This Ph. D dissertation describes the discovery of several new classes of inhibitors of nucleotide pyrophosphatase phosphodiesterase 1 enzymes of two different origins. About, 2500 compounds, natural in origin or synthesized by a number of collaborating researchers, were systematically screened for their inhibitory potential and mechanism of action.
As a result, a total of ninety three (93) compounds were identified as potent inhibitor of snake venom NPPl. Out of these, thirty four (34) secondary metabolites were isolated from different medicinal plants, forty one (41) compounds were of synthetic origin belonging to two different classes, while eighteen (18) were organo metallic complexes. Most of these compounds were identified as members of new classes of NPPl inhibitors.
The most active compounds of various classes were further studied against pure human recombinant NPP1 enzyme. Eleven (11) natural constituents from different plants, thirty six (36) synthetic organic compounds and seventeen (17) organometallic complexes have been identified as new inhibitors of human recombinant NPPl enzymes.
The enzyme inhibition activity was calculated as IC50 (the concentration of compound that inhibits the enzyme activity by 50%) and Ki values (the dissociation constant). These values were determined to evaluate and compare the potency of various inhibitors. The majority of these new compounds were found to inhibit the enzyme in micro molar concentrations.
The inhibition kinetic experiments were performed on these compounds and the results of these studies were employed to investigate the mechanism of inhibition of individual compounds. It was discovered that these compounds inhibit NPPl in a concentration dependent manner. All of them were also found to be representative of reversible non-competitive inhibitors.
Structure activity relationships (SAR) were studied for all 8 different classes of new nucleotide pyrophosphatase phosphodiesterase 1 inhibitors on the basis of the experimental data. In most of the cases, multiple functional groups present at different positions of compounds were found to be responsible of their inhibitory properties. Majority of these compounds have shown Ki in micro and nano molar ranges.
Selected compounds were also evaluated for their cytotoxicity in a neutrophil -based assay. WST-l Tetrazolium salt is readily reduced to formazan dye in the presence of cellular enzymes of metabolically active human neutrophils. Neutrophils are defensive cells against bacterial and viral infections or any type of external factors such as proteins, allergens, etc. They provide a good model for cytotoxicity assessment. Most of the compounds were found to be non toxic up to a concentration of 500 µM/mL, as >90% cells were remain viable after three hours of incubation.