Sponge cystine knot peptides exhibit potential utility as an oral peptide drug carrier

In our continuing search for bioactive compounds from marine sponges, new cystine knot peptides asteropsins [1 – 3] were isolated from a sponge Asteropus sp. The structures of asteropsins were determined by Edman degradation and NMR analysis. The crystal structure of asteropsin A was determined by X-ray at a high resolution of 0.87 Å. Two additional new additional cystine knot peptides, asteropsins F (ASPF) and G (ASPG), were isolated and their tertiary structures were determined by NMR. ASPF and ASPG shared the properties characteristic of the asteropsin family, such as, N -terminal pyroglutamate modification, incorporation of cis prolines, and a highly acidic nature, which distinguish them from other knottin families such as conotoxins and spider toxins. ASPF and ASPG were found to be remarkably stable to digestive enzymes, and stable in human plasma. Furthermore, asteropsins A-F were highly resistant to thermal degradation, non-hemolytic to fish and human red blood cells at concentrations of ≤50µM, non-cytotoxic, and non-stimulatory to RAW 264.7 macrophage, suggesting their potential utility as safe and stable oral peptide-drug scaffolds for the design and synthesis of drug-delivery tool. Utilization of robust stability of asteropsin as an oral peptide carrier may be enabled by artificial synthesis. Further study on recombinant expression of asteropsins in Escherichia coli was performed. The artificial DNA sequence was obtained by back-translation based on the amino acid sequence of asteropsin C. A synthetic oligonucleotide encoding 35-amino acid residues of asteropsin C was cloned and fused in-frame to the His 6 -tagged DsbC gene in the bacterial expression vector pET-40b. The fusion protein was overexpressed in the bacterial host Escherichia coli strain BL21(DE3). Acknowledgements: Dr. David J. Craik (University of Queensland, Australia) is acknowledged for kind donation of kalata B1. NMR data (900 MHz) were measured at Korea Basic Science Institute.