Periodic Reporting for period 1 - BactoDrug (The Bacteroides dual-pumping membrane-integral pyrophosphatase: a novel drug target)
Periodo di rendicontazione: 2015-06-01 al 2017-05-31
The goal of this project was to determine the molecular structure of a Bacteroides mPPase protein, or a closely related mPPase, for the purpose of designing molecules that will specifically inhibit this protein. I solved the structure of the Clostridium leptum mPPase (CpPPase) to 6.8 A, which is a good starting point for further optimization and structural studies. Shortly following the end of this fellowship, data I collected was used to solve a 4.8 A structure of CpPPase, indicating the quick progress that can be made in working towards an atomic resolution structure. In collaboration with Dr. Sarah Harris, I generated and analyzed an atomistic molecular dynamics simulation of Thermotoga maritima mPPase (TmPPase) crystal structures to explore the different intermediate states of mPPases. I also identified a small molecule inhibitor of CpPPase that could lead to a broad-range mPPase-targeted drug, since this molecule was first identified due to activity against TmPPase.
A second major project goal was to design small molecule inhibitors of mPPase from human pathogens. Though we have not yet solved the structure of a dual pumping mPPase, the structure of a Na+-pumping TmPPase and the H+-pumping mPPase from Vigna radiata, have been solved via x-ray crystallography in various conformations throughout the reaction cycle. We, along with our collaborators, have exploited this to design anti-mPPase drugs that target mPPases, including those found in pathogenic bacteria and protozoan parasites, such as the etiological agents of malaria and leishmaniasis. The TmPPase structures have been used to identify viable binding pockets for structure-based drug design. Two approaches have been implemented: in silico screening of small molecules against specific binding sites to select top hits for in vitro inhibition testing and synthesizing novel small molecules that are designed to bind a specific site on the protein. We are currently screening molecules that are most inhibitory against TmPPase in vitro against other mPPases, such as BvPPase. From the initial top 11 TmPPase hits, we found two compounds, one that is active against BvPPase, but not against Plasmodium falciparum mPPase, and vice versa, at the low M level. This suggests that we will be able to design highly-specific mPPase inhibitors that will reduce the risk of horizontal gene transfer and so the spread of resistance.
Since crystallographic structures only give a snapshot of the enzyme at one point during the enzymatic cycle, we employed molecular dynamics simulations to gain insight into intermediate conformations throughout this cycle and, thus, into the mechanism of mPPases. For our purposes, we needed to set up an atomistic simulation of the 140 kDa TmPPase within a lipid bilayer (Figure), which is not a common task within the molecular dynamics field. In our paper (Shah N. et al. 2016. Struct Dyn), we outline our successful TmPPase atomistic molecular dynamics simulation, and our insights into the loop-closing conformational changes that occur upon substrate binding. This molecular simulation of TmPPase will also be used to assess binding dynamics of different drugs in silico during the small molecule structure optimization steps of drug design. Furthermore, the various protein conformations that are revealed by the simulations can be used to identify pockets that can be targeted via structure-based drug design.