MICA: Mechanistic understanding of cell wall biosynthesis to combat antimicrobial resistance

The discovery of the antibiotic penicillin opened the door to the treatment of a wide range of infections. It works by stopping bacteria from making the polymer in the cell wall (peptidoglycan, PG) that holds them together. This is assembled by specialised proteins (called penicillin-binding-proteins or PBPs, which are present in all bacteria) that either have the ability to stitch together both the sugar backbone and the peptides (these are known as bi-functional enzymes), or either just the sugar back bone or just the peptide-crosslinks (mono-functional enzymes). We know little about how the polymerization and cross linking activities are controlled or co-ordinated, or how they truly interact with their natural substrates. Furthermore, 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 and resistance rarely develops by altering the PBP target (with the notable exception of bacteria that can acquire altered PBP genes from other species that are poor targets for the antibiotic). Unfortunately, many bacteria have acquired resistance to penicillin by other mechanisms. Primarily this has been due to the acquisition of enzymes that degrade the antibiotic (beta-lactamases), or reduce penetration (influx) of antibiotic into the bacterium or increasing the rate of efflux out of the bacterium. We urgently need to fight back and the strategy of exploring PBPs to make better versions of current antibiotics that are more active, can evade beta-lactamases or resistance due to changing influx or efflux. Global pharmaceutical companies have a real interest in progressing such developments, however, they need better mechanistic insight into how PBPs work. We attend to address these fundamental gaps in our understanding.

Why can we succeed where others have failed?

1. Progress in achieving this mechanistic insight has been hampered by past inability to routinely synthesise the key chemical components or precursors that make this polymer. From past MRC and BBSRC funding we can now make key chemical components at Warwick, and have developed an exceptional track record of providing reagents to study peptidoglycan biosynthesis to academia worldwide.

2. Having studied how to synthesise all of the chemical precursors used by different PBPs we have developed completely new continuous assays that will now help us to understand how PBPs polymerise precursors or how they crosslink these. We have one assay to finalise, which would bring together our ability to study polymerization and crosslinking in one reaction. Alongside a continuous crosslinking assay, these new technologies represent a 70 year long breakthrough and world first.

3. Super high resolution imaging is now available so that we can see how PBPs work inside bacteria and in the test tube, how they interact with each other and other proteins or lipids within bacterial cells. We can also study PBP structure at ultra-high resolution to understand how PBPs interact at the molecular level with their natural substrates and different well known antibiotics. We also have access to new chemical approaches, which along with our assays and structural biology will help direct us to new ways to stop these enzymes.

4. Finally, we have brought together international academic experts from across the UK with skills in microbiology, chemistry and physics to work in synchrony and closely with many industry experts and a wider scientific advisory panel. This concentration of effort across a wide skill base with new technology will help ensure rapid progress and results with broad application that will be valuable for future programs of antibiotic discovery and development.

Penicillin and the wider family of beta-lactams are the single most important family of antibiotics. They target the final stage of bacterial cell wall biosynthesis, specifically cross-linking the structural polymer peptidoglycan (PG) by a family of enzymes called penicillin-binding-proteins (PBPs). Given this pivotal importance of PBPs we know little about how they interact with their natural substrates, precisely what these substrates are for different PBPs, and, astonishingly, how beta-lactam antibiotics interfere with this process. The SWaN Alliance brings together a multidisciplinary team of the UK's leading experts in PG biology, imaging and PG focussed chemistry to tackle these outstanding questions. Our aim is to build an integrated, multi-centre, multidisciplinary research programme as the UK hub of activity in cell wall biosynthesis. This hub will, in the first instance, develop new insight, and open up new ways to target PBPs, to sidestep the current mechanisms of resistance. We intend to decipher the fundamental mechanisms of PBPs at the structural, functional and cytoplasmic levels, including how PBPs interact with their substrates, and how the two PBP enzymatic (transpeptidase and transglycosylase) activities are co-ordinated. This will include, for the first time, determination of kinetic constants for the TP reaction of PBPs and the development of a dual quantitative continuous TP/TG assay. We will extend this new functional and structural insight, to explore interactions of PBPs with landmark beta-lactams, recently developed novel non-lactam PBP inhibitors, and control proteins. In doing so we will develop a transformative understanding of how PBPs interact with beta-lactams from which we will better understand the role of PBP alterations and substrate alterations in the emergence of target mediated resistance. All of this activity will reinvigorate PBPs as targets for drug discovery and development by industry and academia.

Antibiotics have been a mainstay of human healthcare for over 70 years. However, the inexorable spread of antibiotic resistance limits their ability to prevent and to cure life-threatening diseases. One of the most important and enduring targets for antibiotics is the biosynthesis of the bacterial cell wall. The predominant structural component of the wall is peptidoglycan, the synthesis of which is the target of penicillin and other clinically-relevant antibiotics such as methicillin, cephalosporins and carbapenems. Peptidoglycan is polymerized and cross-linked by a family of enzymes called penicillin-binding-proteins (PBPs) that are the single most important family of antibiotic targets known. Given the pivotal importance of PBPs in human healthcare, it is astounding that we still understand little of how peptidoglycan is made, how this process is controlled and how antibiotics interfere with it at the biochemical, structural and cellular levels. This information is vitally important to (1) help inform global pharma with current lactam projects with new mechanistic and structural insight for further development and (2) underpin the search for non-lactam antibiotics that target PBPs, with the bold objective of side-stepping decades of beta-lactamase evolution in one leap.

This proposal brings together a globally unique group of recognised world leaders in complementary aspects of bacterial biochemistry, chemistry, genetics, physics and physiology in the area of peptidoglycan metabolism, structure and architecture. Our aim is to build from this proposal a nationally integrated, multi-centre, multidisciplinary programme of research to address the critical and unresolved understanding of PBPs in relevant Gram negative and Gram positive pathogens that is essential for future antibiotic discovery.

This will extend key competencies and capabilities of academia to support and engage effectively with the global biotechnology and pharmaceutical sector. As such the project will have diverse impacts within the UK and internationally. Some of the expected impacts will be relatively short-term (i.e. within the life-time of the grant itself) while we expect others to be medium- to long-term in nature. The PI and industry advisory panel, populated with world leading industry consultants, with recognised track records in antibiotic discovery, and senior representatives from current pharma partners, will ensure that impact activities are considered and acted upon throughout the project. This will provide exceptional opportunities to engage with industry and through workshops to expand this across the wider academic community.

Extension of a unique multidisciplinary multi-institutional training environment in cell wall biosynthesis to a wide cohort of students will be another important impact, including the development of training across the antibiotic discovery pipeline with a clear industry focus and specialist input from our industry consultants. The results will be of widespread academic and pharmaceutical interest, because of almost all efforts to date on PBP inhibition have focused on beta-lactams.

We have unprecedented support from global pharma, from PhD support to running screens and providing access to probe compounds, and engaging as members of our exceptional scientific advisory panel. All have a strong interest in using the pre-competitive information that will be generated. To help achieve this engagement we have well defined objectives and routes for further exploitation.

We have help from a political consultant to help engage and inform regional and national government alongside national activities with Antibiotic Research UK, Antibiotic Action and Antibiotic Discovery UK, and international activities via the Pew Trust and the Wellcome Trust.