Developing mechanistic understanding to improve the activity of bicyclic peptides as novel antimicrobials

Each year, millions of people die from bacterial infections and tens of millions suffer from the consequences of these infections. The discovery of the drug penicillin once opened the door to treat these infections. However, over time, bacteria have evolved resistance to these essential drugs, making them ineffective. For example, resistance to penicillins can occur when the bacteria produce an enzyme called beta-lactamase that acts by breaking down the penicillin before it kills the bacteria. The World Health Organisation's Director-General has called antimicrobial resistance a "slow tsunami". Left unchecked, many common medical treatments will become useless and many more people will die from infectious diseases. Research is needed to find new drugs. Already, most species of bacteria exhibit resistance to penicillin.

Our vision is to create a new class of inhibitors called "Bicycles" (bicyclic peptides), which act similarly to penicillin but are not vulnerable to beta-lactamases. Bicycles are chemically very different to penicillin and were discovered using proprietary "Phage-display" technology at BicycleTx, a medium sized UK pharmaceutical company. This grant will support an expert researcher, with relevant expertise, from the University of Warwick to work with BicycleTx to improve the activity of these Bicycles.

Bacteria are protected from the outside world by surrounding themselves with a cell wall. If we can stop this cell wall being made, the bacteria quickly die. Penicillin binding proteins (PBPs) are a family of specialised proteins used by all bacteria to produce the cell wall. As the name suggests, penicillin can bind to PBPs inhibiting their action, thereby preventing cell wall formation and killing the bacteria. Because PBPs are found in all species of bacteria, drugs like penicillin can be used to treat many different bacterial infections and are therefore extensively used by doctors.

Bicycles also inhibit PBPs, but they are very new and before they can be used in a clinical setting, they need further optimisation. Several important questions about how they work need to be answered:
- How do they interact with PBPs?
- What are the methods that bacteria might use to become resistant to Bicycles?
- How do we make sure the Bicycles can break into the bacterial cell in order to have their effect on PBPs?

To answer these questions, knowledge about PBPs and a number of different techniques are needed. One of the methods used is X-ray crystallography, which allows scientists to observe the 3D, atomic structures of proteins and Bicycles. These structures can be used to make improved versions of Bicycles which have an even stronger ability to inhibit bacteria.
If bacteria are grown in the laboratory in the presence of Bicycles, they will slowly evolve resistance. The Bicycle can then be modified in anticipation of the same evolution happening in a clinical setting. To study how well a Bicycle can enter a bacterial cell, we can use a new test developed by BicycleTx. Different Bicycles will be designed and tested for their ability to penetrate bacterial cells.

The University of Warwick and the secondee have expertise highly relevant to these fields of study, knowing how penicillin interacts with PBPs and developing new ways to study this and how new inhibitors such a Bicycles might work. By transferring knowledge to BicycleTx, we can accelerate the development of Bicycles.

This project addresses the global demand for effective new antibiotics to combat the rising threat of antimicrobial resistance. A new class of antibiotics would help safeguard healthcare systems. The project also boosts the exchange of knowledge between universities and UK pharmaceutical enterprises which will help the UK become a world leader in this field. Finally, the project will boost the career of a young scientist, providing them with the skills, knowledge and professional network for a career in antibiotic innovation.

Antibiotic resistance poses a serious threat to public health and requires novel therapeutic approaches. The most important class of antibiotics, beta-lactams, target penicillin binding proteins (PBPs). These are essential components of bacterial cell wall biosynthesis, inhibition of which leads to cell death. PBPs remain an attractive target but the prevalence of beta-lactamase driven resistance and, rarely, target-based mutations, necessitates new classes of PBP-targeting drugs.

BicycleTx have recently developed PBP-inhibiting bicyclic peptides (Bicycles) using their proprietary phage-screening technology. Now, further work is needed to understand the interactions of these compounds to PBPs, whether target mediated resistance impacts upon Bicylcle binding, and how cell-permeating properties can be optimised.

The company has recently obtained a high-resolution (~1.5A) PBP3:Bicycle X-Ray co-structure to understand how Bicyles bind and to develop structural-activity relationships (SAR) using analogues and iterative cycles of medicinal chemistry.
I will look at developing and characterising Bicycle resistance mutants in target pathogens. Identifying and better understanding any potential mechanisms of resistance to Bicycles will be used to reduce vulnerabilities.

The challenge of drug permeation is common to all Gram negative antibiotic discovery work. Usefully, BicycleTx have developed a split luciferase luminescence assay to monitor Bicycle accumulation in the periplasm. This assay will be used to screen for cell permeating peptides which can be integrated into the peptide warhead to give effective and broad-spectrum Gram negative inhibition.

The end goal of this work will be a further improved and characterised new class of PBP-targeting compounds, de-risked against resistance and able to permeate important clinical pathogens.