Biology subjects

Cheloha, R.

Publications and source records attributed to Cheloha, R..

3 recordsLinked to original sources

bioRxiv2025

Nanobody-based conjugates targeting small molecule-binding GPCRs and exhibiting logic-gated signaling

G protein-coupled receptors (GPCRs) are the largest family of plasma membrane embedded signaling proteins. These receptors are involved in a wide array of physiological processes, marking them as attractive targets for drug development. Bitopic ligands, which are comprised of a pharmacophore that targets the receptor orthosteric site and a linked moiety that binds to a separate site, have considerable potential for addressing GPCR function. Here, we report the synthesis and evaluation of novel bitopic conjugates consisting of a small molecule pharmacophore that activates the adenosine A2A receptor (A2AR) linked to antibody fragments (nanobodies, Nbs). This approach leverages the high affinity and specificity binding of Nbs to non-orthosteric sites on engineered A2AR variants to provide bitopic Nb-ligand conjugates that stimulate strong and enduring signaling responses. We further demonstrate that such bitopic conjugates can induce activation by spanning two distinct receptor protomers. This property enables the selective targeting of receptor pairs over either individual receptor, as a form of "logic-gated" activity. We showcase the broad applicability of bitopic conjugates in this context by demonstrating their activity in targeting several pairs of co-expressed receptors, including GPCR monomers from different classes. Furthermore, we demonstrate that this dual-targeting strategy initiates signaling responses that diverge from those induced by monovalent ligands. The ability to target receptor pairs using nanobody-ligand conjugates offers a powerful strategy with potential for cell type-selective signaling and implications for GPCR drug discovery efforts more broadly.

pharmacology and toxicology↗

bioRxiv2023

Highly biased agonism for GPCR ligands via nanobody tethering

Ligand-induced activation of G protein-coupled receptors (GPCRs) can initiate signaling through multiple distinct pathways with differing biological and physiological outcomes. There is intense interest in understanding how variation in GPCR ligand structure can be used to promote pathway selective signaling ("biased agonism") with the goal of promoting desirable responses and avoiding deleterious side effects. Here we present a new approach in which a conventional peptide ligand for the type 1 parathyroid hormone receptor (PTHR1) is converted from an agonist which induces signaling through all relevant pathways to a compound that is highly selective for a single pathway. This is achieved not through variation in the core structure of the agonist, but rather by linking it to a nanobody tethering agent that binds with high affinity to a separate site on the receptor not involved in signal transduction. The resulting conjugate represents the most biased agonist of PTHR1 reported to date. This approach holds promise for facile generation of pathway selective ligands for other GPCRs.

pharmacology and toxicology↗

bioRxiv2023

Semi-synthetic nanobody-ligand conjugates exhibit tunable signaling properties and enhanced transcriptional outputs at neurokinin receptor-1

Antibodies have proven highly valuable for therapeutic development; however, they are typically poor candidates for applications that require activation of G protein-coupled receptors (GPCRs), the largest collection of targets for clinically approved drugs. Nanobodies (Nbs), the smallest antibody fragments retaining full antigen-binding capacity, have emerged as promising tools for pharmacologic applications, including GPCR modulation. Past work has shown that conjugation of Nbs with ligands can provide GPCR agonists that exhibit improved activity and selectivity compared to their parent ligands. The neurokinin-1 receptor (NK1R), a GPCR targeted for the treatment of pain, is activated by peptide agonists such as Substance P (SP) and neurokinin A (NKA), which induce signaling through multiple pathways (Gs, Gq and {beta}-arrestin). In this study, we investigated whether conjugating NK1R ligands with Nbs that bind to a separate location on the receptor would provide chimeric compounds with distinctive signaling properties. We employed sortase A-mediated ligation to generate several conjugates consisting of Nbs linked to NK1R ligands. Many of these conjugates exhibited divergent and unexpected signaling properties and transcriptional outputs. For example, some Nb-NKA conjugates showed enhanced receptor binding capacity, high potency partial agonism, prolonged cAMP production, and an increase in transcriptional output associated with Gs signaling; whereas other conjugates were virtually inactive. Nanobody conjugation caused only minor alterations in ligand-induced upstream Gq signaling with unexpected enhancements in transcriptional (downstream) responses. Our findings underscore the potential of nanobody conjugation for providing compounds with advantageous properties such as biased agonism, prolonged duration of action, and enhanced transcriptional responses. These compounds hold promise not only for facilitating fundamental research on GPCR signal transduction mechanisms but also for the development of more potent and enduring therapeutics.

biochemistry↗