Structure and function of a dual antagonist of the human growth hormone and prolactin receptors with site-specific PEG conjugates

Abstract

Human growth hormone (hGH) is a pituitary derived endocrine protein that regulates several critical postnatal physiologic processes including growth, organ development, and metabolism. Following adulthood, GH is also a regulator of multiple pathologies like fibrosis, cancer, and diabetes. Therefore, there is a significant pharmaceutical interest in developing antagonists of hGH action. Currently there is a single FDA approved antagonist of the hGH receptor (hGHR) prescribed for treating patients with acromegaly and discovered in our laboratory almost three decades ago. Here, we present the first data on the structure and function of a new set of protein antagonists with the full range of hGH actions - dual antagonists of hGH binding to the GHR as well as that of hGH binding to the prolactin receptor. We describe the site-specific poly-ethylene-glycol (PEG) conjugation, purification, and subsequent characterization using MALDI-TOF, size-exclusion chromatography, thermostability, and biochemical activity in terms of ELISA-based binding affinities with GHR and PRLR. Moreover, these novel hGHR antagonists display distinct antagonism of GH induced GHR intracellular signaling in vitro and marked reduction in hepatic insulin-like growth factor 1 output in vivo. Lastly, we observed potent anti-cancer biological efficacies of these novel hGHR antagonists against human cancer cell lines. In conclusion, we propose that these new GHR antagonists have potential for development towards multiple clinical applications related to GH associated pathologies.