Structural Analysis and Comparison of Active Site Architecture from Ancient Bacteria to Human Phosphatases: A Novel Approach to Identification of Lead Compounds with Increased Specificity and Potency for Drug Discovery

Abstract

Background: Ancient bacterial enzymes have remarkable similarity with modern bacterial and mammalian enzymes. The AF2372 and MJ0109 gene products that cleave two sugars inositol-1-phosphate and fructose 1,6, bisphosphate are hyperthermophilic enzymes that are remarkably stable and easily purified. These ancient bacterial enzymes are highly homologous with human enzymes that cleave the same substrates. Methods: In this study inhibitors for E. coli Aspartate Transcarbamoylase (ATCase), a distant relative of the enzymes that cleave sugar phosphates, were synthesized and tested in the AF2372 and MJ0109 active sites in kinetic inhibitory assays. Structural comparisons and molecular docking were done with Human Inositol Monophosphatase (IMPase) and Fructose 1,6 bisphosphatase (FBPase) to predict possible conformations of the inhibitors in the phosphatase active sites. Results: The ATCase inhibitor Inh_3 (3,5-Bis(2-phosphonoacetamido)benzoic Acid) was most effective with AF2373 and MJ0109 gene products in the enzymatic inhibitory assay with a Ki of 0.0850 and 0.110 µM respectively. Docking studies produced binding constants with Inh_3 with human FBPase and FBPase within the low micromolar range. Conclusion: Taken together, enzymatic inhibitory assays, structural comparisons of the ancient bacteria and human species, and molecular docking of the putative inhibitors with Human IMPase and FBPase reveal that these ATCase inhibitory molecules may have relevance as potential inhibitors or lead compounds for these drug targets. Keywords: Allosteric regulation, drug design, enzyme kinetics, inhibition, molecular docking, site directed mutagenesis, structural biology.