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Pfleger: NanoBRET, Receptor Complexes, and the Translation of GPCR Pharmacology

GPCR pharmacology has long depended on recombinant systems in which receptors are expressed and studied as isolated proteins. But in living tissue, GPCRs do not operate alone - they form dynamic complexes with other receptors, scaffold proteins, and cytoskeletal components, and those interactions reshape conformation, ligand binding, and signaling output in ways that isolated assays cannot detect.

Kevin Pfleger's research at the University of Western Australia sits at the intersection of assay technology, receptor pharmacology, and translational medicine. His group developed BRET and NanoBRET-based live-cell platforms that capture protein-protein interactions and signaling dynamics in real time, preserving the kinetic information that endpoint assays routinely discard. Those platforms have been applied across arrestin biology, receptor complex pharmacology, and the angiotensin and chemokine receptor systems, including CCR2.

The conversation also covers what it means when cell biology reaches the clinic. Pfleger co-founded a biotech company that completed phase 2 clinical trials in focal segmental glomerulosclerosis and diabetic kidney disease - and watching protein levels fall in patients' urine is, for him, the reason most scientists enter medical research in the first place.

About the Guest

Kevin Pfleger is Professor, Director of Biomedical Innovation, and Head of Molecular Endocrinology and Pharmacology at the University of Western Australia and the Harry Perkins Institute of Medical Research. His research centers on GPCR functional interactions, with a focus on arrestin signaling, receptor complex pharmacology, and the angiotensin and chemokine receptor systems. He developed BRET and NanoBRET-based live-cell assay platforms now widely adopted across the GPCR field, and serves as Deputy Director of the Australian Research Council Centre for Personalised Therapeutics Technologies. Pfleger co-founded a publicly listed biotech company that has completed phase 2 clinical trials in focal segmental glomerulosclerosis and diabetic kidney disease, and holds leadership roles across Australian biomedical innovation and entrepreneurship.

Scientific Themes of the Conversation

Real-time vs. endpoint assays - why kinetics change the pharmacological picture
GPCR receptor complexes and the limits of isolated receptor screens
Bias signaling as a kinetic and time-dependent phenomenon
NanoBRET and the evolution of live-cell assay design for GPCR biology
Academic entrepreneurship and the path from bench to phase 2 clinical trials
Funding diversification and the transferable skills embedded in PhD training

Key Insights from the Conversation

1. Endpoint Assays Can Miss the Biology Entirely

A single measurement at a fixed time point collapses all the kinetic information in a signaling event into one number. Pfleger describes profiles where the peak of a response occurs early and then subsides - and where an endpoint assay taken after that peak would return a flat or misleading result. The problem is not the assay format itself; it is the assumption that one time point is enough.

2. Bias Signaling Is a Kinetic Problem, Not Just a Pathway Problem

Spider plots comparing signaling bias across multiple pathways are a standard output in GPCR pharmacology. Pfleger challenges a silent assumption embedded in most of them: that the time point chosen for each measurement is irrelevant. If bias has a kinetic component - and the evidence suggests it often does - then what gets called "biased" may be an artifact of when you looked, not what the receptor is actually doing.

3. Receptors in Complexes Behave Differently Than Receptors in Isolation

Standard drug screens present a receptor as a discrete, isolated target. In the body, that receptor is complexing with other receptors, scaffold proteins, and cytoskeletal elements - and those interactions alter its conformation and drug-binding properties. Pfleger argues this is not a theoretical complication; it is a pharmacological reality that current screening approaches largely bypass. Understanding receptor complexes is where the next layer of selectivity and specificity in drug discovery lives.

4. BRET Went From a Handful of Labs to the Go-To Platform

When Pfleger began working with bioluminescence resonance energy transfer in GPCR research, it was a niche technology used by a small number of groups worldwide. That has changed. The development of NanoBRET - including live-cell ligand binding assays built on CRISPR-expressed receptors - contributed to an adoption curve that now spans most GPCR pharmacology labs. For Pfleger, the reach of the technology is not a source of pride as much as a signal that the field recognized what real-time, live-cell measurement offers that nothing else does.

5. The Long Road From HEK Cells to a Patient's Urine

Pfleger describes a particular moment from the phase 2 clinical trial his spin-out company ran in kidney disease: seeing urinary protein levels fall in patients on the compound. He had spent years characterizing arrestin interactions with angiotensin and CCR2 receptors in cell-based systems, working through the logic of functional selectivity in disease-relevant pathways. That cell biology eventually became a clinical hypothesis, then a compound, then a trial result. What made it humbling, he notes, were the calls from patients' families asking to get into the trial.

6. The PhD Trains You for Far More Than the Bench

The skills built during doctoral training - managing time, coordinating teams, working within resource constraints, communicating under uncertainty - map directly onto the skills required in industry, entrepreneurship, and scientific leadership. Pfleger made this case from personal experience: spending years pitching to rooms of executives taught him that the win-win logic he needed there was not foreign to scientific collaboration. It was the same logic, applied to different stakeholders. The analytical discipline of the PhD, he argues, is not a niche credential. It is a transferable toolkit most PhD students do not realize they have built.

Episode Timeline

Timestamps are AI-generated from the transcript and may require minor adjustment after final audio editing.

00:00 Introduction
01:36 Pfleger's philosophy: science as a team sport
03:54 From Cambridge to Edinburgh - an accidental PhD and the origin of a career
06:10 Working on biosignaling before the field had a name for it
09:12 Postdoc pivot: from GnRH receptors to arrestin interactions in Australia
12:47 The spin-out - how a discovery became a phase 2 clinical trial in kidney disease
16:56 Why grant-only funding is becoming unsustainable and what to do instead
22:21 What a PhD actually trains you for
25:42 NanoBRET, live-cell assay design, and the CRISPR ligand binding platform
27:54 Why endpoint assays miss the peak - the case for real-time kinetics
29:44 Bias signaling as a time-dependent measurement
32:26 Screening receptors in complexes, not in isolation
40:03 When cell biology reached the patient

Selected Quotes

"The number of times we see very interesting profiles and if you were to do an endpoint assay, you would completely miss the point because you've missed the peak. A lot of the time you get a signature, and you can compare those signatures and see a lot of nuance."

"Traditionally screening has happened with receptors in isolation, but in reality, in the body, these receptors are complexing. They're interacting with the cytoskeleton, with other proteins, with other receptors, and that will change their conformation. That will change their ability to bind."

"We were actually seeing the protein levels in the urine going down in the patients. That is really why I think most people get into medical research. It's very humbling."

"A PhD is not just a training in working at a bench and writing a thesis. It's so many other things - whether it's about time management, people management, managing a budget. These are all business skills. And these are all skills that can be transferred."

About this episode

Dr. Pfleger trained as a pharmacologist and obtained his Ph.D. at the University of Edinburgh. I sat down with Kevin to chat about GPCRs, pharmacology, and his contributions to the field in both the academic and biotech worlds.

Professor Pfleger has developed extensive expertise in profiling receptor binding and function at the molecular and cellular levels over the last 20 years, particularly involving GPCRs. He also has globally-recognized expertise in bioluminescence resonance energy transfer (BRET) technology, including his patented Receptor-Heteromer Investigation Technology (Receptor-HIT) for studying heteromers.

Kevin is also Director, Biomedical Innovation at The University of Western Australia (UWA) and the MTPConnect Western Australian Life Sciences Innovation Hub. He is Head of Molecular Endocrinology and Pharmacology at the UWA Centre for Medical Research and Harry Perkins Institute of Medical Research, Deputy Director of the Australian Research Council Centre for Personalised Therapeutics Technologies, Chief Scientific Advisor to Dimerix, and co-founder of RAGE Biotech.

He currently serves on the Board of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists and is a member of the British Pharmacological Society International Advisory Group.

Join me and learn more about Kevin’s work and how he manages all his responsibilities.

Dr. Kevin Pfleger on the web