIALS AND METHODS albumin (Fraction V, Sigma, St. Louis, MO) as a standard. To avoid the interference caused by C~e~~c~~.~. Ahphatic and aromatic 3-substituted precipitation of Triton X-100, 0.1 mL of 24% (w/v) thio-l,l,l-tri~uoropropanones (TFKs) used in this sodium dodecyl sulfate (SDS) was added to samples study were synthesized previously by the reaction containing Triton X-100 (final assay vol. 1.3 mL) of the appropriate thiol with 3-bromo-l,l,l- WI. trifluoropropan-2-one, purified, and analyzed Preparation of microsomal membranes from [l&20]. Mipafox (N,N’-diisopropylphosphoro- chicken embryo brains. Brains from day 19 chicken diamidic fluoride) and phenyl valerate were embryos were homogenized for 25 set in 10 mL of synthesized as previously described [21] according ice-cold Buffer A (50 mM Tris-HCl, 0.5 M NaCl, to the methods of Johnson [17]. The purity of 2 mM EDTA, 2 mM EGTA, pH 7.2, at 21”) per g mipafox was monitored by determination of its of tissue with a Polytron homogenizer on setting 7 melting point (60.5”, white crystals), and the (Brinkmann Instruments, Westbury, NY). Homo- structures of mipafox and phenyl valerate were genates were centrifuged in an SS34 rotor (Sorvall, analyzed by NMR and i.r. Paraoxon (phosphoric Wilmington, DE) for 10 min at 4” and 1100~ (rav acid diethyl 4-nitrophenyl ester, Aldrich Chemical 8.26 cm). The upper layer of foam was aspirated and Co., Milwaukee, WI) was analyzed for interfering the supernatant was isolated. The low speed contaminants by thin-layer chromatography and gas supernatant was centrifuged in a Type 60 Ti rotor phase chromatography, and by determination of its (Beckman, Palo Alto, CA) for 20 min at 4” and Isa to NTE (600pM) as recommended by Johnson 100,OOOg (rsv 6.15 cm) to obtain a crude microsomal [22]. Stock solutions of mipafox (10-l M) in 50 mM membrane fraction. Membrane pellets were resus- Tris-citrate, pH 6.0 and paraoxon (10-l M in acetone) pended in Buffer B (50mM Tris-HCl, 0.2mM were prepared and stored in a desiccator at -25”.