Découverte et optimisation d’inhibiteurs pour des enzymes DfrBs impliquées dans la résistance 
bactérienne

Trimethoprim (TMP) is a widely used antibiotic, both clinically as well as preventively in livestock farming and aquaculture. This broad usage causes selective pressure on bacteria. The target of this antibiotic is the chromosomal bacterial dihydrofolate reductase enzyme (Dfr), which is essential in the folate biosynthesis pathway and, therefore, in cell proliferation. Several types of resistance to TMP have been identified, including the transfer of an antibiotic-resistant plasmid between pathogenic bacteria. In clinical environments, this plasmid is of great concern, since this environment is conducive to a greater selective pressure causing the proliferation of TMP-resistant bacteria. TMP-resistant pathogenic bacteria with the plasmids carrying Dfr type B genes (dfrB) have been identified in aquaculture, livestock and wastewater. The dfrBs genes are normally tracked indirectly by the identification of integron elements underestimating the prevalence of dfrBs genes. Because it is imperative to determine the prevalence of these genes, we have demonstrated the possibility to determine their prevalence by the direct search of these genes in a North American library of TMP-resistant clinical samples. To date, no antibiotics are known to overcome the resistance caused by DfrBs. In fact, there is limited data on this emerging family. We know that DfrBs are evolutionarily and structurally unrelated to prokaryotic and eukaryotic chromosomal Dfrs. It is of great importance to develop selective inhibitors for these enzymes that could subsequently serve as potential novel antibiotics. Eventually, dual therapy with TMP and this new antibiotic could increase the antibacterial effectiveness of TMP. In order to develop such molecules, we have deployed several approaches including a follow-up of previously reported fragment-based lead design (FBLD), the design of substrate analogs as inhibitors targeting two therapeutic enzymes, as well as high-throughput screening of a library of marine invertebrate extracts. Since DfrB1 is the best studied among the DfrB family, it was used as a model for these approaches. In the optimization of inhibitors from FBLD, we studied the inhibitor’s mode of binding to direct synthetic efforts for the next generation of potential inhibitors. Thus, knowledge in the fields of enzymology and pharmacology enhances our understanding of the mode of binding of these inhibitors. Members of the DfrB family share 77% to 100% protein sequence similarity. To our knowledge, no kinetic studies of DfrB members other than DfrB1 have been performed. For most members, the correlation of TMP resistance and their source have not been confirmed. We confirm their dihydrofolate reductase activity as well as their resistance to TMP. With these encouraging results, we propose a similar mode of inhibition for all DfrBs. We demonstrate that inhibitors of DfrB1 also inhibit other DfrBs. Our advances indicate that it would be possible to have a lead compound that inhibits all members of this family.

Keyphrases: inhibition, Dihydrofolate reductases B, antibiotic resistance, trimethoprim, inhibition, X-ray crystallography, enzymatic kinetics, in silico low molecular dynamics, prevalence of genes