Dual targeting of GPCR assemblies using Nanobody-ligand conjugates

Authors

• S Roy
• S Sachdev
• RW Cheloha

Abstract

G-protein-coupled receptors (GPCRs), the largest family of cell surface receptors were long hypothesized to exist as monomeric entities but the evidence of GPCR assembly has sparked interest in addressing how receptor oligomerization modulates signaling. Targeting GPCR assemblies with bivalent ligands offers the possibility of designing potent and selective ligands with reduced side effects.
We targeted GPCR assemblies from different classes using a novel bivalent design where a antibody fragment (nanobody) specific to a certain GPCR (NbPTHR1 for PTHR1, NbGLP1R for GLP1R) is linked to a small molecule agonist of a different GPCR (CGS21680 for A2AR) by enzymatic labeling followed by click chemistry. We targeted putative assemblies formed between GPCRs of different classes by co-expressing either a class A (NK1R) or a class B GPCR (PTHR1, GLP1R) with class A GPCR (A2AR). We monitored ligand-induced receptor activation through Gs pathway in live cells. The NbPTHR1-CGS showed activity only when both PTHR1 and A2AR were expressed, whereas the CGS was active with or without PTHR1 co-expression. The expression of PTHR1 and A2AR at different levels led to marked variation in nanobody-CGS potency and efficacy. Interestingly, the activity of NbPTHR1-CGS conjugates showed little dependency on linker length. Similar trends were observed with GLP1R and A2AR using NbGLP1R-CGS. When two class A GPCRs were co-expressed, it showed unexpectedly complex behavior where A2AR expression altered the signaling properties of NK1R, even in the absence of bivalent conjugates. This approach offers promise for highly targeted activation of signaling responses only in cells co-expressing pairs of GPCRs.

Scientific Focus Area: Chemical Biology

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