Medicinal and pharmaceutical chemistry, enzymology, and synthesis

Our research takes advantage of chemical tools to study and manipulate biological systems. We are especially interested in antibiotic resistance, P450 enzymes and biocatalysis (towards green chemistry).

The Auclair research group has expertise in synthesis, medicinal chemistry, enzymology, biophysical analyses, and protein purification, expression and engineering (including bioconjugation). The main goal of our research is to understand how enzymes work and how they can be harnessed or perturbed, especially for pharmaceutically relevant proteins such as antibiotic targets, resistance-causing enzymes, drug activation and drug metabolism. The engineering of enzymes for use in asymmetric synthesis is also of interest. Results of these studies have implications in fields as varied as medicine, biotechnology, industrial processing, agriculture and food science.

Current projects fall under two areas:

1) Antibiotics and antibiotic resistance

2) P450 enzymes

1) Antibiotics and Antibiotic Resistance

Antibiotics are some of the pharmaceutical agents that have improved human life expectancy the most, yet they have one of the shortest utility lifespan of all drugs. By Darwinian selection and horizontal transfer, bacteria eventually develop resistance to all antibacterial agents, and as a result, antibiotic resistance is currently one of the most rapidly spreading health threats worldwide.

The strategies pursued in my group to overcome antibiotic resistance include:

1) study enzymes involved in antibiotic resistance

2) design inhibitors of the resistance mechanisms

3) look for new antimicrobial agents active against resistant strains

The specific goals of this research program include:

1) use biophysical tools to study mechanisms of antibiotic resistance (including aminoglycoside antibiotics)

2) rationallly design molecules that block antibiotic resistance

3) identify novel antibacterial agents with new modes of action

4) elucidate the mode of action of these antimicrobials

5) use medicinal chemistry to improve the biological properties of our molecules

An example: Aminoglycoside N-6'-acetyltransferase

Aminoglycoside N-6'-acetyl transferases (AAC6's) are important enzymes involved in the resistance to aminoglycoside antibiotics. AAC6's are also in the same familly as many histone acetyltransferases which are involved in gene regulation and in cancer. AAC6's specifically acetylate aminoglycosides on the nitrogen at position 6' (see Figure below).

Although this protein had been crystallized (see Figure below) with its cofactor coenzyme A (in green), little was known about the structural elements involved in binding the aminoglycoside substrates and about the mechanism of this enzyme. We have prepared substrate derivatives that show nanomolar competitive inhibition of AAC6'Ii. Some of these molecules have been crystallized with the enzyme and the structure of the complex has provided crucial information about this familly of enzymes. Our synthetic studies towards the preparation of these inhibitors have led to many important discoveries, including: 1) a novel strategy to regioselectively derivatize aminoglycosides at the N-6' , 2) allosteric effects taking place with substrates and inhibitors, 3) novel inhibitors that block aminoglycoside resistance in cells, 4) resistance inhibitors that are prodrugs activated by the CoA biosynthetic pathway.

Overall, this project consists of designing, synthesizing and testing novel substrates and inhibitors for mechanistic and structural studies (X-Ray and NMR) of AAC6' enzymes..


2) P450 enzymes

The cytochrome P450 enzymes (P450s or CYPs) form a ubiquitous family of heme proteins involved in xenobiotic metabolism, biosynthesis of steroids, lipids, vitamins, and natural products. Human P450s are involved in the metabolism of more than 90% of current pharmaceuticals. The P450 enzyme CYP3A4 alone is responsible for the metabolism of ~50% of all drugs. P450 enzymes therefore play a key role in drug-drug interactions. These monooxygenases catalyze a variety of reactions such as hydroxylations, epoxidations, N- or O-dealkylations, Baeyer-Villiger oxidations, and more. P450 enzymes are of considerable interest in synthetic organic chemistry because of their impressive ability to catalyze the insertion of oxygen into non-activated C-H bonds, one of the most challenging transformation for organic chemists.

My research interest for P450 enzymes encompasses two aspects:

1) Optimization of P450 enzymes for biocatalytic applications

2) Drug metabolism

3) Enzyme mechanism and function


An example: P450 product predictability and orthogonal conjugation

Asymmetric synthesis remains one of the most challenging area of organic chemistry. The preparation of chiral molecules is essential to the pharmaceutical industry. P450 enzymes are powerful catalysts capable of regio- and stereo-selective hydroxylation at unactivated C-H bonds. Interestingly, many human P450 isoforms accept a large variety of substrates which makes them ideal catalysts for asymmetric organic synthesis. The use of these enzymes in synthesis suffers from a number of disadvantages: need for 2 expensive cofactors, incompatibility with organic conditions and lack of stability. Moreover, although many isoforms are highly promiscuous towards substrates, it remaims impossible to predict the products of these enzymes. We have developed ways to avoid the use of the expensive cofactors and have also shown that some of these enzymes can be active in organic solvents. There is still room for improved activity and stability and these are the focus of our research. In addition, we are developing novel tagging systems that will allow prediction of the products. Finally, we are developing chemical methods to derivatize P450 enzymes regioselectively and orthogonally. We hope to demonstrate the utility of these systems in various applications.




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