At low rubreserine concentrations, folate biosynthesis appeared as a main target in Arabidopsis and Toxoplasma. However, at high rubreserine concentrations, the growth activity could not be fully restored by the presence of pABA or 5-FTHF, raising the question of the inhibitor specificity. GAT-ADCS belongs to the family of class I glutamine amidotransferases (, ), which contains six other members. The GAT domain of GAT-ADCS does not share strong homologies with the GAT domains of the other members of this class, and the best scores were obtained with AS (about 28% identity) GMPS (about 13% identity) and carbamoyl phosphate synthetase II (involved in UMP synthesis, about 15% identity). To test the effect of rubreserine on other members of this group, we attempted to produce these recombinant Arabidopsis activities. We failed to produce active recombinant carbamoyl-phosphate synthetase, but we produced AS (a heterodimeric protein combining the activities of β- and α-subunits, respectively, equivalent to TrpG and TrpE in prokaryotes ()) and GMPS (a bifunctional enzyme with fused GAT and synthase domains (), like GAT-ADCS). We determined these activities with the same GDH-coupled assay that we used for GAT-ADCS. As shown in , rubreserine also inhibited GMPS and AS activities, although inhibition of GMPS required higher concentrations of inhibitor. Toxoplasma, but not Plasmodium, is an auxotroph for tryptophan because of the lack of AS activity (, ). However, the enzymes involved in the synthesis of GMP (GMPS, a bifunctional enzyme, as in plants ()) and UMP (carbamoyl-phosphate synthetase II, a heterodimer in plants (), but a single protein in apicomplexan parasites ()) was present in these organisms. When a mixture containing pABA, anthranilate, UMP, and GMP (50 μ each) was added to the culture medium of rubreserine-treated plants or was present in the proliferation assay of Toxoplasma, the growth recovery for both organisms was not markedly improved compared with that obtained with pABA or 5-FTHF alones (see B for the experiments with T. gondii).
Two single-base-pair substitutions causing desensitization to tryptophan feedback inhibition of anthranilate synthase and enhanced expression of tryptophan genes of Brevibacterium lactofermentum.
Developments in the design and synthesis of calpain inhibitors.
Regardless of the controversies associated with the virulence/avirulence of lysine auxotrophic strains, several specific inhibitors of enzymes of the α-aminoadipate pathway were designed and tested for their antifungal activity. Particularly, the homoisocitrate dehydrogenase was especially exploited as a possible target. It catalyzes oxidation of homoisocitric acid to oxoglutarate followed by the loss of carbon dioxide to yield the α-ketoadipic acid. Yamamoto et al. () based on the knowledge of two-step mechanism of the homoisocitrate dehydrogenase-catalyzed reaction claimed that a properly designed dead-end inhibitor, which cannot be decarboxylated, may remain bound at the enzyme active site and thus block it. Another possibility is to form a covalent bond between nucleophilic residues present at the active site and analogs of the intermediary enolate (Yamamoto et al. ). According to the results of substrate recognition experiments, two potential inhibitors, namely 3-hydroxypropylidenemalate and 3-carboxypropylidenemalate (Fig. a, b), were designed and synthesized (E and Z isomers) as potent antifungal agents. These compounds appeared as moderate competitive inhibitors of homoisocitrate dehydrogenase from S. cerevisiae; however, there was no time-dependent inactivation of the enzyme. Moreover, it should be noted that there was a major difference in activity between geometric isomers of these compounds. The Z isomers were remarkably more active. The Ki value determined for (R,Z)-3-carboxypropylidenemalate was 72 µM against homoisocitrate dehydrogenase from S. cerevisiae, while that of the (S,E)-3-carboxypropylidenemalate was 790 µM (Yamamoto et al. ). To increase the stability of the intermediary enolate form, another group of homoisocitrate dehydrogenase substrate analogs containing a heteroatom such as sulfur or oxygen at the α-position were synthesized (Yamamoto and Eguchi ). Among them, the thiahomoisocitrate (Fig. c) showed a strong competitive inhibitory effect, with Ki as low as 97 nM toward the homoisocitrate dehydrogenase from S. cerevisiae. This was the first successful example of design and synthesis of a highly active inhibitor of homoisocitrate dehydrogenase. Unfortunately in preliminary tests, the thia-analog did not affect growth of S. cerevisiae, probably due to its low permeability into cells.