Eleonora Comeo: Fluorescent Ligands and the Pharmacology of Adenosine Receptors

Adenosine receptors are among the most studied GPCRs in the human body - and among the most difficult to drug. The four subtypes, A1, A2A, A2B, and A3, are expressed across virtually every tissue and implicated in conditions ranging from Parkinson's disease to ischemic heart disease to cancer. Over 40 crystal structures of the A2A receptor alone have been deposited in the Protein Data Bank. Clinical trials have run for decades. And yet approved therapies that selectively target these receptors remain remarkably scarce.

One reason is that selectivity in this family is not just a matter of receptor subtype. It is also a matter of G protein coupling, tissue context, and the signaling assay used to measure affinity in the first place - measurements that shift depending on the experimental system.

Eleonora Comeo, PhD candidate at the University of Nottingham and the Monash Institute of Pharmaceutical Sciences, is developing fluorescent ligands to address that gap directly - tools designed to visualize adenosine receptor pharmacology at the single-cell level, in environments that better reflect where these receptors actually work. For Comeo, who entered GPCR research without any background in the field and built her PhD around chemistry she could hold in her hand and watch working in a cell, the tools are both scientific instruments and the reason she stayed.

ABOUT THE GUEST

Eleonora Comeo is a PhD candidate jointly enrolled at the University of Nottingham and the Monash Institute of Pharmaceutical Sciences, working across the groups of Barrie Kellam and Steve Hill. A trained medicinal chemist and pharmacist, her research focuses on the design and synthesis of fluorescent ligands for adenosine A1 and A2A receptors, with the goal of developing chemical tools capable of interrogating receptor pharmacology in physiologically relevant cellular environments. Her work integrates organic synthesis with NanoBRET, SNAP-tag labeling, confocal microscopy, and super-resolution imaging to study receptor localization, internalization, and binding at endogenous and heterologously expressed levels.

SCIENTIFIC THEMES OF THE CONVERSATION

1. Fluorescent ligands as pharmacological tools - how synthetic chemistry produces molecules that make receptor behavior visible
   

2. Adenosine receptor subtype pharmacology - the four subtypes, their G protein coupling profiles, and why ubiquity creates selectivity problems
   

3. The tumor microenvironment and adenosine-mediated immune suppression - why A2A and A2B are being targeted in oncology
   

4. Assay dependency and the instability of affinity measurements across experimental systems
   

5. Endogenous receptor expression in heterologous cell systems - how HEK cell biology confounds fluorescent ligand screening
   

6. Receptor dimerization - from A2A/dopamine D2 in Parkinson's to A1/A2A in cardio protection, and what remains unresolved

KEY INSIGHTS FROM THE CONVERSATION

1. Visibility changes what pharmacology can ask

Fluorescent ligands do more than confirm receptor binding - they allow researchers to follow receptors through internalization, track membrane localization, and observe behavior at the single-cell level using super-resolution imaging. Comeo describes the moment she saw her synthesized ligand labeling the cell membrane as one of the most rewarding points in the PhD, precisely because it closed the loop between synthesis and function in a way that binding numbers alone cannot.

2. Ubiquity is adenosine pharmacology's greatest liability

The same properties that make adenosine receptors scientifically compelling - their presence in nearly every tissue, their involvement in nearly every major disease class - are what make selective targeting so difficult. Activating or blocking an adenosine receptor for one indication routinely produces off-target effects in another tissue, because the receptor is already doing something important there. The clinical attrition rate in this family reflects that problem more than it reflects a failure of chemistry.

3. Affinity is not a fixed property - it is assay-dependent

The apparent affinity of adenosine for its own receptors shifts depending on which downstream signaling endpoint is being measured. A cAMP assay and a binding assay using the same receptor and the same ligand can return different affinity values. Comeo points to this as an underappreciated complication in the field - one that matters not just for scientific interpretation but for how pharmacological classifications like "low affinity" and "high affinity" are assigned across the A2A and A2B subtypes.

4. Tumor cells use adenosine receptors to silence the immune response

In the tumor microenvironment, adenosine concentrations are abnormally high. That excess adenosine activates A2A and A2B receptors expressed on immune cells - and that activation suppresses the immune response, allowing tumors to grow unchallenged. Dual A2A/A2B antagonists are now in clinical trials specifically to interrupt this mechanism. Comeo describes this as one of the areas where she most hopes the field can finally validate the receptors as clinical targets, after years of trials that have not delivered.

5. HEK cells are a known confound that still catch researchers out

HEK cells endogenously express A2A and A2B receptors at meaningful levels - a fact that complicates fluorescent ligand screening, binding assays, and any experiment where receptor selectivity is the question. Comeo's work uses selective antagonist controls and NanoBRET-tagged receptor constructs to distinguish transfected receptor signal from endogenous background. She also describes recent work in her group using CRISPR-edited cell lines to express receptors at endogenous promoter levels, producing reliable NanoBRET signal even at physiological expression - a methodological advance with implications for how the field studies GPCRs in native-like contexts.

6. Receptor dimerization raises questions the standard pharmacology framework does not address

Adenosine receptors form both homodimers and heterodimers with other GPCRs, including A2A with dopamine D2 (a complex investigated extensively in the context of Parkinson's) and A1 with A2A (under investigation for cardioprotection in ischemic heart disease). The biological implications of many of these complexes are still being resolved. During the conversation, an unresolved question surfaced: when a tagged receptor is transfected into HEK cells that endogenously express the same or related subtypes, can the exogenous and endogenous receptors dimerize - and if so, does that alter the pharmacological readout? Neither Comeo nor the host claimed an answer. The question remains open.

7. The hardest transition in a PhD is learning that not knowing the answer is the point

Comeo describes the shift from undergraduate training - where not knowing the answer is a liability - to doctoral research, where not knowing the answer is the entire reason the project exists. It is, she notes, a more difficult transition than it sounds, and one that takes time to internalize. The advice she offers to incoming PhD students centers not on technique but on disposition: stay curious about what others are working on, ask for help without embarrassment, and accept that the project is a shared endeavor even when it feels isolating.

EPISODE TIMELINE

Timestamps are AI-generated from the transcript and should be verified against the final edited audio before publication.

• 00:00 Introduction

• 00:44 From Bologna to Nottingham - following her heart into GPCR research

• 06:49 One year at Monash - starting fresh on the other side of the world

• 10:02 Adenosine receptor pharmacology and the challenge of targeting four subtypes

• 16:56 A2A vs A2B - affinity differences, tumor microenvironment, and immune escape

• 22:08 Why affinity measurements shift depending on the assay

• 27:09 Synthesizing fluorescent ligands - from crystal structures to glowing molecules

• 31:03 Following receptors into the cell with super-resolution imaging

• 36:25 Receptor dimerization - A2A/D2 in Parkinson's, A1/A2A in cardioprotection, and one open question

• 41:26 Finishing the PhD - what comes next and what she'll miss

• 42:42 Advice for PhD students - resilience, collaboration, and not fearing the unknown

• 54:44 The dopamine rush that keeps scientists coming back

SELECTED QUOTES

About this episode

Eleonora Comeo is a doctoral candidate in Medicinal Chemistry and Drug Discovery in the joint program of the University of Nottingham in the UK and Monash University in Australia. We sat down to chat about GPCRs, synthesizing labeled ligands, and her unique position that allows her to work with GPCR scientists on 2 continents. We also touched on how COVID-19 affected her Ph.D. work.

Eleonora Comeo on the web

• LinkedIn

• ResearchGate

• Pubmed

• Google Scholar

• Dr. GPCR Ecosystem