Anti-doping chemistry

Drug metabolism and metabolite profiling

Medicinal chemistry

Nicotinic acetylcholine receptors

Drug Checking

Professor Mal McLeod graduated from Monash University with a BSc (hons) and received a DPhil from the University of Cambridge. He has since held positions at The Australian National University (1997-1998), The University of Sydney (1998-2007) before his appointment as Senior Lecturer at The Research School of Chemistry. He was promoted to Professor in 2023. Mal was awarded the Athel Beckwith Lecutreship (2003), the Rennie Memorial Medal of the Royal Australian Chemical Institute (2005), the Biota Medal for Medicinal Chemistry of the Royal Australian Chemical Institute (2007) and Churchill Fellowship in 2022.

1)    The Chemistry of Sports Drug Testing

My group employs a wide range of techniques to study drug metabolism. These include chemical and enzymatic synthesis of drugs and their metabolites, methods of in vitro metabolism coupled with analysis by GC-MS-MS or LC-MS-MS and molecular biology to engineer improved enzymes with anti-doping applications.

Novel methods to detect designer steroids

Designer steroids are steroidal compounds that have been developed to evade methods of drug detection. Typically containing unusual structural features, these compounds have never been studied for use as therapeutic agents and little if anything is known about their safety or efficacy. Given the absence of toxicological data, ethical considerations usually preclude the in vivo study of these compounds in humans. Work in collaboration with Racing NSW - Australian Racing Forensic Laboratory (Drug Test. Anal. 2015, 00, 0000 see here) has developed a pipeline involving the in vitro metabolism, chemical synthesis and characterisation to study designer steroid metabolism leading to the development of new detection methods for these compounds (ARC Linkage Project). The detailed study of designer steroid metabolism offers the potential to develop untargeted testing strategies that will lead to the early detection of new designer agents.

Pipeline to study designer steroid metabolism with local capabilities highlighted in red and designer steroid hemapolin inset.

New enzymes for the analysis of sulfate metabolites

Sulfate esters are a major class of drug metabolite. Recent research shows that some steroid sulfates can act as long term indicators of steroid administration or serve as markers to distinguish between steroids of exogenous and endogenous origin. Despite the emerging importance of steroid sulfates in drug detection there are no generally accepted methods for the hydrolysis of steroid sulfates, which is required prior to employing many methods of analysis. Recently (Drug Test. Anal. 2015, 00, 0000 see here), we discovered that the arylsulfatase from Pseudomonas aeruginosa serves as a catalyst for the mild hydrolysis of a range of steroid sulfates (Commonwealth of Australia, Anti-Doping Research Program). This purified enzyme is available on large scale and provides an ample supply of sulfatase activity or anti-doping applications. The enzyme also serves as a useful starting point to engineer improved variants of the parent sulfatase with greater activity and broader substrate scope (WADA Scientific Research Grants).

Hydrolysis of testosterone 17-sulfate to testosterone by the P. aeruginosa arylsulfatase and a cartoon of the sulfatase (PDB 1HDH) looking into the enzyme active site.

Other projects within the group include the development of new chemical and enzymatic methods for the synthesis of drug metabolites and the discovery of innovative strategies for drug metabolite detection based on phase II conjugates.

2)    The Medicinal Chemistry of Nicotinic Acetylcholine Receptors (nAChRs)

The group is also active in the area of medicinal chemistry of nicotinic acetylcholine receptors (nAChRs). Here our goal is the development of new drugs to treat neurological disorders and the application of tools like covalent trapping, an extension of the substituted cysteine accessibility method, to better understand where drugs bind on nAChRs.

New nAChR positive allosteric modulators (PAMs) to treat ADHD

Positive allosteric modulators offer great promise as drugs because they potentiate the effect of the endogenous neurotransmitter where it is released rather than overruling natural signalling patterns. NS9283, is one of only a few α4β2 PAMs that have been reported, let alone tested in a human clinical trial, and very little is known about its structure activity relationships (SAR). We are working with collaborators at the University of Sydney to describe the SAR and the efficacy determinants of α4β2 nAChR PAMs such as NS9283 (NHMRC Project Grant). Our goal is to develop new high affinity and high potency compounds to be used as drug candidates and pharmacological tools for (α4)₃(β2)₂ receptors containing a unique α4-α4 interfacial binding site.

Positive allosteric modulator NS9283 (grey) bound at the interface of the L. stagnalis AChBP (PDB 4NZB)

Interrogating nAChR binding sites with covalent trapping

Establishing the binding site for non-competitive inhibitors or allosteric modulators in ligand-gated ion channels presents significant challenges. We have developed the method called covalent trapping to reveal ligand binding sites that employs cysteine mutagenesis in combination with synthetically derived reactive probes (Chem. Commun. 2012, 48, 6699 see here). Covalent trapping of the probe depends on binding in proximity to the cysteine mutant and provides strong evidence of binding site location. We recently showed for the first time that MLA binds to the α4-α4 interface but not the β2-α4 interface of the (α4)₃(β2)₂ nAChR leading to non-competitive inhibition of the receptor (J. Biol. Chem. 2013, 288, 26521 see here). Ongoing work is targeting other allosteric ligands to establish ligand binding sites. Once a binding site is revealed and a structural model developed then improved ligands can be designed, synthesised and tested.

Structural model of MLA maleimide covalently trapped at the α4-α4 interface of the (α4)₃(β2)₂ nAChR

Other project within my group involve the efficient total synthesis of nAChR-active natural products and using structural models combined with synthesis to afford ligands with greater selectivity for different receptor subtypes (α7/α4β2) or stoichiometries ((α4)₃(β2)₂/(α4)₂(β2)₃).

Graduate, honours and undergraduate projects are available in a range of areas including, the synthetic medicinal chemistry of nicotinic acetylcholine receptors, and the development of new chemical, enzymatic and analytical approaches to detect drugs in sport.