Enzyme mechanisms and inhibition

Over 300 drugs in clinical use are enzyme inhibitors, including anti-cancer, anti-hypertension, anti-diabetes, anti-HIV drugs, and many antibiotics. The influenza drugs oseltamivir and zanamivir and the anti-cancer drug forodesine are all transition state mimics that were designed based on transition state structures derived from KIEs.

Antibiotic resistance

The WHO calls antibiotic resistance a "slow motion tsunami". It is expected to cause 10 million additional deaths per year by 2050 (vs. ~1 million per year from HIV at its peak) on top of the burden of already hard-to-treat diseases like tuberculosis, which killed 1.6 million in 2017. In Canada, some types of antibiotic resistance are increasing 10-fold per decade. We aim to address this crisis by creating new inhibitors against essential bacterial enzymes.

α-Carboxyketose synthases

The α-carboxyketose synthases (αCKSs) are essential for bacterial virulence/survival, and are attractive broad-spectrum antibiotic targets. The three main αCKSs are DAHP synthase (DAHPS), KDO8P synthase (KDO8PS) and NeuB.

We have developed potent inhibitors against all three αCKSs. The first inhibitors in this class, oxime-based molecules, are potent competitive inhibitors and transition state mimics. They bind tightly and dissociate slowly, with NeuNAc oxime binding to NeuB being effectively irreversible.

These inhibitors have some good characteristics (very high affinity, long residence times in the active site), and some undesirable ones (slow binding to some proteins, not binding to all the active sites in the tetrameric protein). We have performed mutagenesis studies, determined multiple crystal structures, are studying protein dynamics by hydrogen-deuterium exchange (in collaboration with Derek Wilson's lab at York University), and are performing protein NMR studies to understand how inhibition works, and how binding information from one active site is transmitted tens of Ångstroms to the neighbouring active sites.

We are continuing our inhibitor development in several directions — exploring the binding affinity of related inhibitors, using fragment-based inhibitor design (wiki) to explore new functional groups, and searching for new transition state mimics. We now have inhibitors with K_i values of 10 nM, and inhibitors (labelled "0") that stop bacterial growth in culture by inhibiting DAHPS.