Use of transferred nuclear‐Overhauser‐effect spectroscopy to measure the bound conformation of a disulphide‐replaced analogue of glutathione disulphide as an inhibitor of yeast glutathione reductase

Abstract

The analogue of glutathione disulphide (GSSG) in which the disulphide bridge of GSSG is replaced by -CH,-S-was synthesised from L-cystathionine using t-butoxycarbonyl and t-butyl ester protection with triethylsilane-promoted deprotection. This analogue (GCSG) was found to be a linear, competitive inhibitor of yeast glutathione reductase (K, value 981 pM at pH 7.0), a very poor substrate and not to act as an irreversible inhibitor of glutathione reductase. The weak binding of GCSG to glutathione reductase permitted the use of transferred nuclear Overhauser effect spectroscopy (TRNOESY) to investigate the bound conformation of GCSG in its complex with glutathione reductase. The solution structure of free GCSG was investigated by NMR spectroscopy using a range of NMR techniques. The TRNOESY experiment allowed a range of conformations to be determined for the central bridge region (containing the -CH,-S-replacement) of GCSG bound to yeast glutathione reductase. Using the nuclear Overhauser effect constraints thus derived, in combination with molecular graphics and energy minimisation based on the known crystal coordinates of glutathione disulphide (GSSG) bound to human erythrocyte glutathione reductase, allowed an explanation of the lack of substrate activity of GCSG, its inactivity as a suicide inactivator and its relatively weak binding in terms of the enforced mislocation of the -CH,-S-bridge with respect to the catalytic residues (relative to GSSG). Thus, the simple replacement of -S-by -CHZ-, common in medicinal chemistry, can lead to poor receptor binding if the replacement occurs in a central, rather than peripheral, part of the ligand under modification.The recognition of glutathione disulphide by glutathione reductase has been elegantly probed in a series of X-ray diffraction analyses at high resolution [l -31 and the binding contacts between substrate, or substrate analogues, and the enzyme carefully mapped out. The redox defences of trypanosomatids and leishmanias, the organisms causing trypanosomiasis and leishmaniasis, do not use glutathione reductase to regenerate reduced glutathione, GSH, from its oxidised form, GSSG, as in Eqn (l), but involve a pathogen-specific enzyme called trypanothione reductase with its cognate substrate trypanothione disulphide (I) [4 -

Structural Determinants of the Catalytic Reactivity of the Buried Cysteine of Escherichia coli Thioredoxin

“…The similarity between sulfur and carbon in terms of both van der Waals radii and electronegativity has been commonly exploited in design of substrate analogs. In particular, alkyl sulfide derivatives of glutathione have been used to demonstrate specific substrate binding of thioltransferase/glutaredoxin (Hoog et al, 1982), while a methylene substitution for one of the sulfurs in the disulfide bridge of glutathione has been used to generate an inhibitor of glutathione reductase (Embrey et al, 1994).…”

Novel glutathione analogues containing the dithiol and disulfide form of the Cys-Cys dyad

“…Although a number of chemical modifications have been performed on this molecule (Chen et al, 1986;Douglas, 1989;Xie et al, 1991;Embrey et al, 1994;GrOger et al, 1996), literature examination revealed that no data are available on GSH analogues containing a Cys residue replacing the C-terminal Gly and giving rise to a -Cys-Cys sequence.…”

Predictability of Designing Specific Binding Interactions for DNA Minor Groove Ligands from NMR Spectroscopy and Molecular Modeling: A Copper(II)-Activated DNA Cleaver Based on Hoechst 33258

“…Even for enzyme inhibitor design, a small change in the structure of an inhibitor can lead to major changes in the details of binding to the active site. This is lucidly illustrated for dihydrofolate reductase by the 180°twist found in the pteridine ring binding conformation for dihydrofolate compared to methotrexate (Matthews, 1978;Fontecilla-Camps, 1979), and the observation that simply replacing one sulfur atom of the disulfide bridge of GSSG by -CH2-(to give a -CH2S-bridge) leads to a major twist in the conformation accepted by the active site of glutathione reductase (Embrey et al, 1994). Experience in rationally designing ligands for DNA is considerably less than for proteins.…”