FDA-approved drugs and other compounds tested as inhibitors of human glutathione transferase P1-1.

OBJECTIVE: Glutathione transferase P1-1 (GST P1-1) is often overexpressed in tumor cells and is regarded as a contributor to their drug resistance. Inhibitors of GST P1-1 are expected to counteract drug resistance and may therefore serve as adjuvants
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  FDA-approved drugs and other compounds tested as inhibitors of humanglutathione transferase P1-1 Yaman Musdal a,b , Usama M. Hegazy a,c , Yasemin Aksoy b , Bengt Mannervik a,d, ⇑ a Department of Chemistry-BMC, Uppsala University, Box 576, SE-75123 Uppsala, Sweden b Department of Biochemistry, Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey c Molecular Biology Department, Genetic Engineering and Biotechnology Division, National Research Centre, Dokki, 12311 Cairo, Egypt  d Department of Neurochemistry, Stockholm University, SE-10691 Stockholm, Sweden a r t i c l e i n f o  Article history: Received 5 April 2013Received in revised form 25 May 2013Accepted 3 June 2013Available online 13 June 2013 Keywords: Glutathione transferase P1-1FDA-approved drugsEnzyme inhibitionAdjuvant chemotherapeuticsEthacrynic acid a b s t r a c t Objective:  Glutathione transferase P1-1 (GST P1-1) is often overexpressed in tumor cells and is regardedasacontributortotheirdrugresistance.InhibitorsofGSTP1-1areexpectedtocounteractdrugresistanceand may therefore serve as adjuvants in the chemotherapy of cancer by increasing the efficacy of cyto-static drugs. Finding useful inhibitors among compounds used for other indications would be a shortcuttoclinical applications andasearch for GSTP1-1inhibitors amongapproved drugs andother compoundswas therefore conducted. Methods:  We tested 1040 FDA-approved compounds as inhibitors of the catalytic activity of purifiedhuman GST P1-1 in vitro. Results:  We identified chlorophyllide, merbromine, hexachlorophene, and ethacrynic acid as the mosteffective GST P1-1 inhibitors with IC 50  values in the low micromolar range. For comparison, these com-pounds were even more potent in the inhibition of human GST A3-3, an enzyme implicated in steroidhormone biosynthesis. In distinction from the other inhibitors, which showed conventional inhibitionpatterns, thecompetitive inhibitor ethacrynic acidelicited strong kinetic cooperativity inthe glutathionesaturation of GST P1-1. Apparently, ethacrynic acid serves as an allosteric inhibitor of the enzyme. Conclusion and practical implications:  In their own right, the compounds investigated are less potent thandesired for adjuvants in cancer chemotherapy, but the structures of the most potent inhibitors couldserve as leads for the synthesis of more efficient adjuvants.   2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Glutathione transferases (GSTs; EC are a group of detoxication enzymes, involved in the biotransformation of xeno-biotic and endogenously produced compounds [1]. They catalyzethe conjugation of reduced glutathione (GSH) to electrophilicatoms (C, N or S) of various nonpolar compounds assisted by low-ering the pKa of thiol group of GSH from9.2 in solution to below7in the active site of the enzyme [2]. Besides their role in detoxica-tion,theyareinvolvedinisomerizationreactionsleadingtosteroidbiosynthesis [3], cell signaling [4], and as ‘‘ligandins’’ in intracellu- lar transport of steroids, heme and bilirubin [5].In human cells there are seven classes of soluble cytosolic GSTs(termed alpha, mu, pi, sigma, zeta, omega, and theta) occurring indimericforms[6].Oneoftheenzymes,GSTP1-1,firstisolatedfromerythrocytes [7] and placenta [8,9] is of particular interest since it is expressed at high levels in many tumor cell lines [10,11]. Itspresence in drug-resistant tumors such as human malignant mel-anoma [12] and murine osteosarcoma [13] suggested that GST P1-1 could contribute to the resistance phenotype. Overexpressionof GST P1-1 is often associated with progression of drug resistance[14,15].Theroleoftheenzymeindrugresistancehasconsequentlybeen investigated by numerous investigators [16] as a promisingtherapeutic target in the treatment of malignancies. One approachto overcome this resistance phenotype is to discover and maketherapeutic use of novel GST P1-1 inhibitors.A variety of GST P1-1 inhibitors are described in the literature,such as the diuretic drug ethacrynic acid (EA) and EA derivatives[17], GSH analogs [18,19], polyphenolic compounds [20], and anti- malarial drugs [21]. Chlorambucil and EA, in addition to theirinhibitory effects, also act as substrates for GST P1-1 [22,23]. EAenhances the cytotoxicity of alkylating chemotherapeutic drugsin cells that are resistant to these drugs [24,25] and has thereforebeensubjectedtomoredetailedinvestigations.Nevertheless,therearecontradictoryreportsabouthowEAworksandinhibitsGSTP1-1 [23,26,27]. 0009-2797/$ - see front matter    2013 Elsevier Ireland Ltd. All rights reserved. ⇑ Corresponding author at: Department of Neurochemistry, Stockholm Univer-sity, SE-10691 Stockholm, Sweden. Tel.: +46 08 164196 ; fax: +46 08 161371. E-mail address: (B. Mannervik).Chemico-Biological Interactions 205 (2013) 53–62 Contents lists available at SciVerse ScienceDirect Chemico-Biological Interactions journal homepage:  The US Drug Collection is a set of 1040 FDA-approved com-pounds containing a wide variety of compounds including chemo-therapeutics, herbicides, antibacterial, antifungal, and anti-inflammatory compounds, etc. In the search for novel inhibitorsthis set was screened as inhibitors of human GST P1-1, and furtherkinetic studies were performed with selected compounds. Theinhibition characteristics of the most potent inhibitors chlorophyl-lide, merbromin, hexachlorophene, and EA were compared. 2. Materials and methods  2.1. Materials The US Drug Collection set comprising 1040 compounds waspurchasedfromMicroSourceDiscoverySystems, Inc. andthe otherchemicals were purchased from Sigma–Aldrich or Merck.  2.2. Protein expression and purification Recombinant human GST P1-1 and GST A3-3 were expressed inEscherichiacoli strainXL-1Blue andRosetta(DE3), respectively, inthe presence of 0.2mMisopropyl-1-thio- b -D-galactopyranoside at37  C as described by Kolm et al. [28]. The cells were harvested bycentrifugation, resuspended in ice-cold lysis buffer [1mg/ml lyso-zyme in 10mM Tris/HCl (pH 7.8 for P1-1 and pH 8.4 for A3-3),1mM EDTA, 10mM 2-mercaptoethanol, 0.1% Triton X-100 (forA3-3) and Complete™ protease inhibitors], disrupted by ultrason-ication and the cell debris were removed by centrifugation at20,000  g   for 45min. GST P1-1 and GST A3-3 were purified in a sin-gle chromatographic step by affinity chromatography using S-hex-ylglutathione-Sepharose 6B as affinity matrix [29]. GST P1-1 waseluted with 5mM S-hexylglutathione in 10mM Tris–HCl (pH 7.8)containing 0.2M NaCl, 0.2mM dithiothreitol, 1mM EDTA and0.02% (w/v) NaN 3  and dialyzed against the same elution bufferwithout S-hexylglutathione at 4  C. By contrast, GST A3-3 waseluted with 50mM glycine-NaOH (pH 10), fractions of 2ml werecollected into tubes already containing 0.2ml of 2M Tris/HCl (pH7.2) in order to lower the pH immediately after elution and theprotein-containing pool was dialysed against 10mM Tris/HCl (pH7.8) containing 5mM 2-mercaptoethanol at 4  C. A total yield of 40mg GST P1-1 and 92mg A3-3 per liter culture were obtained.The purity of enzyme was analyzed by polyacrylamide gel electro-phoresis (SDS–PAGE) and isoelectric focusing using precast gels(Pharmacia LKB PhastSystem). The stock solutions of purified GSTP1-1 and A3-3 containing 5.47 and 47mg/ml, respectively, weredivided into 20 l l aliquots and stored at   80  C.2.3.  GST activityassays. GST activity was measured spectrophotometrically by measur-ing the initial rate of absorbance change at 340nm as describedby Habig et al. [30]. Standard enzymatic assays were performedin 0.1M phosphate buffer, pH 6.5 containing 1mM EDTA in thepresence of 1mM GSH and 1mM 1-chloro-2,4-dinitrobenzene(CDNB) at 30  C. The molar absorption coefficient for CDNB is D e 340  =9.6mM  1 cm  1 . Correction for the spontaneous nonenzy-matic reaction was obtained by subtracting the reaction rate of GSHwithCDNBintheabsenceofenzymefromtherateinthepres-ence of enzyme.  2.3. Inhibition studies Preliminary screening of the US Drug Collection for GST inhibi-tion was performed in 96-wells microtiterplates monitored photo-metrically in Spectramax PLUS 384 (Molecular Devices). The USDrug Collection comprised compounds dissolved at 10mM con-centration in DMSO. In all enzyme assays the DMSO concentrationwas 1% (v/v), which gave negligible inhibition of enzyme activity.IC 50  values of the most potent inhibitors were determined withCDNBas electrophilicsubstrate ona UV-2501PC(Shimadzu) spec-trophotometerinthestandardassayusingaseriesofinhibitorcon-centrations. TheIC 50  valueis definedastheinhibitorconcentrationthatgives50%inhibitionoftheenzymaticactivity.Fordeterminingthe inhibition type and  K  i  values of compounds, one of the sub-strates (GSH or CDNB) was kept at near-saturating concentrationswhile the concentration of the other substrate and the inhibitorwere varied. In all the experiments 12 nM of GST P1-1 and 25nM of GST A3-3 (calculated as monomer concentration) was usedin the assays.  2.4. Data analysis All measurements weremadeintriplicateandeachpointinthegraphsis givenasmeanandstandarderrorofthemean(SEM).IC 50 and  K  i  values of the compounds were determined by using Graph-pad Prism 4.0 software. IC 50  values were obtained by using thenonlinear regression analysis-one site competition option of thesoftware by fitting the relationship between the log 10  of at leastsixdifferentinhibitorconcentrationsversusthepercentageofinhi-bition. Forobtainingthe K  i  values,thesuitablerateequationfittingthe data was selected from the nonlinear analysis, comprising theoptions competitive, noncompetitive, uncompetitive or linearmixed inhibition. 3. Results  3.1. Screening the inhibitory effect of US drugs library compounds TheUSDrugCollectioncompoundswerescreenedforinhibitionof the GST P1-1 enzyme activity. The preliminary screening wasdone at 33 l M compound concentration. The most effective 49compoundswereselected,26oftheseshowedmorethan70%inhi-bition, while the rest of these showed 20–70% inhibition (Supple-mentary Table 1). In addition, a ranking of the less potentinhibitors was made (Supplementary Table 2). The most potent26 compounds were tested further at 3.3 l M concentration. Thepercent inhibition and the structure of the potent inhibitors areshown in Table 1. A complete list of all substances tested is pro-vided in Supplementary Table 3. The most potent nine compoundswere chosen for determination of the IC 50  values (Table 2). Chloro-phyllide, merbromin, EA, and hexachlorophene were the most po-tentinhibitorsofGSTP1-1withIC 50 values2.3 l M,3.1 l M,4.9 l M,and 9.7 l M, respectively. To evaluate enzyme specificity of inhibi-tors, the selected 49 compounds were also screened for inhibitionof GST A3-3 in four different concentrations (0.16, 0.33, 0.66 and3.3 l M). As compared with GST P1-1, chlorophyllide, merbromin,EA and hexachlorophene were found more potent toward GSTA3-3 with the IC 50  values 0.33 l M, <0.66 l M,   0.4 l M, and<0.16 l M, respectively. Although GST P1-1 was not inhibited withbithionate sodium, nisoldipine, flufenamic acid and sanguinarinesulfate, these compounds were highly potent inhibitors for GSTA3-3 as shown by their IC 50  values (Table 2).  3.2. Kinetic inhibition parameters The  K  i  values and inhibition type characterization of the mostpotent inhibitors were determined with respect to GSH and CDNBas varied substrates (Table 3). Chlorophyllide, the most potentinhibitor,showsmixedinhibitionwithrespecttoGSHandcompet-itiveinhibitionagainstCDNBwiththe K  i  values1.7 l Mand4.2 l M,respectively (Fig. 1). Merbromin, the second most effective inhibi- 54  Y. Musdal et al./Chemico-Biological Interactions 205 (2013) 53–62  tor, is a competitive inhibitor for GSH with the  K  i  value 0.73 l M,and a noncompetitive inhibitor against CDNB with the  K  i  value20 l M (Fig. 2). By marked contrast, inhibition studies with EA shows apronounceddeviationfromlinearitywithvaryingconcen-tration of GSH at 10 and 30 l MEA, while there is no obvious devi-ation from linearity when EA is absent or with varyingconcentration of CDNB under GSH-saturation condition [31]. EAshows nonlinear competitive inhibition [32] with GSH and linearnoncompetitive inhibition with CDNB. The  K  i  value of EA is13.8 l Mfor CDNB, but no value for varying GSH could be obtained  Table 1 Structure and percent inhibition of GST P1-1 by the most potent compounds at 3.3 l M. Compound Inhibition (%) StructureChlorophyllide 51Merbromin 44Chloramphenicol palmitate 38Hexachlorophene 34Ethacrynic acid 25Meclizine hydrochloride 25 ( continued on next page ) Y. Musdal et al./Chemico-Biological Interactions 205 (2013) 53–62  55  duetothenonlineareffect. Inthelattercasetheexperimental datawere adequately fit by a rational 2:2 function in GSH concentra-tion, but the parameters obtained by regression analysis have noobvious meaning in the absence of an underlying kineticmechanism.  3.3. Double inhibition experiments It is known that two inhibitors added simultaneous can showmutual interactions in binding to an enzyme [33–35]. Fig. 3 shows Yonetani-Theorell plots of double inhibition experiments with EA  Table 1  ( continued ) Compound Inhibition (%) StructureMitotane 25Fenofibrate 24Iodoquinol 24Amphotericin B 23Anthralin 22Clofazimine 2256  Y. Musdal et al./Chemico-Biological Interactions 205 (2013) 53–62   Table 1  ( continued ) Compound Inhibition (%) StructureErgotamine tartrate 21Avobenzone 19Clotrimazole 17Temefos 17Estradiol valerate 15Sanguinarine sulfate 15Nisoldipine 14Atovaquone 12 ( continued on next page ) Y. Musdal et al./Chemico-Biological Interactions 205 (2013) 53–62  57
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