Design, synthesis and characterization of bivalent glutathione S-transferase inhibitors using combinatorial chemistry

Dimeric glutathione S-transferases (GSTs) are pharmacological targets for several diseases including cancer. Isoform specificity has been difficult to achieve due to their overlapping substrate selectivity. Here we demonstrate the utility of bivalent GST inhibitors, and their optimization via combinatorial linker design. A combinatorial library with dipeptide linkers emanating symmetrically from a central scaffold (bis-3,5-aminomethyl benzoic acid, AMAB) to connect two ethacrynic acid moieties was prepared and decoded via iterative deconvolution, against the isoforms GSTA1-1 and GSTA1-1. The library yielded high affinity GSTA1-1 selective inhibitors (70-120 fold selectivity) and with stoichiometry of 1 inhibitor: 1 GSTA1-1 dimer. Saturation Transfer Difference (STD) NMR with one of these inhibitors, with linker structure - (Asp-Gly-AMAB-Gly-Asp) and KD = 42 nM for GSTA1-1, demonstrates that the Asp-Gly linker interacts tightly with GSTA1-1, but not P1-1. H/D exchange mass spectrometry was used to map the protein binding site, and indicates that peptides within the intersubunit cleft and in the substrate binding site are protected by inhibitor from solvent exchange.; A model is proposed for the binding orientation of the inhibitor, which is consistent with electrostatic complimentarity between protein cleft and inhibitor linker as the source of isoform selectivity and high affinity. A striking feature of the model is the V-shaped conformation of the inhibitor instead of the expected extended conformation. To investigate the preference for a V-shaped conformation in both solution state as well as bound state we utilized 1 D-steady state Nuclear Overhauser Enhancement NMR and molecular dynamic simulations. Preliminary studies indicate that the free energy minimized solution state conformation of the inhibitor also has a V-shaped conformation similar to the bound conformation obtained by computational docking. There appears to be no significant change in linker conformations between the solution state and bound state of the inhibitor. This might amount to a minimal entropic conformational energy cost upon binding which in turn could contribute to the high affinity observed with these inhibitors. In conclusion, we have identified selective and potent GSTA1-1 inhibitors using a combinatorial or "irrational" linker design and utilized different methods to determine the inhibitor binding epitope as well as the binding site conformation.