Resistance to natural antibiotics is a phenomenon that has existed for millions of years, due to competitive interactions within microbial communities. The recent development of synthetic antibiotics and their widespread use have contributed to the emergence of new resistance mechanisms. Trimethoprim is a synthetic antibiotic commonly used to treat urinary tract infections and as a preventive measure in livestock and fish farming. Dihydrofolate reductase type B (DfrB) effectively confers resistance to trimethoprim: the latter targets the ubiquitous bacterial dihydrofolate reductase, FolA, but does not interact with DfrB due to significant structural differences between these two dihydrofolate reductases. DfrB is a homotetrameric enzyme, whose efficiency is 100 times lower than that of FolA, a monomeric enzyme. The Pelletier laboratory is interested in the evolution of the multimerization capacity of DfrB and its function (Lemay-St-Denis et al. (2025)). At the same time, we are investigating the evolutionary pathways that have led to the recruitment of DfrB genes into the modern resistome: we have reported that functional DfrBs are found in various environments that are not linked to human activities (Lemay-St-Denis et al. (2021), Cellier-Goetghebeur et al. (2022)). 

Although DfrB confers resistance to the antibiotic trimethoprim, there is still no antibiotic that targets DfrB. We have also reported the first specific inhibitors of this emerging drug target, obtained through fragment-based design (Bastien et al. (2012)). We observed that a variety of small aromatic compounds offer millimolar inhibition of DfrB1, which is consistent with the proposed “primitive” nature of its relatively promiscuous binding site (Schmitzer et al. (2004)). By these means, small aromatic molecules of 150-250 g/mol (fragments) selectively inhibiting DfrB1 in the low millimolar range were identified. The weakly inhibitory molecules served as a basis for testing more complex compounds, which allowed an increase in affinity from the millimolar to the micromolar range. More complex symmetrical bis-benzimidazoles and a bis-carboxyphenyl allowed selective inhibition of the target in the low micromolar range (Ki = 2-4 μM) (Yachnin et al. (2011)). We are currently working on the next generation of DfrB inhibitors, testing both covalent and competitive inhibitors that combine our previous work and our knowledge of natural ligands for this enzyme. 

P450 monooxygenases are heme-containing enzymes used in biocatalysis. In recent years, there has been increasing interest in using P450 enzymes as biocatalysts due to their ability to perform highly selective reactions under mild conditions, reducing the environmental impact of chemical processes. P450 BM3 from Bacillus megaterium hydroxylates various substrates using NADPH and molecular oxygen. Mutations near the heme have expanded its substrate range beyond fatty acids. Colorimetric methods, such as bacterial colony-based assays, accelerate substrate screening. Expanding P450 BM3's substrate range for industrially relevant reactions would improve efficiency and sustainability by reducing reliance on harsh chemical catalysts. This versatile enzyme could be used in pharmaceuticals, agrochemicals, and fine chemicals, providing access to new chemical scaffolds and stereoisomers for high-value products.

Adapting the P450 BM3 enzyme to a flow reactor system could address certain challenges such as costly cofactor dependency, enzyme reusability and uncoupling efficiency. Flow chemistry is the process of performing chemical reactions in a flow reactor, such as a capillary or micro- structured device, then, the residence time determines the reaction time. The reactants are first pumped through a mixing device and then driven into a controlled temperature flow reactor; the technology of the flow chemistry makes it possible to better control the reaction parameters, such as the mixing, concentrations, the temperature or the reaction time. This means that we can take advantage of more productive, selective and cleaner reactions. It is an approach that offers better reaction control and reduces side reactions, therefore radically different from the traditional chemistry of performing reactions in glass vials or Continuous stirred-tank reactors.