Identifying new drug targets within Mycobacterium tuberculosis

Tuberculosis (TB) is the leading cause of death worldwide and reflects a serious global health challenge. This bacterial pathogen kills more people than malaria and HIV combined, and is increasingly developing resistance to many of the current front-line drugs. We therefore urgently need to develop new therapeutics.

To do this we need to understand the structural and dynamic basis of the macromolecular drug targets with the causative agent of TB: Mycobacterium tuberculosis (Mtb). The outer coat of Mtb is unusally complex and forms the front-line to this bacterium’s defence. Interwoven within this bacterial cell envelop is a complex mixture of proteins, lipids and sugars.

The overall aim of this PhD project is to provide a structural and dynamic description of bacterial cell envelope within Mtb; with especial focus on the membrane protein components, which are the foremost targets for novel medicines.

The development of the protein-folding software, AlphaFold2, has enabled the computational determination of over 130 million monomeric protein structures, and therefore we now have structural information for the 4,083 proteins in Mtb; captured in isolation. Of these proteins, 729 are proposed to be membrane-embedded, fulfilling many key tasks, e.g., selective uptake of nutrients, processing of secreted proteins or expulsion of toxic compounds, such as antibiotics. Membrane proteins therefore form current and future targets for novel antimicrobials, through inhibition of these processes.

Membrane proteins, however, do not exist in isolation. They engage with other proteins, form tight contacts with lipids and exist in multiple conformations. The objectives of this proposal are therefore to:

1. Build and identify novel homo- and heteromeric complexes of membrane proteins from Mtb.
2. Assemble mycobacterial membranes around the complexes to capture key lipid interactions and to study the molecular dynamics of these macromolecular machines.
3. Identify potential drug binding sites for small molecules using cavity detection tools in surface-exposed regions of the membrane protein complexes.
4. Determine the membrane permeability of known and novel small molecule inhibitors of Mtb.

By understanding the three-dimensional details of how these complexes form and move, we have a better grasp of the fundamental processes performed by these proteins. This therefore provides an improved understanding of how one can develop novel antimicrobial inhibitors.

References:

1. Brown CM, Corey RA, Gao Y, Choi YK, Gilleron M, Destainville N, Fullam E, Im W, Stansfeld PJ, Chavent M. From Molecular Dynamics to Supramolecular Organization: The Role of PIM Lipids in the Originality of the MycobacterialPlasma Membrane. Biorxiv/PNAS (minor revisions).
2. Ashraf KU, Nygaard R, Vickery ON, Erramilli SK, Herrera CM, McConville TH … Trent S, Stansfeld PJ, Mancia F. Structural basis of Lipopolysaccharide Maturation by the WaaL O-Antigen Ligase. Nature 604:371-376.
3. Maloney FP, Kuklewicz J, Corey RA, Bi Y, Ho R, Mateusiak L, Pardon E, Steyaert J, Stansfeld PJ, Zimmer J. Structure, substrate-recognition, and initiation of hyaluronan synthase. Nature 2022; 604:195-201
4. Oluwole AO,Corey RA,Brown CM, Hernández-Rocamora VM, Stansfeld PJ, Vollmer W, Bolla JR, Robinson CV. Peptidoglycan biosynthesis is driven by lipid transfer along enzyme-substrate affinity gradients. Nature Comms.
5. Song W, Corey RA, Ansell TB, Cassidy CK, Horrell MR, Duncan AL, Stansfeld PJ, Sansom MSP. PyLipID: A Python Package for Analysis of Protein-Lipid Interactions from Molecular Dynamics Simulations. JCTC 2022;18:1188-1201.
6. Corey RA, Song W, Duncan AL, Ansell TB, Sansom MSP, Stansfeld PJ. Identification and assessment of cardiolipin interactions with E. coli inner membrane proteins. Science Adv. 2021 7:eabh2217
7. Vickery ON, Stansfeld PJ. CG2AT2: An Enhanced Fragment-based approach for Serial Multi-scale Molecular Dynamics simulations. JCTC
8. Fiorentino F, Sauer JB, Qiu XY, Corey RA, Cassidy CK, Mynors-Wallis B, Mehmood S, Bolla JR, Stansfeld PJ, Robinson CV. Ligand induced conformational dynamics of the LPS translocon LptDE. Nature Chemical Biology 2021
9. 
   

BBSRC Strategic Research Priority: Understanding the rules of life – Structural Biology, and Microbiology, Sustainable Agriculture and Food - Microbial Food and Safety, and Animal Health and Welfare, and Integrated Understanding of Health - Pharmaceuticals.

Techniques that will be undertaken during the project:

• AlphaFold2-based protein folding.
• Molecular Dynamics (MD) simulations.
• Structural Bioinformatics.
• Python-based programming.
• Ligand Docking
• Structure-based Drug Design