Bräuner-Osborne, H. | Search bioRxiv

bioRxiv2024

Discovery and pharmacological characterization of nanobodies acting as potent positive allosteric modulators of the calcium-sensing receptor

The calcium-sensing receptor (CaSR) is responsible for sustaining a stable blood calcium concentration. Consequently, genetic and acquired changes in this G protein-coupled receptor can give rise to various calcium homeostasis disorders. Synthetic positive allosteric modulators targeting CaSR are currently used to treat hypercalcemia, but their usage is highly limited due to the high risk of severe hypocalcemia and gastrointestinal intolerance. In this study, we aimed to generate pharmacologically active CaSR-specific nanobodies that could be employed as a new generation of pharmacological tools to investigate the receptor function and potentially serve as a new drug modality for effective treatment of CaSR-related disorders. Nanobodies were generated by immunization of a llama with CHO cells recombinantly overexpressing a myc-epitope-tagged human CaSR. Following construction of a phage display library representing the repertoire of nanobody genes, nanobodies binding to the CaSR were isolated by FACS of whole HEK293 cells recombinantly overexpressing HA-epitope-tagged human CaSR. Based on sequence comparison, 37 nanobodies from 25 different sequence families were purified and subsequent characterized in vitro for modulation of CaSR signaling. The nanobodies were screened for agonist, as well as positive and negative allosteric modulators activity in in vitro cellular assays downstream of CaSR activation. We identified eight pharmacologically active nanobodies acting as positive allosteric modulators that could be divided into five main families based on their sequence identity. The most potent nanobody (Nb4) binding to the extracellular domain of CaSR was slightly more potent than the reference small molecule PAM NPS R-568. This study describes the discovery and pharmacological characterization of nanobodies acting as potent CaSR positive allosteric modulators. These nanobodies are a new class of pharmacological research tools for the CaSR, which potentially can be developed into new therapeutics in the treatment of CaSR-related disorders.

biochemistry↗

bioRxiv2020

Dissecting the roles of GRK2 and GRK3 in μ-opioid receptor internalization and β-arrestin2 recruitment using CRISPR/Cas9-edited HEK293 cells

Most G protein-coupled receptors (GPCRs) recruit {beta}-arrestins and internalize upon agonist stimulation. For the -opioid receptor (-OR), this process has been linked to development of opioid tolerance. GPCR kinases (GRKs), particularly GRK2 and GRK3, have been shown to be important for -OR recruitment of {beta}-arrestin and internalization. However, the contribution of GRK2 and GRK3 to {beta}-arrestin recruitment and receptor internalization, remain to be determined in their complete absence. Using CRISPR/Cas9-mediated genome editing we established HEK293 cells with knockout of GRK2, GRK3 or both to dissect their individual contributions in {beta}-arrestin2 recruitment and -OR internalization upon stimulation with four different agonists. We showed that GRK2/3 removal reduced agonist-induced -OR internalization and {beta}-arrestin2 recruitment substantially and we found GRK2 to be more important for these processes than GRK3. Furthermore, we observed a sustained and GRK2/3 independent component of {beta}-arrestin2 recruitment to the plasma membrane upon -OR activation. Rescue expression experiments restored GRK2/3 functions. Inhibition of GRK2/3 using the small molecule inhibitor CMPD101 showed a high similarity between the genetic and pharmacological approaches, cross-validating the specificity of both. However, off-target effects were observed at high CMPD101 concentrations. These GRK2/3 KO cell lines should prove useful for a wide range of studies on GPCR function.

pharmacology and toxicology↗