Development and validation of new reagents and assays to exploit the final steps of peptidoglycan construction

Millions of people die each year from bacterial infections and tens of millions suffer from the consequences of these infections. The discovery of the antibiotic penicillin once opened the door to treat these infections. It did this by stopping bacteria from making the polymer in the cell wall that holds them together. This polymer, called peptidoglycan (PG), is made up of an interlocking network of sugars and strings of amino acids (peptides). Specialised proteins (called penicillin-binding-proteins or PBPs, which are present in all bacteria) either have the ability to stitch together the sugar backbone and peptides. The construction of peptide cross-links by PBPs is famously the target inhibited by penicillin which stops cell wall construction and kills the bacterium. Penicillin has been an excellent antibiotic, not least because it targets multiple PBPs simultaneously within a bacterium.

Unfortunately, many bacteria are no longer killed by penicillin and other antibiotics that attack the production of peptidoglycan. Bacteria have changed by evading the action of these antibiotics by modifying the target PBPs and producing enzymes that degrade the antibiotic. We need to fight back and the strategy of exploring PBPs for new inhibitors is widely recognised as an important well validated option.

Progress in achieving this has been hampered by our inability to routinely synthesise the key chemical components that make this polymer. We can now do this at Warwick, and have an exceptional track record of providing reagents to study peptidoglycan biosynthesis to academia worldwide. Having studied how the precursors of these reagents are produced by enzymes in the PG pathway, we intend to exploit the opportunities we have discovered to develop completely new reagents with bespoke components. This is exciting for both the academic and industrial communities as we will become able to produce tailor-made intermediates for specific functions. For example, we can include radioactive sugars or amino acids, fluorescent labels, or modifying sugars or amino acids in ways which alter their ability to polymerise. These reagents will enable us, and the wider community, to explore fundamentally important unanswered questions about these targets andhow bacteria grow and control the production of peptidoglycan. We will be in a position to use these reagents to develop ambitious new assays, not only to characterise the activities of these targets, but also to explore the translation of these assays into formats for industry to use them in the search for completely new classes of inhibitors, overcoming current problems of resistance to penicillin and related antibiotics.

To achieve this we will use our academic expertise gathered over the past decade of funding with enzymologists, chemists, engineers, mathematicians and physicists, and use this in a new closer partnership with industry. This partnership will provide open access for us to develop the work more widely, to increase the platform of reagents we can produce, extend our capability into new assays to study the complex, difficult, final stages of peptidoglycan construction. All of this will work towards fundamentally new biological insights. It will also underpin opportunities to further develop these reagents and assays for use by industry. To do this we will have to refine current methods to scale up production and develop robust industry quality assays. Our partnership consists of scientists and technical support at Warwick University with complementary skills and specialist knowledge to acomplish these tasks, along with Astra Zeneca, who are committed to supporting open access to this new underpinning technology and helping to develop novel approaches to high throughput screens. This heralds an era where academics and industry can work closely together in the search for new antibiotics.

Penicillin-binding- proteins (PBPS) involved in the construction of peptidoglycan (PG) are the target of important classes of antibiotics, the b-lactams and glycopeptides. Although resistance has arisen to these classes of antibiotic, the formation of peptide cross-links between the glycan backbone of PG by transpeptidation (TP) remains an excellent target for antimicrobial development. Advances have been made in understanding mechanisms underlying the activity of PBPs, however, assay development (let alone high throughput screening) for these enzymes has remained difficult, involving extensive product isolation and characterization.

We will extend our development and scale up of novel reagents and target enzymes. Reagents will encompass the chemical diversity that exists in PG structure across bacteria, including, non-cognate substitutions within the linear muramyl pentapeptide, the production of polymeric lipidII where required to prime reactions, and truncation of the C55 lipid tail (using C35 or C20 lipids etc.) to improve in assay solubility and assay function. Interestingly, the required acceptor transpeptidation substrate can be as small as a single amino acid with D-chirality around the alpha-carbon atom. This observation opens up the development of a range of novel more complex (linear/branched) acceptor molecules for use in assays to probe the TP acceptor site structurally and to format assays to identify novel non-lactam TP inhibitors.

These reagents will ultimately enable a systematic characterization of the transpeptidase activity of PBPs, the development of co-crystal structures, and provide the foundation for previously unavailable robust assays with direct readouts. Astra Zeneca will work with us to help scale up reagent production, format assays for use in high throughput screens, test sensitivity, and help validate these for industry though IP free access to trial compound libraries.

1) Impacts: relate to the exploitation of new reagents and assays generated by this project enabling the kinetic and structural exploration of terminal stages of peptidoglycan (PG) biosynthesis. Specifically the targets of penicillin (penicillin-binding-proteins PBPs) which remain difficult to assay yet represent fundamental targets for research and exceptional targets for drug discovery to identify new classes of antiinfectives using these radically new, bespoke, assays.

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: Training and skills into the development and use of assays and reagents will extend beyond the PDRAs and technician employed by LINK by inviting at least UK (6), EU (5), US (2) and CA (6) research groups to i) research workshop yr 1 (of about 50-100 people) including members of the £20M 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 include a competition and prize for the best PUS poster(s) these will be used within host institutions and available via a NEW PUS section of the well used BACWAN web site.

Workshop i) will also include invitations to: UK and global industry ( AZ, Basilea, CBV, Cubist, GSK, Novacta, Novartis); Health Protection Agency; NHS clinical microbiologists; representatives from Antibiotic Action; TSB; Warwick Corporate Affairs and Communications teams. Together these constituencies will help to produce media outputs during yr 1 (post workshop) and yr 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.

We will help convene 1 additional international workshop to promote policy for funding and to attract industry investment and showcase activities at MRCT ELRIG drug discovery each year (planned UK/Canada meeting with academics, industry, HPA, government representatives and national funding agencies at the Canadian Embassy).

Applications for reagents and assays will be developed across UK, EU, US and CA academic research groups and in consultation with AZ 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 (yr 1), Follow on funding (yr 2) and the TSB (yr 2) or earlier (into yr 1) if rapid progress is made with reagents and the identification of an industry partner to progress applications.

If this LINK is awarded CGD will apply, yr 1, for a BBSRC Flexible Interchange Programme (FLIP) for secondment allowing closer engagement with industry, and industry focussed research groups in the field, to improve and extend these relationships, identify current applications (and specifications) for industry and future needs. The aim being to foster long term collaboration and investment. Additionally, reagents and assays developed by this LINK will be promoted through 3 national and international industry outsourcing conventions such as Bio Trinity, Bio Chicago, Bio Europe

Community: Posters and media presentations will be used in outreach to at least 2 local schools and Cafe Scientifique B'ham