Report
Teaser, summary, work performed and final results
Periodic Reporting for period 1 - BactoDrug (The Bacteroides dual-pumping membrane-integral pyrophosphatase: a novel drug target)
Teaser
Antibiotic resistance is a growing global problem, with resistance developing against even the ‘drugs of last resort’. This highlights the need for new antibiotics, and one strategy is to pursue novel targets in the organisms that cause disease. We proposed membrane-bound...
Summary
Antibiotic resistance is a growing global problem, with resistance developing against even the ‘drugs of last resort’. This highlights the need for new antibiotics, and one strategy is to pursue novel targets in the organisms that cause disease. We proposed membrane-bound pyrophosphatases (mPPases) are a viable new target in many pathogens, from bacterial pathogens, such as Bacteroides species, to protist parasites. mPPases are important for survival under low-energy or stress conditions, as would be encountered by Bacteroides species when inhabiting alternate environments in the body. Furthermore, mPPases are not found in humans, therefore decreasing the chance of harming human cells.
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.
Work performed
One main project goal was to determine the atomic structure of a dual-pumping membrane-bound pyrophosphatase, which pumps both H+ and Na+, to determine how these enzyme differ from their single ion-pumping homologs as well as facilitate structure-based drug design against this class of enzyme. This part of the project began with testing and optimizing the expression of five different dual-pumping membrane-bound pyrophosphatases (mPPases). These five genes were expressed using three different bacterial plasmid constructs in five Escherichia coli expression strains under various growth and expression conditions. Two mPPase proteins, originating from Bacteroides vulgatus (BvPPase) and Clostridium leptum (CpPPase), were successfully expressed in an active form and localized to the membrane fraction. Further optimization of the purification of these two enzymes resulted in a final detergent-solubilized protein with pyrophosphatase activity. Cyrstallization trials were conducted for both proteins, after optimization of promising hit conditions, BvPPase crystals diffracted to 13 Ã…, whereas CpPPase crystals from initial sparse matrix screens diffracted to 6.8 Ã…. The 6.8 Ã… data set of CpPPase was solved using molecular replacement with a homologous protein, in which a monomer search template was placed as a dimer in the correct orientation. This showed that the overall structure of the dual-pumping mPPases is indeed very similar to that of the Na-pumping mPPases, like Thermotoga maritima mPPase (TmPPase). The crystal contacts that allow for packing of the CpPPase protein are evident in the 6.8 Ã… solution, and this information can be used to optimize the CpPPase construct, to facilitate tighter crystal packing, which often leads to better diffracting crystals.
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 ïM 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 th
Final results
The work accomplished during the span of this fellowship will lead to an atomic resolution structure of a novel class of membrane-bound pyrophosphatases (mPPase), which play a role in stress resistance in bacterial species, including the human pathogen Bacteroides vulgatus. At present, we have a 6.8 Ã… structure of the dual-pumping mPPase from Clostridium leptum, and further work in crystallisation condition optimization and construct optimization will lead to an atomic resolution structure. Solving this structure will not only answer several basic science questions, such as how this enzyme pumps two different ions, but also provide the basis for structure-based drug design to specifically target dual-pumping mPPase-expressing human pathogens. This will open up the possibility to specifically target certain bacterial pathogens, such as Bacteroides species, without causing substantial perturbation to the gut microbiota, as many of these organisms do not express this enzyme. In the other direction, designing anti-mPPase drugs that target different classes of mPPases would also help in the treatment of protozoan parasites, such as malaria and leishmaniasis. Both of these protozoan pathogens utilize mPPases to adapt to different host conditions, which can be very important during the transition between the insect vector and the human host. Therefore, developing anti-mPPases drugs holds great promise for a targeted but also broad-ranged antibiotic against several human pathogens.
Website & more info
More info: http://www.astbury.leeds.ac.uk/people/staff/staffpage.php.