Combining biology, chemistry, mathematics, and physics to develop novel antimicrobials

Antibiotic-resistant infections are top threats to global public health and development. There is a global drive to develop alternative treatments that act differently to traditional antibiotics. Amongst the most promising are "anti-virulence" compounds that inhibit the ability of the bacterial pathogen to colonise the human host, making the pathogen more susceptible to clearance by the human immune system.

This project aims to develop anti-virulence compounds against Neisseria gonorrhoeae, a WHO Priority Pathogen. This bacterium causes ~100 million cases per year worldwide, with widespread antibiotic resistance and significant morbidity (pelvic inflammatory diseases, infertility), particularly in low socio-economic communities. Since infection does not elicit a protective immune response, no vaccine is available.

A metal-dependent enzyme on the Neisseria surface has been identified as a target for anti-virulence compounds. There are already efforts to block enzyme activity. Here we propose to block enzyme assembly.

All >200 published X-ray crystal structures of this enzyme and its homologues show a homo-oligomeric form of the assembled enzyme, with bound metal atoms in the active sites. Last year, our group discovered that newly translated protein, before any metal is bound, is monomeric. No monomer structure exists, but initial biochemical analyses hint that the monomer-to-oligomer transition involves a substantial rearrangement of protein structure.

We hypothesise that it is possible to interrupt the transition of this enzyme from the inactive, monomeric form to the active, oligomeric form, and therefore inhibit Neisseria virulence and combat Neisseria infection. This approach represents a novel anti-virulence strategy that can be applied to other virulence enzymes in other bacterial pathogens.

This project will examine the monomer-to-oligomer transition of this respiratory enzyme and use this knowledge to develop compounds that block this transition.

Aim 1: What is the monomer structure? We can already produce pure protein monomers in large quantities. We will generate high-quality, X-ray diffracting monomer crystals for structural determination. In parallel, we will subject the monomer to Small-Angle X-Ray Scattering and mathematical modelling of protein topology, to predict the monomer structure in the solution state, which will be additionally useful in case a crystal structure is not obtained.

Aim 2: What is the mechanism of monomer-to-oligomer transition? Initial biochemical analyses suggest that oligomerisation is initiated by metal binding. This metal-binding step appears to orient a flexible region in the monomer to favour oligomer formation.

We will identify this flexible region using molecular dynamics modelling and, in parallel, lysine-based chemical cross-linking. Using methods already established in the lab, We will then generate protein variants lacking this flexible region to verify its role in oligomer formation and enzyme activation.

Aim 3: What chemical strategies can be employed to interrupt the monomer-to-oligomer transition? We will employ unbiased, high-throughput screens. If crystals are obtained in Aim 1, we will use crystallographic fragment screening to find molecules that bind to the flexible region (from Aim 2) and thus block the monomer from becoming oriented correctly for oligomer formation.

If crystals are not obtained, we will use solution-based fragment screening instead. Secondly, we will use structural information (Aim 1) and mechanistic information (Aim 2) to design a peptide inhibitor. This peptide will mimic the flexible region and therefore interrupt the monomer-oligomer transition.

We will validate compound hits using methods already established in the group, namely measurements of: (i) oligomer formation in vitro and (ii) enzyme activity in vitro and in vivo.