Exploiting prokaryotic nucleotide excision repair as a novel antimicrobial target for combinatorial therapies in cancer patients

Infections are a group of pathologies which are increasing in severity due to the lack of new therapeutic approaches and the concomitant rise of antimicrobial resistance. This issue is particularly concerning in cancer patients who are often immunocompromised as a side effect of chemotherapy.

Most anticancer treatments target rapidly dividing cancerous cells but also hit immune system cells and bacteria. Bacteria, however, possess efficient DNA repair systems that allow them to escape otherwise lethal damage. In this thesis, we evaluated strategies to extend the cytotoxic activity of DNA-damaging agents to bacteria by inhibiting nucleotide excision repair (NER), a central prokaryotic DNA repair system responsible for recognising various damages.

We began our study with ATBC, the first effective NER inhibitor in mycobacteria, to understand its mechanism of action and molecular target. We confirmed its activity on Escherichia Coli and used in silico tools to predict its binding to UvrA, the first responder in the NER pathway. We then confirmed this activity in vitro by directly measuring UvrA's ATP turnover and its compromised DNA binding ability. Furthermore, we evaluated the compound's ability to inhibit the activity of the entire UvrABC complex through an incision assay. This data provided the rationale for moving forward in our study.

We designed two screening approaches to develop novel NER inhibitors to be used alongside the widely used anticancer drug cisplatin. We screened an FDA-approved drug library composed of ~3000 compounds in combination with cisplatin as well as virtually screening ~120000 compounds directly against UvrA ATP-binding pockets. The hits that emerged from this approach were subjected to a wide range of in vivo, biochemical, in silico and single molecule studies to confirm NER as a molecular target and to elucidate their mechanism of action.

We highlighted 8 principal candidates, among which 5 showed increased antibacterial activity against a clinically relevant E. coli strain responsible for multidrug-resistant infections. In doing so, we have taken the first step in developing a novel antimicrobial approach that could represent a valuable tool in fighting co-infections in cancer patients.