WEHI PhD Completion Seminar hosted by Professor Aaron Jex

Alex Lam

PhD Student – Jex Laboratory, Infection & Global Health division, WEHI

Chemoproteomic Target Deconvolution in Giardia duodenalis

Davis Auditorium

Join via SLIDO enter code #WEHIphdcompletion

Including Q&A session

Followed by refreshments in Tapestry Lounge

The gastrointestinal parasite Giardia duodenalis causes ~200 million symptomatic infections annually, disproportionately in lower socioeconomic tiers and children. Chemotherapeutic interventions are limited to nitroheterocyclic antibiotics such as metronidazole. However, high doses are toxic and drug-resistant treatment failures occur in up to 20% of cases, highlighting the urgency of novel and safer chemotherapeutics.

We previously identified a series of drug-like kinase inhibitors via high-throughput, phenotypic screening with sub-micromolar anti-giardial activity. To identify its high affinity kinase target(s) in Giardia, we performed a high-throughput thermal proteome profiling experiment using the proteome integral solubility alteration assay coupled with data-independent acquisition (PISA-DIA) mass spectrometry (MS), to probe thermal-stabilised proteins and shortlisted candidate drug binders. After stringent filtering, we identified 49 ‘hit’ proteins, of which four were selected based on in silico characterisation and kinase domain-containing achitectures. Alongside, we immobilised a representative compound to azide-agarose supports through “Click” chemistry and performed a conjugated target pulldown experiment. Using uncoupled beads and competitive elution as negative controls, we excluded low-affinity/non-specific binders, and identified only 10 hit proteins. From these, two contained a protein kinase domain, which were additional candidates to the PISA-DIA experiment. We have since biochemically validated target engagement of the shortlisted hits. We recombinantly expressed 3/6 of the kinase domains, and performed differentially scanning fluorimetry (DSF) and native MS. In the DSF, we demonstrated ligand-induced stabilisation of a candidate under thermal challenge, a hallmark of protein-ligand interactions. Subsequently, native MS, validated this ligand-target association via a mass shift. As a final step, we demonstrated the kinase activity of a recombinantly expressed candidate, based on its ability to phosphorylate myelin basic protein, and that binding of our ligand competitively inhibited this phosphorylation event. Together this workflow incentivises high-throughput, target-centric screening campaigns for repurposing clinically-approved kinase inhibitors as chemotherapeutic interventions against G. duodenalis.

All welcome!