Ligand-based protein alignment and isozyme specificity of glutathione S-transferase inhibitors.

Glutathione S-transferases (GST, E.C.2.5.1.18) comprise a family of detoxification enzymes. Elevated levels of specific GST isozymes in tumor cells are thought responsible for resistance to chemotherapeutics, which renders selective GST inhibitors potentially useful pharmaceutical agents. We discuss the development of a structure activity model that rationalizes the isozyme selectivity observed in a series of 12 glutathione (GSH) analogues. Enzymatic activity data was determined for human P1-1, A1-1, and M2-2 isozymes, and these data were then considered in light of structural features of these three GST proteins. A survey of all GST structures in the PDB revealed that GSH binds to these proteins in a single "bioactive" conformation. To focus on differences between binding sites, we exploited our finding of a common GSH conformation and aligned the GST x-ray structures using bound ligands rather than the backbones of the different proteins. Once aligned, binding site lipophilicity and electrostatic potentials were computed, visualized, and compared. Docking and energy minimization exercises provided additional refinements to a model of selectivity developed initially by visual analysis. Our results suggest that binding site shape and lipophilic character are key determinants of GST isozyme selectivity for close GSH analogues.

Predicting ligand binding to proteins by affinity fingerprinting.

BACKGROUND: There are many ways to represent a molecule's properties, including atomic-connectivity drawings, NMR spectra, and molecular orbital models. Prior methods for predicting the biological activity of compounds have largely depended on these physical representations. Measuring a compound's binding potency against a small reference panel of diverse proteins defines a very different representation of the molecule, which we call an affinity fingerprint. Statistical analysis of such fingerprints provides new insights into aspects of binding interactions that are shared among a wide variety of proteins. These analyses facilitate prediction of the binding properties of these compounds assayed against new proteins. RESULTS: Affinity fingerprints are reported for 122 structurally-diverse compounds using a reference panel of eight proteins that collectively are able to generate unique fingerprints for about 75% of the small organic compounds tested. Application of multivariate regression techniques to this database enables the creation of computational surrogates to represent new proteins that are surprisingly effective at predicting binding potencies. We illustrate this for two enzymes with no previously recognizable similarity to each other or to any of the reference proteins. Fitting of analogous computational surrogates to four other proteins confirms the generality of the method; when applied to a fingerprinted library of 5000 compounds, several sub-micromolar hits were correctly predicted. CONCLUSIONS: An affinity fingerprint database, which provides a rich source of data defining operational similarities among proteins, can be used to test theories of cryptic homology unexpected from current understanding of protein structure. Practical applications to drug design include efficient pre-screening of large numbers of compounds against target proteins using fingerprint similarities, supplemented by a small number of empirical measurements, to select promising compounds for further study.

Glutathione-S-transferase activates novel alkylating agents.

Alkylating agents which are activated by glutathion-S-transferases (GSTs) have been designed and synthesized. The model compound gamma-glutamyl-alpha-amino-beta-[(2-ethyl N,N,N',N'-tetraethylphosphorodiamidate) sulfonyl]propionylglycine (1) and the nitrogen mustards gamma-glutamyl-alpha- amino-beta-[[2-ethyl N,N,N',N'-tetrakis (2-chloroethyl)phosphorodiamidate] sulfonyl]propionylglycine (2) and gamma-glutamyl-alpha-amino-beta-[[2-ethyl-N,N,N',N'-tetrakis(2- chloroethyl)phosphorodiamidate]sulfonyl]-propionyl-(R)-(-)-phenylg lycine (3) were prepared via multistep chemical synthesis. The compounds were tested with recombinant human A1-1, M1a-1a and P1-1 GSTs. HPLC studies showed that the compounds were differentially and catalytically cleaved by biologically relevant concentrations of the GSTs. Mass spectral studies of the cleavage mixture of 2 showed that M1a-1a GST liberated the cytotoxic phosphate moiety needed for efficacy as an alkylating agent. Cell culture studies with MCF-7 breast cancer cells showed that 1 was not toxic at 200 microM, while 2 and 3 showed IC50S of 40.6 and 37.5 microM, respectively, for the same cell line. MCF-7 cells transfected to overexpress P1-1 GST showed enhanced sensitivity with 2 and 3, with IC50S of 20.9 and 9.5 microM, respectively. This result correlates well with the rates of cleavage of 2 and 3 by P1-1 GST observed in vitro and demonstrates that higher levels of cellular P1-1 GST will give increased sensitivity to these drugs.

Isozyme-specific glutathione-S-transferase inhibitors: design and synthesis.

Glutathione-S-transferase (GST) isozyme-selective inhibitors were designed by an empirically guided strategy. In the first phase, literature data were used to select C-terminal modifications which generated maximum variation in the catalytic efficiency (Vmax/Km) for glutathione (GSH) analogs used as substrates with different rat GSTs. Also, on the basis of literature data, the sulfhydryl group was functionalized with a selection of alkyl and aryl groups to maximize potential isozyme specificity. Affinity chromatography sorbents were prepared from these which showed isozyme selectivity for both rat tissue and recombinant human GST isozymes. Some of these compounds also showed selective inhibition of GST activity in catalysis of the reaction of 1-chloro-2,4-dinitrobenzene with GSH. In the second phase, electronic effects were explored through synthesis of an isostructural series of S-benzyl GSH ligands with different substituents on the aromatic ring. GST isozyme specificity for these ligands, measured by binding to derivatized sorbents, varied substantially, with hydrophobic substituents favoring the human GST M1a isozyme and electronegative moieties favoring GST P1. In the third phase, information obtained from testing both series of compounds was combined and used to prepare GSH analogs with chemical features responsible for isozyme specificity at both the C-terminus and the sulfur. This approach gave two new compounds which showed improved potency while still maintaining selectivity in the inhibition of GSTs. A detailed discussion of the logic used in the selection of functional groups for maximum potency and selectivity is included.

Isozyme specificity of novel glutathione-S-transferase inhibitors.

A systematically diversified set of peptide analogs of the reaction product of glutathione with an electrophilic substrate have been tested as isozyme-specific inhibitors of human glutathione-S-transferase (GST). The potency of the best of the inhibitors is in the 0.5 to 20 micromolar range, with kinetics indicative of competitive inhibition with glutathione at the active site. The specificity observed among three recombinant-derived GST isozymes at both low and high potency ranged from negligible to high (at least 20-fold over the next most sensitive isozyme). These results define a novel strategy for the design of drugs targeting cells with elevated levels of particular GST isozymes, such as tumor cells for which elevated levels of GST are believed to be an important cause of chemotherapeutic drug resistance.