Isolation of a Novel Kunitz Family Protease Inhibitor in Association with Tethya Hemolysin from the Sponge Tethya ingalli

• Published in: Journal of natural products (Washington, D.C.) Vol. 60; no. 11; pp. 1094 - 1099
• Main Authors: O'Keefe, Barry R, Beutler, John A, Cardellina, John H, Prather, Tanya R, Shoemaker, Robert H, Sowder, Raymond C, Henderson, Louis E, Pannell, Lewis K, Boyd, Michael R
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01-11-1997; Glendale, AZ American Society of Pharmacognosy
• Subjects: AGENTES ANTINEOPLASTICOS; Amino Acid Sequence; Amino Acids - analysis; Animals; ANTINEOPLASTIC AGENTS; BIOASSAYS; Biological and medical sciences; Cell Survival - drug effects; CHEMICAL COMPOSITION; CHEMICAL STRUCTURE; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; CYTOTOXIC COMPOUNDS; ENSAYO BIOLOGICO; EPONGE; Erythrocyte Membrane - metabolism; ESPECTROMETRIA; ESPONJAS; ESTRUCTURA QUIMICA; General pharmacology; HAEMOLYSINS; Hemagglutination; HEMOLISINAS; HEMOLYSINE; Hemolysis; Humans; In Vitro Techniques; INHIBIDORES DE PROTEINASAS; INHIBITEUR DE PROTEINASES; Isoelectric Focusing; Medical sciences; MEDICAMENT CYTOSTATIQUE; MEDICINAL PROPERTIES; Molecular Sequence Data; NEW ZEALAND; NOUVELLE ZELANDE; NUEVA ZELANDIA; PARAZOA; Pharmacognosy. Homeopathy. Health food; Pharmacology. Drug treatments; Porifera - enzymology; PROPIEDADES MEDICINALES; PROPRIETE PHARMACOLOGIQUE; Protease Inhibitors - chemistry; Protease Inhibitors - isolation & purification; Protease Inhibitors - pharmacology; PROTEINAS; PROTEINASE INHIBITORS; PROTEINE; PROTEINS; SPECTRAL ANALYSIS; SPECTROMETRIE; SPECTROMETRY; SPONGES; STRUCTURE CHIMIQUE; SUBSTANCE TOXIQUE; SUSTANCIAS TOXICAS; TEST BIOLOGIQUE; TOXIC SUBSTANCES; Tumor Cells, Cultured; New Zealand; Oceania; Antineoplastic agent; Toxicity; Cytotoxicity; Marine environment; Established cell line; N terminal-Sequence; Tumor cell; Aminoacid sequence; Human; Hemolysis; Kunitz inhibitor; Purification; Serine endopeptidases; Enzyme; Red blood cell; Enzyme inhibitor; Animal origin; In vitro; Biological activity; Protein; Peptidases; Trypsin; Porifera; Hydrolases; Isolation; Invertebrata; Comparative study
• ISSN: 0163-3864; 1520-6025
• DOI: 10.1021/np970242l

Aqueous extracts from the New Zealand sponge Tethya ingalli (Hadromerida) displayed potent cytotoxicity in the NCI's 60-cell-line human tumor panel. Fractionation of the extract by ammonium sulfate precipitation, gel filtration, ultrafiltration, and both hydrophobic interaction and reversed-phase chromatography resulted in the isolation of two biologically active proteins. The first protein, Tethya protease inhibitor (TPI), which was purified to homogeneity, inhibited trypsin with an EC50 of 65 nM. TPI had a molecular mass of 11 431 Da, and an isoelectric point of 8.2. A partial N-terminal amino acid sequence determined for TPI showed significant homology with protease inhibitors of the Kunitz family. The second isolated protein displayed potent cytotoxicity, with pronounced selectivity for certain tumor cell lines (e.g., ovarian, renal, CNS, and breast). The latter protein, which had an apparent molecular weight of 21 kDa (SDS−PAGE), also lysed human red blood cells (EC50 of 39 nM) and was similar to a hemolysin previously isolated from the sponge Tethya lycinurium.