Ross Cheloha: Nanobody-GPCR Conjugates and the Engineering of Receptor Selectivity

The parathyroid hormone receptor sits at the intersection of calcium homeostasis, bone metabolism, and a set of pharmacological questions that have resisted clean resolution for decades. How does receptor conformation shape the duration of downstream signaling? Why do some ligands continue activating from the endosome while others don't? And can selectivity for one receptor subtype be engineered without redesigning the ligand from scratch?

Ross Cheloha approaches these questions from a chemical biology perspective - using synthetic peptide analogs, camelid single-domain antibodies (nanobodies), and bifunctional conjugates that split the binding event into two independently tunable pieces. In this conversation, he describes how attaching a moderately active peptide to a receptor-targeted nanobody can boost potency by up to 10,000-fold, and how the selectivity of the nanobody - not the ligand - is what engineers receptor subtype specificity.

For Cheloha, the motivation traces back to a habit he carried from childhood: writing reports on exotic diseases and what scientists were doing to treat them. The experiment that mattered most was not a drug candidate or a clinical advance - it was a designed conjugate that worked when there was no clear reason it had to, every control behaving exactly as expected.

About the Guest

Ross Cheloha is a tenure-track investigator at the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) at the National Institutes of Health. His research combines peptide chemistry, chemical biology, and nanobody engineering to build mechanistic tools for studying GPCR signaling, with a particular focus on the parathyroid hormone receptor and its downstream consequences for calcium homeostasis and bone biology.

Trained in the Gelman lab at the University of Wisconsin-Madison and later in the Ploegh lab at Harvard Medical School and Boston Children's Hospital, he developed methods for covalently functionalizing camelid single-domain antibodies with synthetic ligands to probe receptor conformation, endosomal activity, and the duration of signaling responses. His work has demonstrated that nanobody-ligand conjugates can rescue pharmacologically weak peptides, engineer receptor subtype selectivity, and open mechanistic questions that conventional ligand design cannot easily address.

Scientific Themes of the Conversation

1. Peptide analog engineering and the stabilization of GPCR ligands using non-natural amino acids
   

2. Receptor conformation selectivity: G-protein coupled versus uncoupled states and their downstream consequences
   

3. Camelid single-domain antibodies (nanobodies) as a platform for GPCR chemical biology
   

4. Nanobody-ligand conjugates as bifunctional tools for potency rescue, selectivity engineering, and mechanistic dissection
   

5. Duration of signaling and endosomal GPCR activity: what wash-and-measure paradigms have been missing
   

6. Tissue-specific receptor targeting and the pharmacological logic of reducing GPCR side effect profiles

Key Insights from the Conversation

1. Conformation selectivity changes the duration of the signal

PTH receptor exists in G-protein coupled and uncoupled states, and peptide analogs incorporating non-natural amino acids show differential affinity for each conformation. The analogs that preferentially bind the uncoupled state produce markedly prolonged signaling responses - in cells and in animal experiments - compared to ligands that do not. The biology may have been using this distinction all along: two natural ligands for the same receptor already show subtly different conformation preferences and different physiological profiles in vivo.

2. One nanobody, four orders of magnitude

Connecting a short, weakly active PTH fragment (11 amino acids, approximately 100 nanomolar potency) to a receptor-targeted nanobody produced sub-nanomolar potency in cell-based assays - an improvement of up to 10,000-fold from a single engineering step. The working model is that the nanobody anchors the conjugate at the receptor surface, positioning the ligand for activation that would otherwise be too transient to drive a strong response. The selectivity comes from the nanobody's binding specificity, not from chemical modification of the peptide itself.

3. Selectivity without redesigning the drug

PTH 1-34 activates both PTHR1 and PTHR2 with high potency, and engineering subtype selectivity through chemical modification of the peptide is technically demanding. Attaching a short PTH fragment to a PTHR1-selective nanobody produces a ligand selective for that subtype; the same fragment attached to a PTHR2-selective nanobody redirects it entirely. The implication generalizes: wherever a selective nanobody exists for a GPCR target, it can be used to introduce specificity into an otherwise promiscuous ligand without restructuring the pharmacophore.

4. Endosomal signaling is being missed

Conventional GPCR assay design - stimulate, wash, measure endpoint - systematically misses signaling that continues after receptor internalization into the endosome. Cheloha's position is that the standard assumption linking tighter binding to longer signaling is incomplete. Endosomal activity is a distinct mechanistic process, and the tools to dissect it - bifunctional conjugates with independently tunable affinity components - are not available with conventional single-piece ligands. This is the central mechanistic priority he is bringing to his NIH lab.

5. Tissue-specific targeting as a strategy for reframing side effects

The primary dose-limiting side effect of PTH-based therapies is hypercalcemia, driven in part by receptor activity in kidney tissue rather than bone. Connecting a ligand to a tissue-targeted nanobody could restrict receptor activation to bone and reduce unwanted calcium release. The pharmacological logic is sound, and the engineering framework to test it exists. Whether it holds in vivo remains to be demonstrated - Cheloha is careful to say so - but it represents a genuinely new angle on a long-standing problem in PTH pharmacology.

6. The experiment that had no reason to work - and did

Among three scientific aha moments Cheloha described, the one with the clearest forward momentum was the first successful nanobody-PTH conjugate - designed not because a clear rationale existed, but because he wondered whether it was possible. No hypothesis guaranteed the result. Every positive and negative control performed exactly as designed. The conjugate worked. That single experiment, Cheloha said, opened more mechanistic doors than anything he had produced before - and it came directly from experience-built intuition rather than hypothesis-first design.

Episode Timeline

Timestamps are AI-generated from the transcript and are approximate. Verify against the final edited video before publishing.

• 00:00 Introduction

• 01:58 From pharmacy school to chemical biology: how the research path took shape

• 08:12 Academia versus industry - why freedom to ask non-translatable questions matters

• 09:43 The academic job search: 65 applications, six interview trips, and landing NIH

• 14:20 How a peptide chemist found the PTH receptor - and a collaborator who changed everything

• 17:20 Non-natural amino acids, protease stability, and receptor conformation selectivity

• 21:40 PTH receptor pharmacology: calcium spikes, bone biology, and why long-acting isn't always better

• 23:22 A detour into immunology and the discovery of camelid nanobodies

• 25:45 The first nanobody-GPCR ligand conjugate and an unexpected 10,000-fold potency gain

• 30:32 Engineering receptor subtype selectivity without redesigning the pharmacophore

• 36:10 Mechanistic priorities at NIH: endosomal signaling and duration of signaling

• 40:25 Are GPCRs still a good drug target? Biased agonism, endosomal signaling, and what remains

• 47:13 Three aha moments: first data, an email from Gardella, and the conjugate that worked

Selected Quotes

About this episode

Dr. Ross Cheloha is an Investigator at the National Institutes of Health in the Laboratory of Bioorganic Chemistry in Bethesda, MD, where he started in October 2020. He completed his postdoctoral training at MIT and Harvard Med School in the lab of Hidde Ploegh, where he developed new applications of single-domain antibodies (nanobodies). He earned his Ph.D. in Chemistry at the University of Wisconsin-Madison in the lab of Sam Gellman on the study of analogs of the GPCR peptide ligand parathyroid hormone. Work in his independent laboratory is focused on developing new pharmacological tools via chemistry and protein engineering to interrogate GPCR signaling.

Ross and I chatted about his work and transition to an independent investigator; join me to learn more about class B GPCRs and Dr. Cheloha’s work.

Dr. Ross Cheloha on the web

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