DISCOVERY AND PHARMACOLOGICAL EVALUATION OF NOVEL CAERIDINS WITH SMOOTH MUSCLE ACTIVITIES, ISOLATED FROM THE SKIN SECRETION OF THE AUSTRALIAN WHITE’S TREE FROG, LITORIA CAERULEA

Lei Li

School of Pharmacy, Queen's University, Belfast, UK

Abstract

Abundant biological active peptides were discovered from frog skin secretion. The diversity of bioactivities of these peptides have made frog skin secretion a rich source of therapeutic compounds for drug discovery, which have attracted more attention from the pharmaceutical industry. A combination of transcriptomic and peptidomic analysis was employed here in the discovery and identification of two novel bioactive peptides, Caeridin-1 and S5-Caeridin-1, from the skin secretion of the Australian white’s tree frog, Litoria caerulea. The full-length cDNAs respectively encoding prepropeptide Caeridin-1 and prepropeptide S5-Caeridin-1 were obtained by a rapid and random amplification through “shotgun” cloning. The putative mature peptide Caeridin-1 and S5-Caeridin-1 were subsequently fractionated from the skin secretion of Litoria caerulea by reversed-phase high performance liquid chromatography (RP-HPLC) and further determined by tandem mass spectrometric (MS/MS fragmentation) sequencing analysis. The primary structure of Caeridin-1 and S5-Caeridin-1 showed resemblance to caeridins, a peptide family catergorised from the skin secretion of Australian frogs of the genera, Litoria, but with no ascribed bioactivities, through comparisons to protein database of National Center of Biotechnology Information (NCBI) on-line portal with Basic Local Alignment Search Tool (BLAST) programme. Chemically-synthetic versions of each natural caeridin demonstrated bioactivities on rat smooth muscles. Caeridin-1 was found to produce a contraction of rat bladder smooth muscle with an EC50=14.38 nM, and S5-Caeridin-1 was found to induce a relaxation of rat ileum smooth muscle with an EC50=6.26 nM. Hardly any haemolytic effects were observed after testing on horse red blood cells. The promising ex vivo pharmacological discovery of these two caeridins will encourage further intensive and systematic studies of frog skin secretion to promote the discovery of natural templates as lead compounds for drug discovery and therapeutic application.

Keywords: Frog skin secretion, drug discovery, caeridin, rat smooth muscles.