Structural identification and bioactivity assessment of QUB-2999, an antimicrobial peptide from the skin secretion of Pelophylax nigromaculatus

Abstract

Amphibians have evolved various skin secretions to fight against pathogens and their predators. These secretions also have medicinal functions, which some ancient people utilised to cure diseases. With more pathogens developing resistance to clinical antibiotics nowadays, there is an urgent need for new antibiotic resources, and amphibian skin secretions can be a treasure trove for bioactive peptide discovery. In this study, an antimicrobial peptide was obtained from the frog skin secretion of Pelophylax nigromaculatus by molecular cloning, which mature peptide sequence was GILKDTLKGAATNVAGVLLDKLKCKITGGC. The peptide was named QUB-2999 in this thesis based on its molecular mass and was classified into the Ranatuerin-2 family according to NCBI-BLAST results. Its secondary structure was predicted by the software of RPBS Web-portal and CI-TASSER, indicating there are α-helix and Rana-Box in its structure. Then, QUB-2999 was synthesised by the solid-phase peptide synthesis (SPPS) method, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and identified by Matrix-Assisted Laser Desorption Ionisation Time-of-Flight (MALDI-TOF) MS. After which, this peptide sample was used for a series of assays. The antimicrobial activity of QUB-2999 was tested on three microorganism models, including the gram-negative E. coli NCTC 10418, the gram-positive S. aureus NCTC 10788, and the fungi C. albicans ATCC 10231, whose MIC/MBC values were 4/8 µM, 32/64 µM and 128/256 µM, respectively. Meanwhile, QUB-2999 also showed antiproliferative effects on the H838 lung cancer cell line. Despite being weaker than cisplatin, whose IC50 value was 34.34 µM, QUB-2999 showed the potential of inhibiting cancer cells with an IC50 value of 76.89 µM, and together with a low haemolysis effect that its HC10 value was 113 µM. These results provide evidence that QUB-2999 could be a promising antimicrobial and anticancer clinical candidate and indicate that more tests such as modification, stability, mechanism and bioactivities can be studied in the future. Thesis is embargoed until 31 December 2029.