Discovery of Novel GMPS Inhibitors of Candidatus Liberibacter Asiaticus by Structure Based Design and Enzyme Kinetic

Citrus production is facing an unprecedented problem because of huanglongbing (HLB) disease. Presently, no effective HLB-easing method is available when citrus becomes infected. Guanosine 5′-monophosphate synthetase (GMPS) is a key protein in the de novo synthesis of guanine nucleotides. GMPS is used as an attractive target for developing agents that are effective against the patogen infection. In this research, homology modeling, structure-based virtual screening, and molecular docking were used to discover the new inhibitors against CLas GMPS. Enzyme assay showed that folic acid and AZD1152 showed high inhibition at micromole concentrations, with AZD1152 being the most potent molecule. The inhibition constant (Ki) value of folic acid and AZD1152 was 51.98 µM and 4.05 µM, respectively. These results suggested that folic acid and AZD1152 could be considered as promising candidates for the development of CLas agents.

Assessing the Interactions of Statins with Human Adenylate Kinase Isoenzyme 1: Fluorescence and Enzyme Kinetic Studies

Statins are the most effective cholesterol-lowering drugs. They also exert many pleiotropic effects, including anti-cancer and cardio- and neuro-protective. Numerous nano-sized drug delivery systems were developed to enhance the therapeutic potential of statins. Studies on possible interactions between statins and human proteins could provide a deeper insight into the pleiotropic and adverse effects of these drugs. Adenylate kinase (AK) was found to regulate HDL endocytosis, cellular metabolism, cardiovascular function and neurodegeneration. In this work, we investigated interactions between human adenylate kinase isoenzyme 1 (hAK1) and atorvastatin (AVS), fluvastatin (FVS), pravastatin (PVS), rosuvastatin (RVS) and simvastatin (SVS) with fluorescence spectroscopy. The tested statins quenched the intrinsic fluorescence of hAK1 by creating stable hAK1-statin complexes with the binding constants of the order of 104 M−1. The enzyme kinetic studies revealed that statins inhibited hAK1 with significantly different efficiencies, in a noncompetitive manner. Simvastatin inhibited hAK1 with the highest yield comparable to that reported for diadenosine pentaphosphate, the only known hAK1 inhibitor. The determined AK sensitivity to statins differed markedly between short and long type AKs, suggesting an essential role of the LID domain in the AK inhibition. Our studies might open new horizons for the development of new modulators of short type AKs.

The Vibrio cholerae SpeG Spermidine/Spermine N-Acetyltransferase Allosteric Loop and β6-β7 Structural Elements Are Critical for Kinetic Activity

Polyamines regulate many important biological processes including gene expression, intracellular signaling, and biofilm formation. Their intracellular concentrations are tightly regulated by polyamine transport systems and biosynthetic and catabolic pathways. Spermidine/spermine N-acetyltransferases (SSATs) are catabolic enzymes that acetylate polyamines and are critical for maintaining intracellular polyamine homeostasis. These enzymes belong to the Gcn5-related N-acetyltransferase (GNAT) superfamily and adopt a highly conserved fold found across all kingdoms of life. SpeG is an SSAT protein found in a variety of bacteria, including the human pathogen Vibrio cholerae. This protein adopts a dodecameric structure and contains an allosteric site, making it unique compared to other SSATs. Currently, we have a limited understanding of the critical structural components of this protein that are required for its allosteric behavior. Therefore, we explored the importance of two key regions of the SpeG protein on its kinetic activity. To achieve this, we created various constructs of the V. cholerae SpeG protein, including point mutations, a deletion, and chimeras with residues from the structurally distinct and non-allosteric human SSAT protein. We measured enzyme kinetic activity toward spermine for ten constructs and crystallized six of them. Ultimately, we identified specific portions of the allosteric loop and the β6-β7 structural elements that were critical for enzyme kinetic activity. These results provide a framework for further study of the structure/function relationship of SpeG enzymes from other organisms and clues toward the structural evolution of members of the GNAT family across domains of life.