Research

Medicinal and Pharmaceutical Chemistry, Green Chemistry, Microbiology, Enzymology The Auclair research group has expertise in synthesis, medicinal chemistry, mechanoenzymology, biocatalysis, microbiology, as well as protein purification, expression, and engineering (including bioconjugation). Check out our projects below! Mechanistic Enzymology Mechanoenzymology Medicinal Chemistry Antibacterial Development

Mechanistic Enzymology P450/CPR

Cytochrome P450 enzymes (CYPs) and their redox partner cytochrome P450 reductase (CPR), play important roles in the biosynthesis of steroids and lipids, as well as in the metabolism of small molecule medicines. They do so by catalyzing oxidation reactions, the most common of which is the hydroxylation of inactivated C-H bonds. This is one of the most challenging reactions for chemists, making CYPs an intriguing research focus. The kinetics of human CYP3A4 is, however, complicated by allostery and cooperativity. This impacts drug metabolism and thus often puts this enzyme at the forefront of drug-drug interactions. Moreover, its partner enzyme, CPR, also indirectly affects drug metabolism by acting as an essential accessory protein to CYPs. CPR is a reductase that mediates electron transfer from nicotinamide adenine dinucleotide phosphate (NADPH) to the heme iron of CYPs via two tightly bound flavin co-factors. Proper function of CPR, as well as its interaction with CYPs, is highly dependent on its conformational dynamics. Our group has interest in delineating the mechanism and dynamics of CYPs and CPR. For example, we use various techniques to 1) Investigate the allosteric properties, both kinetically and structurally, of human CYP3A4, and 2) Elucidate the key structural changes involved during electron transfer by CPR, as well as those involved in the proper interaction with CYP3A4. Methods used in these studies are highly diverse, including but not limited to: site-directed mutagenesis, small-molecule bioconjugation, and hydrogen-deuterium exchange mass spectrometry (HDX-MS).

Mechanoenzymology

If you think about how enzymes are traditionally used in research or industry, you’ll probably think of dilute, aqueous conditions, where your enzyme is surrounded by bulk water. However, if you compare this to how enzymes are actually found in nature, you’ll see that their natural environments are drastically different than what we provide them in lab. For example, many fungal enzymes are secreted directly into their environment, where they are on a surface exposed to air moisture. In our lab, we try to provide a better mimic of enzymes’ natural environments by adding only very few equivalents of water to our reactions, resulting in a solid consistency that we refer to as “moist-solid” reaction mixtures. Because we work with this solid-like consistency, we use mechanical mixing methods such as ball milling to stir our reactions. Using enzymes under these mechanically mixed, moist-solid conditions makes them part of the field of “mechanoenzymology”. We have shown that many enzymes can thrive under these conditions and even show increased selectivity, and therefore our group is focusing on using enzymes in the absence of bulk water as a tool for sustainable chemistry.

Mechanoenzymology for the breakdown of natural and synthetic polymers

We have used our mechanoenzymatic technology to depolymerize both biopolymers such as cellulose, hemicellulose, and chitin directly from biomass or paper, but also synthetic polymers such as polyethylene terephthalate (PET) and polylactic acid (PLA) plastics. We have demonstrated that using enzymes under moist-solid conditions has numerous advantages over using enzymes in analogous in-solution reactions, such as: increased selectivity for the desired product (ex monosaccharides from natural polymers or the building blocks of plastics), no requirement for chemical or thermal pre-treatment of the substrate, and a reduction in the production of waste.

Currently, we are focusing on expanding upon this technology to break down other plastics, with the goal of finding efficient, and environmentally friendly recycling methods to help tackle the plastic pollution problem.

Mechanoenzymology for the capture of carbon dioxide

Greenhouse gases are a prominent environmental concern. In respect to the global energy and environmental crises, the development of effective methods to capture and convert carbon dioxide are of profound interest. We have already demonstrated the numerous advantages of using enzymes under moist-solid conditions, including the ability to work with substrates that are poorly soluble under dilute aqueous conditions. For this reason, we are also interested in adapting our mechanoenzymatic technology to gaseous substrates, including the enzymatic capture and conversion of carbon dioxide into useful organic compounds through cascade transformations.

Medicinal Chemistry

Antibiotics are essential to modern medicine, not only to cure infections but also to protect weak patients and those undergoing surgery. Unfortunately, antibiotics do not work so well anymore - a phenomenon known as antibiotic resistance. This is also a problem for antifungal and antimalarial drugs. We are interested in designing new antimicrobial agents that have the potential to become new drugs to treat infections. Our current focus is on pantothenamide-mimicking molecules.

Pantothenamides are a new structural class of antimicrobials with a unique mode of action, making them especially interesting for the development of new pharmaceuticals. They act by utilizing bacterial and parasitic coenzyme A biosynthetic enzymes that bioactivate pantothenamides into corresponding coenzyme A antimetabolites that can infiltrate further downstream coenzyme A utilizing processes, resulting in bacterial/parasitic cell death.

However, pantothenamides are unstable in human blood due to their susceptibility to cleavage by serum enzymes, called pantetheinases. We have identified several strategies to create pantothenamide-mimicking molecules with nanomolar potency and high blood stability. These techniques involve alterations at either the geminal dimethyl group, the β-alanine moiety, or the labile amide group.

Currently, we use both traditional and novel bioisosteres to stabilize the labile amide bond in pantothenamides, which leads to potent growth inhibitors of the malaria parasite and/or some bacteria. This medicinal chemistry project involves organic synthesis and testing of the antibacterial activity in our lab, as well as studies with the malaria parasite that are performed through collaboration.

Antibacterial Development

In the fight against antimicrobial resistance, it is crucial that we not only develop new antibiotics but also alternative treatments with a reduced frequency of resistance development. We have pioneered a new strategy to fight bacterial infections, termed bacterio-modulation, where we resensitize intracellular bacteria to our immune system by inhibiting the mechanisms that bacteria use to survive despite our immune systems’ defences. In this project, we are targeting for inhibition the bacterial itaconate degradation pathway.

During an infection, macrophages engulf bacteria and kill them through a variety of mechanisms, including the use of antimicrobial molecules. Itaconate is one such molecule that blocks a metabolic pathway required for bacterial survival within macrophages, thus selectively killing the intracellular pathogens. Bacteria such as Salmonella enterica ser. Typhimurium, Pseudomonas aeruginosa and Mycobacterium tuberculosis have evolved to express itaconate degrading enzymes that help them to survive intracellularly and lead to difficult-to-treat long term infections. This three-enzyme pathway converts itaconate into molecules that can be used as a food source for bacteria.

This project aims at demonstrating that inhibition of the itaconate degradation pathway is a promising approach for the treatment of intracellular infections. To achieve this goal, we synthesize molecules and test them using a variety of in vitro assays at the enzyme, cellular, and infected-macrophage levels. Our first generation of inhibitors were prodrugs selectively activated by the bacterium’s coenzyme A biosynthetic machinery into substrate-like molecules.

We are continuing to synthesize better inhibitors and develop new ways to test them as our understanding of the structure-activity relationships of these compounds improves. We believe that this strategy will have a reduced potential for resistance development by targeting bacteria only while inside of macrophages, but not in normal non-infection related environments.