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One route to the design of lead compounds for rational drug design approaches to developing drugs against trypanosomiasis, Chagas' disease and leishmaniasis is to develop novel inhibitors of the parasite-specific enzyme trypanothione reductase. A lead inhibitor based on a peptoid structure was designed in the present study based on the known strong competitive inhibition of trypanothione reductase by N-benzoyl-Leu-Arg-Arg-beta-naphthylamide and N-benzyloxycarbonyl-Ala-Arg-Arg-4-methoxy- beta-naphthylamide. In the target peptoid the arginyl residues were replaced by alkylimidazolium units and the benzyloxycarbonyl group by the benzylaminocarbonyl function. The peptoid was synthesised using t-butoxycarbonyl protection chemistry and couplings were activated by 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. The resulting peptoid was shown to be a competitive inhibitor of recombinant trypanothione reductase from Trypanosoma cruzi with a K(i) value of 179 microM and with only weak inhibition of human erythrocyte glutathione reductase (the inhibition of glutathione reductase was at least 291-fold weaker than of trypanothione reductase).

Kinetic data for alternative substrates of recombinant trypanothione reductase from Trypanosoma cruzi were measured for a series of N-substituted-L-cysteinylglycyl-3-dimethylaminopropylamides, in which the cysteine N-substituent was either a variant of the benzyloxycarbonyl group or was L-phenylalanine or L-tryptophan. Replacing the benzylic ether oxygen atom by CH2 or NH had relatively minor effects on kcat, but raised the value of K(m) 4.5- and 10-fold, respectively. Similarly, relative to the carbobenzoxy group, an N-L-phenylalanyl or N-L-tryptophanyl replacement on the cysteine hardly altered kcat, but increased K(m) values by 16.6 and 7.4 fold, respectively. These observations were consistent with the K(m) values referring primarily to binding for this series of nonspecific substrates.

The synthesis of asymmetrical disulfides, based on Zervas' inter-mediate, monocarbobenzoxy-L-cystine, has been developed. A series of substrate analogues of trypanothione disulfide (TSST) and glutathione disulfide (GSSG) are described, where the spermidine ring of (TSST) has been replaced by 3-dimethylaminopropylamine (DMAPA). The free amino group in Zervas' product was condensed with phenylalanyl, tryptophanyl or glutamyl residues, while the carbobenzoxy group was unaffected under the reaction conditions employed. The same synthetic approach was applied in the design of analogues of glutathione disulfide (GSSG).

The synthesis of asymmetrical disulfides, based on Zervas' inter-mediate, monocarbobenzoxy-L-cystine, has been developed. A series of substrate analogues of trypanothione disulfide (TSST) and glutathione disulfide (GSSG) are described, where the spermidine ring of (TSST) has been replaced by 3-dimethylaminopropylamine (DMAPA). The free amino group in Zervas' product was condensed with phenylalanyl, tryptophanyl or glutamyl residues, while the carbobenzoxy group was unaffected under the reaction conditions employed. The same synthetic approach was applied in the design of analogues of glutathione disulfide (GSSG).

By introducing cationic charge sites novel peptide lead inhibitor structures for trypanothione reductase have been designed using molecular modelling methods. The inhibitors showed reversible, linear competitive inhibition and the strongest peptide inhibitor to date was found to be N-benzyloxycarbonyl-Ala-Arg-Arg-4-methoxy-beta-naphthylamide with a Ki value of 2.4 microM and a selectivity for parasitic enzyme (trypanothione reductase) over the host enzyme (human glutathione reductase) of over 3 orders of magnitude.