Tools and Reagents for Next Generation Inhibitor Discovery in Peptidoglycan biosynthesis

Antimicrobial resistance to existing antibiotics threatens future healthcare at multiple levels and has been acknowledged as a worldwide issue with an impact as important as climate change. However, a number of factors has led to a steady decline in the discovery and development of antimicrobials in the pharmaceutical industry despite the clear clinical need. The remaining pharmaceutical company antibacterial research and development in this area has focused generally in the past on either attempts to discover new targets for antibiotics or new compounds with limited activity profiles based on existing targets. Generally this approach has met with very limited success which combined with other factors, means we have a decreasing number of drugs to treat bacterial infection leading to a crisis in healthcare.
It is clear that bacteria are adept at the selection of resistance to drugs for single gene targets (i.e. their protein products) and that the successful antimicrobial chemotherapy of the past, selects biological processes where there are multiple essential activities e.g. protein synthesis at the ribosome, the proteins which are responsible for DNA supercoiling in the cell and mechanisms by which formation and cross-linking of the bacterial cell wall polymer is achieved. The biosynthesis of the bacterial cell wall polymer called peptidoglycan is an excellent target for antimicrobials in this respect since it provides an opportunity for multi-targeting as described above, is not a structure found in animals or humans so there is less chance of adverse chemical sensitivity and the process occurs outside the cell allowing easier access for drug molecules.
Whilst the penicillin group of drugs have been used to exploit these advantages in the past, they target the cross-linking activity of the bacterial enzymes concerned. These enzymes, called penicillin binding proteins, are also responsible for the formation of the peptidoglycan polymer as well in a completely separate reaction that has hitherto been under explored as a target for antibiotics.
Our proposal seeks to gain the essential knowledge required to explore these enzymes for next generation antibiotics. Our proposal links the knowledge and advances in academia and industry in a mutually beneficial effort to get the key information required. Critically, we need to generate a series of specialist chemical probes to interrogate the mechanism by which these enzymes work, to determine the structures of these probes bound to the enzymes to generate new assays that will allow the discovery of future antibiotics.

It is clear that activity within the antibacterial R&D sector to discover new lead compounds, primarily focused on the search for inhibitors of essential single gene targets as been unsuccessful. However consideration of historically successful antibiotic targets suggest that multi-targeting is more successful and should lead to lower resistance rates as a result of mutation in those targets.
The biosynthesis of the bacterial cell wall peptidoglycan molecules is one such target and due to its absence from mammalian genomes, its extracellular location and multi-targeting potential, deserves a 21st century re-evaluation. Beta-lactam drugs successfully target the peptide cross-linking activity of penicillin binding proteins but these enzyme work upon a polymerised glycan chain that is formed primarily by, class A penicillin binding proteins. Whilst resistance mechanisms to beta lactams are a threat to their continued use there are almost no reports of resistance to natural products in agriculture that are known to target the peptidoglycan glycosyl transferase. Moreover, its appears that this glycosyltransfrase activity and the peptide crosslinking activity are linked and coordinated in a way previously unrealised. What is needed at this point is a detailed mechanistic understanding of glycosyl transferase mechanism. To do this we need new mechanistic insight provided by the development of new chemical probes and fragment molecules, structural biology of their interaction with exemplar peptidoglycan glycosyl transferase enzymes and development of biochemical assays and information that will support next generation drug discovery. This is best achieved by close cooperation between academia and industry to enable the exchange of information, training and infrastructure for development of the biochemistry in this space.

1) Impacts: Relate to the potential future exploitation of new reagents, assays, structural and biochemical information generated by this project enabling a molecular level, mechanistic understanding of terminal stage of peptidoglycan biosynthesis. Specifically this information provides the next level of information required to pump prime antimicrobial discovery and addresses both priority and strategic areas for BBSRC impact.

The potential beneficiaries of the impacts are: the resurging UK and global academic research community exploring bacterial cell wall biosynthesis, cell division and drug discovery; national and international pharmaceutical industries; UK capacity building (skills and business) for this Strategically Important and Vulnerable Area of UK Bioscience Expertise, international collaborations; public and understanding of science (PUS) and government policy.

2) Engagement: The immediate impact of this proposal will be to foster academic-industry liaison and skills transfer for the PDRA and applicants on the project. The project will be managed with formal weekly meetings of the UK team followed by monthly teleconference reports and discussion with the US based partners. This intensive project management regime is unusual within an academic setting but engenders a highly focused project engagement and reflection that influences decisions and impact. In addition, training and skills into the development and use of assays and reagents will extend beyond the applicants and PDRAs contracted by the LINK proposal by inviting at least UK (6), EU (5), US (2) and CA (6) research groups to i) and academic-research workshop (of about 50-100 people) including members of the former regional development agency funded Science City Research Alliance (SCRA) in Translational Medicine between Warwick and Birmingham, and ii) smaller experimental workshops yr 2 (~10 - 20 people from as many groups as possible). Workshop i) will also include invitations to: UK and global industry (AZ, Basilea, CBV, Merck, Cubist, GSK, Novacta, Novartis,); Health Protection Agency; NHS clinical microbiologists; representatives from Antibiotic Action; Innovate UK; Warwick Corporate Affairs and Communications teams. Together these constituencies will help to produce media outputs during year 1 (post workshop) and year 2, such as an iCAST video, inform PUS and academic groups to better engage with pharmaceutical industry, influence national and international government policy of the need to support drug discovery activities, specifically teams of academic industry partnerships.

Applications for reagents and assays generated by the proposal will be developed across UK, EU, and North America academic research groups and in consultation with Cubist and other pharma, exploitation and commercialisation will be guided by Warwick Ventures (technology transfer and IP protection) and Warwick Corporate Affairs. Identification of applications will be helped by the planned workshops. Additional funding and training will be sought through BBSRC/MRC CASE awards (year 1), BBSRC Follow on funding (year 2), Innovate UK (year 2) if rapid progress is made with reagents and the identification of an industry partner to progress applications. If the grant succeeds in delivering the information required for drug discovery further appropriate industry-academic partnering could be sought through Welcome Trust seeding drug discovery programmes or MRC Developmental Pathway Funding Scheme (DPFS).