Mechanism-based development of small molecule probes for functional studies of bacterial E3 ubiquitin ligases

Communication within a cell - a process called cell signalling - has to be very tightly regulated to ensure that the cell responds appropriately to any internal or external changes. For example, when a cell is infected with bacteria or viruses, it should produce molecules that alert surrounding cells to the danger and provoke an immune response. If this communication breaks down or the signals are overactive then this can have devastating effects, causing many types of disease.

Proteins are macromolecules that play essential roles in our body. For example, they build muscles, carry out chemical reactions (enzymes) or act as messengers (some hormones). A common way of controlling cell signalling is the modification of proteins with small molecules (chemical 'tags'), which change their behaviour or location within a cell. One such modifier is the small protein ubiquitin that can be attached to other proteins - a process called ubiquitination. Protein ubiquitination regulates many cellular processes including immune responses and DNA damage repair. Defects in the ubiquitination system have been linked to many diseases including cancer, autoimmune and neurodegenerative disorders.

Bacteria do not contain a ubiquitin system. However, some disease-causing bacteria have evolved to produce a type of protein that is able to hijack the human ubiquitin system to destroy host proteins that are important to fight infection. In this project we will study how these bacterial proteins work. We will use this new knowledge to produce small chemical molecules that can specifically inhibit the action of these bacterial proteins and help us to better understand how they hijack the human ubiquitin system. This research may suggest novel ways to treat bacterial infections, something that is urgently needed to deal with the rise of bacteria that are resistant to treatment with antibiotics.

Protein ubiquitination is an essential mechanism in eukaryotes to alter protein function and regulate many cellular processes. It requires a cascade of E1 activating enzymes, E2 conjugation enzymes and E3 ligases to attach ubiquitin to substrate proteins. Although the canonical ubiquitination machinery is absent in bacteria they have evolved proteins with E3 ligase activity that are injected into host cells to hijack the host ubiquitin system and manipulate signalling pathways to enable successful bacterial infection.

Some of these bacterial E3s resemble host E3s in terms of structure and catalytic mechanism whereas others are distinct and have no eukaryotic counterparts. These include the NEL (new E3 ligase) family present in Shigella and Salmonella. NEL proteins contain an N-terminal substrate binding leucine rich repeat (LRR) domain and a C-terminal catalytic NEL domain that contains a conserved Cys residue which forms a thioester intermediate with ubiquitin, similar to HECT and RBR ligases.

We have recently used fragment-based covalent ligand screening to develop small molecule probes for the study of the human RBR ligase HOIP. These probes target the active site cysteine and inhibit catalytic activity. We now want to test if we can target the active site cysteine of NEL family members with a similar approach to enable the development of small molecule probes that will aid the study of their role during bacterial infection. To do so we will combine mechanistic and structural approaches to gain insight into the conformational dynamics underlying E3 function and use the knowledge gained to develop specific probes that target the active site of NEL proteins.

The proposed research will provide the first generation of small molecule probes that will enhance our understanding of the function of NELs during infection and could help to validate this protein family as a new target for the development of antibacterial therapeutics.

Economic and Societal Impacts
The rise in antibiotic resistance is becoming a major threat to human health and novel approaches to tackle bacterial infections are urgently needed. Bacteria inject virulence factors (effector proteins) into their hosts that help their replication and survival. Among effector proteins are ubiquitin E3 ligases that target host proteins for degradation to suppress an effective immune response. In this proposal we will characterise the mechanism of a specific family of bacterial E3 ligases, the NEL family, and will aim to develop small molecule probes that target the activity of these enzymes to aid their study in a cellular environment. Such compounds will enable validation of these enzymes as potential novel targets for antimicrobial therapies and may constitute a starting point for inhibitor development.
Through our partnership with GSK, we have access to scientists working on Global Health programmes including Shigella research. If we are successful in our stated aims, there is a clear path towards target validation and eventual translation into drug discovery.

Training workforce for UK economy
The proposed programme will provide the postdoctoral researcher with professional training at the interface of academia and industry. Working in a multi-disciplinary environment, across two organisations will allow them to expand and strengthen their technical capabilities, gain excellent communication and organisational skills and build a diverse professional network. The comprehensive training obtained will ensure that the postdoctoral researcher has all the skills required to be highly competitive when entering the job market and to make important contributions to UK academia and industry.

Education and public engagement
The Francis Crick Institute has an extensive outreach and education programme to engage the general public and inspire young students to follow a career in science. In addition, members from the KR group have participated in non-Crick-based outreach events, most recently the Blue Dot festival, and we will continue to take part in such events. We will also continue to offer one-week placements to A-level students to provide them with first-hand experience of modern biomedical research, and specifically to encourage girls to consider studying STEM subjects. Furthermore, the Crick has a dedicated summer student programme to allow undergraduates to carry out an 8-week laboratory-based research project. Project design, student shortlisting, interviewing and supervision is run by postdoctoral researchers themselves (with guidance provided by PIs) to allow them gain experience in supervision and people management. The KR group has hosted many summer students over the years and the postdoctoral researcher will be encouraged to take part in this programme.