GL12

1: CNRS, Ecole polytechnique; Palaiseau, France

Despite archaea and mycobacteria being evolutionary unrelated, our bioinformatics and experimental studies using the hyperthermophilic archaeon Pyrococcus abyssi have led to the identification of novel thymidylate synthase and mismatch repair enzymes in Mycobacterium tuberculosis (Mtb). In this talk, I will describe how these unexpected discoveries have contributed to fighting and understanding the emergence of anti-microbial resistance against tuberculosis.

Mtb uses the alternative flavin-dependent thymidylate synthase family, ThyX, for the synthesis of an essential DNA precursor. This enzyme does not exist in humans, and it is structurally and mechanistically distinct from human thymidylate synthase, thus making it an ideal drug target. We have exploited the unique nucleotide binding pocket of ThyX proteins to identify non-substrate-based, tight-binding ThyX inhibitors using a target-based screening. The identified inhibitors selectively inhibited the growth of genetically modified bacteria dependent on thyX in a manner mimicking a genetic knockout of thymidylate synthase. Our inhibitors with anti-mycobacterial activity bind within the conserved active site of the tetrameric ThyX enzyme, at the interface of two monomers, partially overlapping with the dUMP binding pocket. Our studies provide new chemical tools for investigating the ThyX reaction mechanism and establish a novel mechanistic and structural basis for inhibition of thymidylate synthesis in Mtb. Currently, we are, in collaboration, developing Mtb-specific AI technologies for optimization of ThyX inhibitors, as well as drug repurposing.

The apparent absence of the canonical mismatch repair (MMR) system in many archaea and actinobacteria led to the discovery of a novel mismatch repair enzyme that uniquely corrects mismatches by creating a double-strand break. NucS. We are currently investigating whether this enzyme is involved in a non-canonical mismatch repair (MMR) mechanism in Mtb. We have also recently performed a large-scale bioinformatics analysis that causally linked the defects of DNA repair to drug resistance in at least 12.5% of clinical isolates with available genome sequences. Notably, a number of the detected single-nucleotide polymorphisms were positively selected during Mtb evolution. Our findings highlight the role of 3 R gene mutations in resistance, emphasizing the need for surveillance to improve early detection and control strategies.