Finding peculiar patterns of kinetoplastida enzymes to be exploited in drug design

If a parasite and host metabolic ways differ, such as an enzyme is present only in parasite, obviously this is a good target in drug design. Also, essential enzymes present in both can be good targets if we exploit selective patterns of the parasite enzyme. At this aim, comparison of the pentose phosphate pathway (PPP) 2nd dehydrogenase, 6-phosphogluconate dehydrogenase (6PGDH), between mammal host and Trypanosoma brucei, has been done in our lab. PPP provides above all necessary NADPH to all cell reduction reactions, including those contrasting both oxidative, parasite environment and host response. Although kinetoplastids 6PGDH shows only 33% amino acid identity with mammal 6PGDH, 3D-structure and general acid-base mechanism are similar, with many conserved residues in the active site. Anyway, by studying enzymes in more detail, several differences were found. Looking for the affinity of some polycyclic compounds, preferential binding of T. brucei 6PGDH to triphenylmethane with either nitrogen or oxygen as substituent in two rings and a sulfonate in the 3rd ring was found compared to the mammal enzyme. A selectivity of 40 was shown by Brilliant Green and this is probably due to the shorter distance between two active site lysines than in mammal. By studying homotropic cooperativity other significant differences were found. In fact substrate binding to one subunit increases homodimer catalytic efficiency; even if the two subunits have an identical sequence they are implied in different steps. This was shown by several ways, for instance in presence of the substrate 6PG there is a NADP half-site reactivity, also 6PG activates decarboxylation following 6PG oxidation, but differently from the liver enzyme the parasite's one is able to catalyse this step even in absence of an activator. Besides, allosteric modulation for the parasite enzyme is not apparent when reverse reaction is studied, while it is shown by yeast enzyme, which is very similar to the liver's one. Thus, diversity exists which allowed to find ligands more than 250-fold selective, as transition state analogues 4-phospho-erythronoxamate and 4-phospho-erythronate. This characterization has disclosed lead compounds for medicinal chemists to transform those into pro-drugs and derivatives with good stability and solubility. Last difference found is that parasite 6PGDH shows a ligand-modulated tetramerization, which is not present in mammal enzyme representing further drug exploitable potential.