The evolution of antibacterial chemotherapy: targeting RecA to sabotage antibiotic tolerance and resistance mechanisms

Antibiotic resistant bacteria are rendering the current supply of available antibacterial drugs ineffective at an alarming rate and there is a dearth of novel drug targets for the treatment of bacterial infectious diseases. New strategies are required to combat pathogenic bacteria and in this context RecA has emerged as an intriguing candidate for inhibition studies. In the bacterial kingdom, the RecA protein is a highly conserved recombinase enzyme that mediates DNA repair and horizontal gene transfer and across all species it almost uniformly regulates the SOS response to DNA damage. Recent evidence suggests that these RecA-controlled processes are responsible for an increased tolerance to antibiotic chemotherapy and they up-regulate pathways which ultimately lead to full-fledged antibiotic resistance. We propose targeting RecA with small molecules to sabotage the molecular mechanisms which are believed to cause antibiotic chemotherapy to fail. Towards the goal of validating RecA as an important and novel target for the chemotherapeutic treatment of bacterial infectious diseases we have studied the interaction of metal-dithiols, nucleotide analogs and drug-like small molecules with the RecA protein. Upon activation RecA binds ssDNA and performs ATP hydrolysis, therefore we observed either a reduction of RecA-ssDNA binding or ATP hydrolysis in the presence of potential inhibitors using fast and efficient screening assays. As the size and complexity of the compound libraries increased in our studies, the methods we employed to identify inhibitors evolved to meet the demand they imposed. In all, more than 64,000 compounds were assayed against RecA and we identified several lead structures which were active against RecA in Escherichia coli cell cultures. We demonstrate that cell-permeable inhibitors of RecA are capable of abrogating the SOS response and potentiate the toxicity of bactericidal antibiotics, e.g. ciprofloxacin. The results of this study suggests that RecA may serve as a novel antibacterial drug target not belonging to any class of currently prescribed antibiotics, and which has not previously been examined in this regard

DPYD and fluorouracil-based chemotherapy: Mini review and case report

5-Fluorouracil remains a foundational component of chemotherapy for solid tumour malignancies. While considered a generally safe and effective chemotherapeutic, 5-fluorouracil has demonstrated severe adverse event rates of up to 30%. Understanding the pharmacokinetics of 5-fluorouracil can improve the precision medicine approaches to this therapy. A single enzyme, dihydropyrimidine dehydrogenase (DPD), mediates 80% of 5-fluorouracil elimination, through hepatic metabolism. Importantly, it has been known for over 30-years that adverse events during 5-fluorouracil therapy are linked to high systemic exposure, and to those patients who exhibit DPD deficiency. To date, pre-treatment screening for DPD deficiency in patients with planned 5-fluorouracil-based therapy is not a standard of care. Here we provide a focused review of 5-fluorouracil metabolism, and the efforts to improve predictive dosing through screening for DPD deficiency. We also outline the history of key discoveries relating to DPD deficiency and include relevant information on the potential benefit of therapeutic drug monitoring of 5-fluorouracil. Finally, we present a brief case report that highlights a limitation of pharmacogenetics, where we carried out therapeutic drug monitoring of 5-fluorouracil in an orthotopic liver transplant recipient. This case supports the development of robust multimodality precision medicine services, capable of accommodating complex clinical dilemmas

Directed molecular screening for RecA ATPase inhibitors

The roles of bacterial RecA in the evolution and transmission of antibiotic resistance genes make it an attractive target for inhibition by small molecules. We report two complementary fluorescence-based ATPase assays that were used to screen for inhibitors of RecA. We elected to employ the ADP-linked variation of the assay, with a Z′ factor of 0.83 in 96-well microplates, to assess whether 18 select compounds could inhibit ATP hydrolyis by RecA. The compounds represented five sets of related inhibitor scaffolds, each of which had the potential to cross-inhibit RecA. Although nucleotide analogs, known inhibitors of GHL ATPases, and known protein kinase inhibitors were not active against RecA, we found that three suramin-like agents substantially inhibited RecA's ATPase activity

A complementary pair of rapid molecular screening assays for RecA activities

The bacterial RecA protein has been implicated in the evolution of antibiotic resistance in pathogens, which is an escalating problem worldwide. The discovery of small molecules that can selectively modulate RecA’s activities can be exploited to tease apart its roles in the de novo development and transmission of antibiotic resistance genes. Toward the goal of discovering small-molecule ligands that can prevent either assembly of an active RecA-DNA filament or its subsequent ATP-dependent motor activities, we report the design and initial validation of a pair of rapid and robust screening assays suitable for the identification of inhbitors of RecA activities. One assay is based on established methods for monitoring ATPase enzyme activity and the second is a novel assay for RecA-DNA filament assembly using fluorescence polarization. Taken together, the assay results reveal complementary sets of agents that can either selectively suppress only the ATP-driven motor activities of the RecA-DNA filament or prevent assembly of active RecA-DNA filaments altogether. The screening assays can be readily configured for use in future automated HTS projects to discover potent inhibitors that may be developed into novel adjuvants for antibiotic chemotherapy that moderate the development and transmission of antibiotic resistance genes and increase the antibiotic therapeutic index