Development of High Affinity Calcitonin Analog Fragments Targeting Extracellular Domains of Calcitonin Family Receptors.

Sangmin Lee

doi:10.3390/biom11091364

Abstract

The calcitonin and amylin receptors (CTR and AMY receptors) are the drug targets for osteoporosis and diabetes treatment, respectively. Salmon calcitonin (sCT) and pramlintide were developed as peptide drugs that activate these receptors. However, next-generation drugs with improved receptor binding profiles are desirable for more effective pharmacotherapy. The extracellular domain (ECD) of CTR was reported as the critical binding site for the C-terminal half of sCT. For the screening of high-affinity sCT analog fragments, purified CTR ECD was used for fluorescence polarization/anisotropy peptide binding assay. When three mutations (N26D, S29P, and P32HYP) were introduced to the sCT(22–32) fragment, sCT(22–32) affinity for the CTR ECD was increased by 21-fold. CTR was reported to form a complex with receptor activity-modifying protein (RAMP), and the CTR:RAMP complexes function as amylin receptors with increased binding for the peptide hormone amylin. All three types of functional AMY receptor ECDs were prepared and tested for the binding of the mutated sCT(22–32). Interestingly, the mutated sCT(22–32) also retained its high affinity for all three types of the AMY receptor ECDs. In summary, the mutated sCT(22–32) showing high affinity for CTR and AMY receptor ECDs could be considered for developing the next-generation peptide agonists.

Highlights

Calcitonin (CT) a 32 amino acid peptide hormone is secreted from thyroid glands and activates the calcitonin receptor (CTR) to control calcium homeostasis
Since the crystal structures of receptor extracellular domain (ECD)-bound CGRPmut appeared similar to the CTR ECDbound Salmon CT (sCT) structure (Figure 1b), those mutations were applied to the corresponding amino acids of sCT
K35F mutation of CGRPmut was not applied to the corresponding sCT G30 since Lee et al reported that the alanine mutation of sCT G30 significantly decreased the sCT(22–32) binding for the CTR ECD suggesting that the flexibility of G30 helped sCT(22–32) binding [18]

Summary

Introduction

Calcitonin (CT) a 32 amino acid peptide hormone is secreted from thyroid glands and activates the calcitonin receptor (CTR) to control calcium homeostasis. Salmon CT (sCT) was developed as a peptide drug targeting human CTR due to its higher affinity and potency than human CT [1,2,3]. The CTR:RAMP complexes gain affinity for the peptide hormone amylin and are known as the amylin receptor (AMY receptor). The AMY receptor is a drug target for diabetes treatment and its activation holds a potential for treating other metabolic diseases including obesity [5,6,7]. Lots of effort has been focused on developing next-generation peptide drugs targeting AMY receptors as exemplified with dual amylin calcitonin receptor agonists (DACRA) and long-acting amylin/calcitonin receptor agonists [9,10,11,12,13]

Methods

Results

Conclusion