Structure and function of Plasmodium falciparum malate dehydrogenase: role of critical amino acids in co-substrate binding pocket.

The malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our laboratory have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal glycine motif, which forms a characteristic Rossman dinucleotide-binding fold in the co-substrate binding pocket, differentiates PfMDH (GlyXGlyXXGly) from other eukaryotic and prokaryotic malate dehydrogenases (GlyXXGlyXXGly). The amino acids lining the co-substrate binding pocket are completely conserved in MDHs from different species of human, primate and rodent malaria parasites. Based on this knowledge and conserved domains among prokaryotic and eukaryotic MDH, the role of critical amino acids lining the co-substrate binding pocket was analyzed in catalytic functions of PfMDH using site-directed mutagenesis. Insertion of Ala at the 9th or 10th position, which converts the N-terminal GlyXGlyXXGly motif (characteristic of malarial MDH and LDH) to GlyXXGlyXXGly (as in bacterial and eukaryotic MDH), uncoupled regulation of the enzyme through substrate inhibition. The dinucleotide fold GlyXGlyXXGly motif seems not to be responsible for the distinct affinity of PfMDH to 3-acetylpyridine-adenine dinucleotide (APAD, a synthetic analog of NAD), since Ala9 and Ala10 insertion mutants still utilized APADH. The Gln11Met mutation, which converts the signature glycine motif in PfMDH to that of PfLDH, did not change the enzyme function. However, the Gln11Gly mutant showed approximately a 5-fold increase in catalytic activity, and higher susceptibility to inhibition with gossypol. Asn119 and His174 participate in binding of both co-substrate and substrate. The Asn119Gly mutant exhibited approximately a 3-fold decrease in catalytic efficiency, while mutation of His174 to Asn or Ala resulted in an inactive enzyme. These studies provide critical insights into the co-substrate binding pocket of PfMDH, which may be important in design of selective PfMDH/PfLDH inhibitors as potential antimalarials.

3-D-QSAR of N-substituted 4-amino-3,3-dialkyl-2(3H)-furanone GABA receptor modulators using molecular field analysis and receptor surface modelling study.

We report the theoretical validation of the experimentally observed structure-activity relationships (SAR) of a set of N-substituted 4-amino-3,3-dialkyl-2(3H)-furanone GABA receptor modulators showing positive allosteric modulatory activity of the GABA(A) receptor similar to that shown by Loreclazole. Efforts were made to explain some of the conclusions drawn during this study based on a solitary instance of occurrence of the observation within the dataset. Some of the conclusions selected for study included (i) the enhanced activity for the R enantiomer of a compound, (ii) enhanced activity for a compound with an amide type functionality vis-à-vis an amine type functionality at C-4, (iii) enhanced activity for a compound with a carboxamide or carbamate type functionality linking the end group at C-4 over a compound with only the end group attached, provided the alkyl groups attached at C-3 are identical in both cases. The 3-D-QSAR method of molecular field analysis along with receptor-ligand complex stability studies were found to be the most suitable for explaining these activities. While the first conclusion was comprehensively proven, significant support was obtained in case of the latter two. Further comprehensive study is underway and we hope to report them shortly.