Design of calcium-sensing receptor targeted thera(g)nostic agents

Objectives: The Ca²⁺-sensing receptor (CaSR) plays a central role in regulating [Ca²⁺]_o homeostasis and many (patho)physiological processes in multiple organs. Numerous diseases are related to mishandling calcium such as familial hypocalciuric hypercalcemia (FHH), neonatal severe hyperparathyroidism (NSHPT), autosomal dominant hypocalcemia (ADH), autosomal dominant hypocalcemic hypercalciuria (ADHH), renal stone disease (nephrolithiasis) and secondary hyperparathyroidism (HPT) by chronic kidney disease (CKD). Therefore, it is critical to develop novel allosteric modulators for various diseases with cellular and organ specificity as well as mapping CaSR in vivo expression and preoperative or intraoperative imaging. Here, we report our effort in the design of a novel class of calcium-sensing receptor-targeted thera(g)nostic agents.

Methods: High-resolution X-ray crystallography was applied to determine the structure of the extracellular domain of calcium-sensing receptor that was mammalian cell expressed, deglycosylated and purified. Mass spectrometry was applied to identify a tryptophan derivative (TNCA) that is co-crystalized with the ECD of CaSR. Functional assay, computational modeling, and docking were further performed to design an array of drug candidates. The binding affinity of synthesized tetrahydro-beta-carblinecarboxylic acids including [¹⁸F] (1) was determined and their efficacies to CASR were examined by an array of functional assays. [¹⁸F] (1) was synthesized and whole body MicroPET-CT images will be performed in rats to examine the biodistribution of CaSR in vivo. The histological analysis of (1) will be performed to determine the targeting specificity of the TNCA agent to CaSR with IHC and IFC.

Results: We have determined the first crystal structure of the human CaSR extracellular domain (ECD) with bound Mg²⁺ and identified a novel agonist tryptophan derivative (TNCA) at 2.1 Å. We have further synthesized compound (1) based on structural modeling and docking of the CaSR-TNCA complex. (1) was shown to have significantly improved potency and efficacy (>1000 fold) to CASR mediated calcium oscillation and IP production than natural amino acid Phe. (1) is also able to rescue cellular functions of CASR with disease mutations. The binding of (1) to CaSR has been further determined by FRET assay and radioactive binding assay. Its ¹⁹F (1) was further shown to has strong agonist activity and specificity to CaSR using CaSR stably transfected cell line. PET imaging and histological analysis indicate this derivative also has strong in vivo CaSR targeting specificity and sensitivity.

Conclusions: We have shown our hypothesis that effective thera(g)nostic agonists of CaSR can be achieved by the development of ¹⁸F/¹⁹F derivatives of TNCA with strong affinity, specificity, and proper in vivo organ and tissue distribution. We have demonstrated a novel avenue in the development of CaSR specific agonists and antagonists for imaging and the treatment of various CaSR associated human diseases using newly available structures and patented methodology. Acknowledgment: This work was supported in part by NIH Research Grants R33CA235319, R42CA183376, R42AA025863 and American Heart Association 16GRNT31210016 to JJY

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