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H2020Individual fellowship2015–2017

BactoDrug · The Bacteroides dual-pumping membrane-integral pyrophosphatase: a novel drug target

Horizon 2020 — Marie Skłodowska-Curie Actions

Duration
2015-06-01 → 2017-05-31
EU contribution
€183,455
Participants
1
Scheme
MSCA-IF

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Results in brief

The Bacteroides dual-pumping membrane-integral pyrophosphatase: a novel drug target

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.

Data: CORDIS, © European Union

Project objective

Membrane-integral pyrophosphatases (M-PPases) couple cleavage of pyrophosphatase to pumping of ions across a membrane to generate membrane potential and play an important role in resistance to stressors. The solved structures of an H+-pumping M-PPase from Vigna radiata and an Na+-pumping M-PPase from Thermotoga maritima show M-PPases form a channel through the membrane, and this channel is plugged by an ion gate formed by three charged residues. Despite these structures, there are still many outstanding questions regarding M-PPases, especially in relation to H+ and Na+ dual-pumping M-PPases.Bacteroides species are a major cause of anaerobic infections, and though they are part of a healthy human gut flora, when these bacteria escape the gut, they can cause bacteremia and abscess formation. Bacteroides species are associated with high antibiotic resistance rates and have a 19% or greater mortality rate. However, they do possess a possible drug target: an H+/Na+-pumping M-PPase.A major goal of this project is to solve the structure of the Bacteroides vulgatus H+/Na+-pumping M-PPase to guide mutational studies to determine how M-PPases select for ions and to explore how the ion gate is opened and closed during ion pumping. Since the ion gate is closed in all M-PPase structures to date, I will also use single molecule fluorescence resonance energy transfer and total internal reflection fluorescence microscopy to determine the kinetics and conformational changes during ion gate movement. Finally, I will use molecular mechanics modeling to simulate ion gate function and design small-molecule drug candidates. Molecules that trap the ion gate in the open conformation will convert M-PPase into a pore in the membrane of Bacteroides species, leading to collapse of the membrane potential. This project will further my career goal of pursuing research in bacterial pathogenesis from various perspectives, utilizing X-ray crystallography and single molecule technologies.

Original text from CORDIS.

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Data: CORDIS, © European Union