Key points are not available for this paper at this time.
Spectroscopic and rapid kinetic experiments were performed to detail the interaction of human glutathione S-transferases GSTA1-1, GSTM2-2, and GSTP1-1 with 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX). This compound is a representative molecule of a new class of 7-nitro-2,1,3-benzoxadiazole (NBD) derivatives (non-GSH peptidomimetic compounds) that have been designed both to give strong GST inhibition and to accumulate in tumor cells avoiding the extrusion mechanisms mediated by the multidrug resistance protein pumps. We have recently shown that submicromolar amounts of NBDHEX trigger apoptosis in several human tumor cell lines through the dissociation of the JNK·GSTP1-1 complex (Turella, P., Cerella, C., Filomeni, G., Bullo, A., De Maria, F., Ghibelli, L., Ciriolo, M. R., Cianfriglia, M., Mattei, M., Federici, G., Ricci, G., and Caccuri, A. M. (2005) Cancer Res. 65, 3751-3761). Results reported in the present study indicated that NBDHEX behaves like a suicide inhibitor for GSTs. It bound to the H-site and was conjugated with GSH forming a σ complex at the C-4 of the benzoxadiazole ring. This complex was tightly stabilized in the active site of GSTP1-1 and GSTM2-2, whereas in GSTA1-1 the release of the 6-mercapto-1-hexanol from the σ complex was the favored event. Docking studies demonstrated the likely localization of the σ complex in the GST active sites and provide a structural explanation for its strong stabilization. Spectroscopic and rapid kinetic experiments were performed to detail the interaction of human glutathione S-transferases GSTA1-1, GSTM2-2, and GSTP1-1 with 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX). This compound is a representative molecule of a new class of 7-nitro-2,1,3-benzoxadiazole (NBD) derivatives (non-GSH peptidomimetic compounds) that have been designed both to give strong GST inhibition and to accumulate in tumor cells avoiding the extrusion mechanisms mediated by the multidrug resistance protein pumps. We have recently shown that submicromolar amounts of NBDHEX trigger apoptosis in several human tumor cell lines through the dissociation of the JNK·GSTP1-1 complex (Turella, P., Cerella, C., Filomeni, G., Bullo, A., De Maria, F., Ghibelli, L., Ciriolo, M. R., Cianfriglia, M., Mattei, M., Federici, G., Ricci, G., and Caccuri, A. M. (2005) Cancer Res. 65, 3751-3761). Results reported in the present study indicated that NBDHEX behaves like a suicide inhibitor for GSTs. It bound to the H-site and was conjugated with GSH forming a σ complex at the C-4 of the benzoxadiazole ring. This complex was tightly stabilized in the active site of GSTP1-1 and GSTM2-2, whereas in GSTA1-1 the release of the 6-mercapto-1-hexanol from the σ complex was the favored event. Docking studies demonstrated the likely localization of the σ complex in the GST active sites and provide a structural explanation for its strong stabilization. Glutathione S-transferases (GSTs, 1The abbreviations used are: GST, glutathione S-transferase; CDNB, 1-chloro-2,4-dinitrobenzene; NBD, 7-nitro-2,1,3-benzoxadiazole; NBD-Cl, 4-chloro-7-nitro-2,1,3-benzoxadiazole; JNK, c-Jun N-terminal kinase; NBDHEX, 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol; GS-NBD, (7-nitro-2,1,3-benzoxadiazol-4-ylthio)glutathione; HPLC, high pressure liquid chromatography; SVD, singular value decomposition. EC 2.5.1.18) are a superfamily of dimeric enzymes present in human tissues and subdivided in at least eight gene-independent classes named Alpha, Pi, Mu, Theta, Zeta, Omega, Sigma, and Kappa (1Mannervik B. Alin P. Guthenberg C. Jensson H. Tahir M.K. Warholm M. Jornvall H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7202-7206Crossref PubMed Scopus (1076) Google Scholar, 2Meyer D.J. Coles B. Pemble S.E. Gilmore K.S. Fraser G.M. Ketter B. Biochem. J. 1991; 274: 409-414Crossref PubMed Scopus (748) Google Scholar, 3Buetler T.M. Eaton D.L. Environ. Carcinog. Ecotoxicol. Rev. 1992; 10: 181-200Crossref Scopus (175) Google Scholar, 4Meyer D.J. Thomas M.R. Biochem. J. 1995; 311: 739-742Crossref PubMed Scopus (142) Google Scholar, 5Pemble S.E. Wardle A.F. Taylor J.B. Biochem. J. 1996; 319: 749-754Crossref PubMed Scopus (264) Google Scholar, 6Board P.G. Baker R.T. Chelvanayagam G. Jermiin L.S. Biochem. J. 1997; 328: 929-935Crossref PubMed Scopus (483) Google Scholar, 7Board P.G. Coggan M. Chelvanayagam G. Easteal S. Jermiin L.S. Schulte C.K. Danley D.E. Hoth L.R. Griffor M.C. Kamath A.V. Rosner M.H. Chrunyk B.A. Perregaux D.E. Gabel C.A. Georghegan K.F. Pandit J. J. Biol. Chem. 2000; 275: 24798-24806Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar). The design or discovery of efficient compounds that may bind to these enzymes modulating their biological activity has become one of the primary aims in cancer research because GST isoenzymes such as GSTP1-1 are overexpressed in many cancer cell lines (8Tew K.D. Monks A. Barone L. Rosser D. Akerman G. Montali J.A. Wheatley J.B. Schmidt D.E. Mol. Pharmacol. 1996; 50: 149-159PubMed Google Scholar) and induce drug resistance by inactivating many chemotherapeutic compounds via GSH conjugation (9Schultz M. Dutta S. Tew K.D. Adv. Drug Deliv. Rev. 1997; 26: 91-104Crossref PubMed Scopus (127) Google Scholar). GSTP1-1 displays an additional antiapoptotic activity based on a protein-protein interaction with c-Jun N-terminal kinase (JNK), a key enzyme in the apoptotic cascade (10Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (962) Google Scholar). Many efforts have been made in the last years to find tight inhibitors of these enzymes to reduce their protective role in vivo (9Schultz M. Dutta S. Tew K.D. Adv. Drug Deliv. Rev. 1997; 26: 91-104Crossref PubMed Scopus (127) Google Scholar). Ethacrynic acid, an inhibitor that lacks class specificity for GSTs, represented a first attempt in this direction; however, its scarce affinity and the deleterious side effects have discouraged its use in clinical practice (9Schultz M. Dutta S. Tew K.D. Adv. Drug Deliv. Rev. 1997; 26: 91-104Crossref PubMed Scopus (127) Google Scholar). Taking advantage of the strong specificity of the transferase G-site for GSH and GSH derivatives, a few GSH peptidomimetic compounds have been designed recently. For example, γ-glutamyl-S-(benzyl)-cysteinyl-R(-)-phenylglycine diethyl ester (TER199) acts as proinhibitor for GSTs. It rapidly enters the cells and is activated by the intracellular esterases. In the active form, it selectively inhibits GSTP1-1, enhancing the effect of alkylating drugs in various cancer cell lines that overexpress this isoenzyme (11Morgan A.S. Ciaccio P.J. Tew K.D. Kauvar L.M. Cancer Chemother. Pharmacol. 1996; 37: 363-370Crossref PubMed Scopus (129) Google Scholar). This specific compound is under investigation for possible use in human therapy, but other GSH derivatives that are good inhibitors of GSTs may not be so efficient in cancer cells. In fact many GSH derivatives are actively extruded from the cell by specific export pumps, such as the multidrug resistance protein, thus avoiding their intracellular accumulation (12Muller M. Meijer C. Zaman G.J. Borst P. Scheper R.J. Mulder N.H. de Vries E.G. Jansen P.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 13033-13037Crossref PubMed Scopus (640) Google Scholar, 13Zaman G.J. Cnubben N.H. van Bladeren P.J. Evers R. Borst P. FEBS Lett. 1996; 391: 126-130Crossref PubMed Scopus (75) Google Scholar). Multidrug resistance protein activity represents a crucial problem for GST inhibition as the estimated concentration of GSTP1-1 in cancer cells may reach 0.05 × 10-3 m, and an equivalent or higher concentration of inhibitor must accumulate in the cell to modulate efficiently the GST activity. To overcome this problem, we have tried to design new molecules that bind efficiently to GSTs but are not GSH derivatives and display suitable lipophilic properties to be able to cross the cell membrane. Besides the G-site, which specifically recognizes GSH and its derivatives, a second binding subsite is present in all GSTs that is able to interact with a lot of different hydrophobic co-substrates. This subsite, termed H-site, is a hydrophobic cavity, located near the G-site, that displays different topography and substrate specificity in GSTA1-1, GSTM2-2, and GSTP1-1. As GSTs evolved to bind many different hydrophobic toxic species, the H-site normally displays moderate affinity for these compounds, i.e. Km values in the millimolar range. A few years ago, we found that 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) is an unusual co-substrate for GSTs showing a Km value in the micromolar range (14Caccuri A.M. Ascenzi P. Antonini G. Parker M.W. Oakley A.J. Chiessi E. Nuccetelli M. Battistoni A. Bellizia A. Ricci G. J. Biol. Chem. 1996; 271: 16193-16198Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). We therefore started from this evidence to design specific derivatives of NBD-Cl that are the object of the present study. We have recently shown that very low levels of NBD thioethers can induce tumor cell death through dissociation of the JNK·GSTP1-1 complex (15Turella P. Cerella C. Filomeni G. Bullo A. De Maria F. Ghibelli L. Ciriolo M.R. Cianfriglia M. Mattei M. Federici G. Ricci G. Caccuri A.M. Cancer Res. 2005; 65: 3751-3761Crossref PubMed Scopus (106) Google Scholar). The NBD derivatives represent an interesting class of GST inhibitors as they interact with the H-site with KD values in the micromolar range for GSTA1-1 and GSTP1-1 and in the nanomolar range for GSTM2-2. A detailed investigation of the binding mechanism of a representative molecule revealed that GSTs promote the formation in the active site of a σ complex between the inhibitor and GSH. Thus, these NBD derivatives act as suicide inhibitors and may represent a new type of potent antitumor agents. Materials—GSH, 1-chloro-2,4-dinitrobenzene (CDNB), NBD-Cl, 6-mercapto-1-hexanol, 4-mercapto-1-butanol, 2-mercaptoimidazole, 3-mercapto-1-propionic acid, 6-amino-1-hexanol, 3-bromopyruvate, RPMI 1640 medium, fetal calf serum, l-glutamine, and were from protein was from and of the NBD and 6-mercapto-1-hexanol were for in of a of and in a at The was and by suitable of the of the the of 6-mercapto-1-hexanol was with of The is a compound that was by and with of The was via The was by a at and the a at The of NBDHEX was on a in The and The of NBDHEX was under an was from 4-mercapto-1-butanol, 2-mercaptoimidazole, 3-mercapto-1-propionic acid, 6-amino-1-hexanol, and GSH a was used for the of and with to the and they were as via and and cell cell by M. Cianfriglia were in RPMI 1640 with fetal calf serum, l-glutamine, and in a at NBDHEX was in the was to the concentration in RPMI 1640 cell with a concentration not In all experiments were to the of cell at different drug was by the P. R. D. Monks A. J. D. H. S. M.R. J. Natl. Cancer 82: PubMed Scopus Google Scholar). The cells were in at a of × in of the cells were to NBDHEX at the concentration and to for the cell was by an in cell by a with B. The the value for NBDHEX concentration used to cell The of apoptosis by NBDHEX was in and cell lines with × was with a by the with the GST GSTM2-2, and GSTP1-1 were in and as M. Battistoni A. P.G. M. Federici G. Ricci G. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). concentration was the protein GST activity was at as reported PubMed Scopus Google Scholar). The × 10-3 × 10-3 CDNB, and × 10-3 in of The activity was at the of from of the of GST is as the of enzyme that the formation of of at of GSTs by NBD derivatives was × GSTA1-1 or GSTP1-1 or × GSTM2-2. The concentration that inhibits of values were in the of various amounts of the NBD The inhibition mechanism with NBDHEX was from of the enzyme activity at different GSH and and in the of different amounts of of NBDHEX to of NBDHEX for human GSTs were in the or of × 10-3 GSH. In a the of the of the protein at and at was in a S. A. at the of amounts of NBDHEX to × GSTs in were both for and for effects and to which the KD value for the NBDHEX bound to GSTs, is the protein in the of a of NBDHEX, and is the at NBDHEX is the and between NBDHEX and GSH was in the of GSTs by at the of NBDHEX × in in the of different of GSH × 10-3 to × 10-3 The dissociation of the complex was from the at the of GSH. The were to and are by and of the was of the with the of NBDHEX × in × 10-3 GSH was at and the of amounts of GSTP1-1 or GSTM2-2, and the of the was by of this with GSTA1-1, the of NBDHEX × was both at and at in the of amounts of the enzyme in × 10-3 GSH. The of × NBDHEX at in × 10-3 GSH was at and the of × GSTA1-1, GSTM2-2, or GSTP1-1. kinetic experiments were performed on an kinetic with a In the of GSTs, amounts of GSH × 10-3 to × 10-3 were rapidly with an of NBDHEX × 10-3 in in the of GSTs were performed by rapidly the GST × in × 10-3 with an of NBDHEX × to × in the were on a to were from and by the different a and were of was performed by of the on an by singular value in with The of is by the of A × × the are the and their is represented by the The values of the the of the in the a is of one of the they have different can be by the to the kinetic of the to kinetic performed the P. Google Scholar, P. Biochem. Sci. 1997; Full Text PDF PubMed Scopus Google Scholar, P. PubMed Scopus Google were by of at of the to the kinetic The of the of the of the can be used to the of the of the J. 1992; Scopus Google Scholar, J. Biochem. 1994; PubMed Scopus Google Scholar, G. A. M. G. Biochem. J. 1996; PubMed Scopus Google Scholar). Docking were performed with the G.M. R. A.J. J. Chem. Scopus Google Scholar). For the was from the and for GSTA1-1, GSTM2-2, and GSTP1-1, were and C. G. S. P. J. Chem. Scopus Google Scholar) were Docking studies of the σ complex were performed with a but the of of the GSH were to the values from the were by the J. M. Scopus Google Scholar). of were in a on the of the side in GSH and in the GSTA1-1, GSTM2-2, and GSTP1-1, Docking was performed the with and a of G.M. R. A.J. J. Chem. Scopus Google and a of and The were to their binding and a of The R. M. J. Mol. 1996; PubMed Scopus Google Scholar) was used for and of of GSTs by NBD NBD derivatives in were as reported under and as possible inhibitors for GSTA1-1, GSTM2-2, and GSTP1-1, representative enzymes of human Alpha, Mu, and the NBD derivatives were strong inhibitors of GSTs showing values in the nanomolar range for and in the micromolar range for GSTP1-1 and GSTA1-1 the of the to be an for tight In all GSTs for which is a structural of NBDHEX but with the by an on GSTs in a new all these compounds, NBDHEX the value GSTP1-1, the isoenzyme overexpressed in cancer cell lines (8Tew K.D. Monks A. Barone L. Rosser D. Akerman G. Montali J.A. Wheatley J.B. Schmidt D.E. Mol. Pharmacol. 1996; 50: 149-159PubMed Google Scholar). This molecule was therefore to detail the interaction of these compounds with human GSTs. The inhibition mechanism of NBDHEX was with GSTP1-1 and GSTA1-1 by GSH or with was not possible to its very high affinity GSTP1-1 and GSTA1-1 NBDHEX like a inhibitor in to that this compound to the hydrophobic of the active site In to GSH NBDHEX a type that NBDHEX with the H-site of GSTs but that the G-site is not of NBDHEX on effect of NBDHEX was on and tumor cell The values were × × and × for and cell of apoptosis by NBDHEX was by with of and cells with NBDHEX and in and which represent the of apoptosis L. S. Ghibelli L. Res. 1996; PubMed Scopus Google Scholar). the values for these cell lines were of the of as the value found for GSTP1-1 × which is overexpressed in these specific cells as as in tumor cell of NBDHEX to binding of NBDHEX to GSTP1-1, GSTA1-1, and was by the of the of the to GSTP1-1 in the of × 10-3 GSH an with an dissociation of × m, which is to the value and reported in In the of GSH the affinity of NBDHEX GSTP1-1 × and the binding strong of NBDHEX to GSTA1-1 in the of × 10-3 GSH a with an dissociation of × This value was the value In the of GSH the dissociation × and a was with an of In the of × 10-3 so strong an affinity NBDHEX that was not possible to the dissociation by binding of the affinity between and the NBDHEX × was by the inhibition of GST activity with both the enzyme and the inhibitor in the nanomolar range and In the of the affinity of for NBDHEX by of and binding experiments a KD value of × and at interaction of NBDHEX with GSTs was by NBDHEX was at in the of a of GSTP1-1 and × 10-3 the of NBDHEX at and a new at were in the of enzyme or GSH was or by × 10-3 not of to the of NBDHEX with enzyme and the at the NBDHEX This a interaction between NBDHEX and GSH that is possible in a active A was for that the new was at and a higher new represent a σ complex between NBDHEX and GSH stabilized in the active site of GSTP1-1 and GSTM2-2. In it is that through a σ complex PubMed Scopus Google Scholar, P. M. J. H. Biochem. Mol. Biol. 1994; Google Scholar) and that σ of NBD have between and M.R. L.M. J. Chem. Google Scholar, M.R. D. Chem. PubMed Google Scholar). A different type of interaction was found in the of In this the NBDHEX at not but was at of not the of of the between NBDHEX and The of NBDHEX × in × 10-3 GSH was at in the and in the of amounts of of NBDHEX were at and at different the of amounts GSTA1-1 the interaction between NBDHEX, and GSTA1-1 was at a at was that was rapidly the at The at the σ complex stabilized in the active site of and GSTP1-1, whereas the at be as the glutathione with NBD that specifically at this (14Caccuri A.M. Ascenzi P. Antonini G. Parker M.W. Oakley A.J. Chiessi E. Nuccetelli M. Battistoni A. Bellizia A. Ricci G. J. Biol. Chem. 1996; 271: 16193-16198Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The formation of in the of GSTA1-1 was by not and by in the of GSTs, of NBDHEX be at × 10-3 GSH. at higher GSH concentration to × 10-3 a at was with a of the at this was but not to that in the of GSTs. of the to the NBDHEX that the new was via a that GSH in the at GSH × 10-3 to × 10-3 a dissociation of × 10-3 for this and This at × 10-3 GSH the σ complex was present as a in of the in the of interaction of NBDHEX with GSH in the of GSTs was detailed by of a rapid of amounts of GSH with NBDHEX at and were from The of an in the reported in that a new this The at different GSH was to This revealed that the of the was in and for of the whereas was to a the of and the at NBDHEX and at the new to a mechanism The found by the were and The dissociation the new GSH and NBDHEX was × 10-3 m, a value that the one from at × 10-3 of the of the and are reported in in the of experiments revealed that a complex interaction NBDHEX and GSH were rapidly in the of GSTs. The was at as at higher it to be In the of and GSTA1-1, were from by the different and were not for GSTP1-1 because of the was the As shown in not in a the of at least the and The at different NBDHEX for isoenzyme was to the of and the at and at and to the in that a first between NBDHEX and the complex to a first that a second The of the from are shown in and In both and GSTA1-1, the first in the and at This was to that at high GSH in the of GSTs. The second was by a at that of the σ complex at of NBDHEX with GSH in the of or GSTP1-1 and with GSTA1-1 at low We that in GSTA1-1 the second was not stabilized but was the This very at and it not be in the rapid kinetic the of M.R. L.M. J. Chem. Google Scholar, M.R. D. Chem. PubMed Google Scholar) the first be as a σ complex with the of GSH bound at the of the benzoxadiazole ring. The may to a σ complex with the of GSH bound at the The kinetic for GSTA1-1 and found by the are in and values with GSTA1-1, whereas and were in these This that both the and the C-4 σ were by the active The KD value for the dissociation of the C-4 σ complex from the active to give and NBDHEX be from the KD The values were × for and × for The KD value for GSTA1-1 to the one by whereas the value found for was This may be to the different used in these experiments and from of NBDHEX × × × × × × in a new The new NBD derivatives in this study are strong and inhibitors of GSTs, showing values of × for GSTM2-2, × for GSTP1-1, and × for GSTA1-1, representative isoenzymes for the human Mu, Pi, and class GSTs. experiments performed on human cancer cell lines demonstrated a strong activity of NBDHEX, the potent inhibitor of GSTP1-1 The between the of GSTP1-1 inhibition × and of on and tumor cell lines × × and × m, that NBDHEX recognizes GSTP1-1 as a in and this interaction can be for we have (15Turella P. Cerella C. Filomeni G. Bullo A. De Maria F. Ghibelli L. Ciriolo M.R. Cianfriglia M. Mattei M. Federici G. Ricci G. Caccuri A.M. Cancer Res. 2005; 65: 3751-3761Crossref PubMed Scopus (106) Google Scholar) a detailed investigation of the apoptosis on the cell lines showing that NBDHEX tumor cell death through the dissociation of GSTP1-1 from the This interesting in cancer and can be by that NBDHEX a crucial in the GST and kinetic reported in the present study indicated that NBDHEX may both the G-site and the It like a suicide in it was conjugated with GSH to a σ complex by a at The formation of a σ complex or complex has been in the active site of GSTs of with GSH PubMed Scopus Google Scholar, P. M. J. H. Biochem. Mol. Biol. 1994; Google Scholar). The NBD derivatives are forming σ with many A of the at the of the benzoxadiazole by a the C-4 has been M.R. L.M. J. Chem. Google Scholar, M.R. D. Chem. PubMed Google Scholar). and rapid kinetic that GSTs the formation of the σ complex at the that in the C-4 σ in the active site of GSTA1-1, the C-4 σ complex rapidly evolved the release of the of all we the dissociation which the affinity of enzyme for the C-4 σ the × the reported in we values of × × and × for GSTA1-1, GSTM2-2, and GSTP1-1, values that the σ complex between NBDHEX and GSH at the C-4 represents one of the inhibitors found for of the dissociation of the σ complex bound to GSTs at was by the × is the dissociation of NBDHEX from the complex from binding experiments × × and × for Alpha, Pi, and GSTs, is the dissociation of GSH from the and it was to be × for the and GSTs and × for the isoenzyme A.M. G. P.G. Parker M.W. M. M. Federici G. Ricci G. Biochem. J. 1999; PubMed Scopus (54) Google and KD is the dissociation of GSH and NBDHEX from the σ complex in × 10-3 Docking a for this tight In the C-4 σ complex bound to a binding site in all isoenzymes with very between and The GSH in the G-site as found in the of the whereas the benzoxadiazole tightly a by and on one side and by the of on the This is present in all and is by a that with the of the It is interesting to that the is in the isoenzymes the different localization of the In the of the is in enzyme The study not the release of 6-mercapto-1-hexanol from the GSTA1-1 active this is likely by structural of the active site not by In we that the design of peptidomimetic molecules as suicide inhibitors for GSTs represents a in the GST and interesting for cancer
Ricci et al. (Thu,) studied this question.