Synthesis and Evaluation of the Sunflower Derived Trypsin Inhibitor as a Potent Inhibitor of the Type II Transmembrane Serine Protease, Matriptase Ya-Qiu Long, a Sheau-Ling Lee, b Chen-Yong Lin, b IstvanJ.Enyedy, b Shaomeng Wang, b PengLi, a RobertB.Dickson b andPeterP.Roller a, * a Laboratory of Medicinal Chemistry, National Cancer Institute, NIH, FCRDC, 376/208, PO Box B, Frederick, MD 21702, USA b Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20007, USA Received 1 February 2001; accepted 9 July 2001 Abstract—We report here the synthesis of a 14-amino acid long bicyclic peptide, previously isolated from sunflower seeds. This peptide, termed sunflower trypsin inhibitor (SFTI-1), is one of the most potent naturally occurring small-molecule trypsin inhibi- tors. In addition to inhibiting trypsin, the synthetic SFTI-1 is also a very potent inhibitor, with a K i of 0.92nM, of the recently identified epithelial serine protease, termed ‘matriptase’. Published by Elsevier Science Ltd. Plant derived protease inhibitors serve in the defense mechanisms of plants against pests and plant patho- gens. 1 These inhibitors can be classified into a number offamiliesbasedontheiractive-sitestructuresandtheir specificities to inhibit the cleavage of specific peptide sequences within proteins. The majority of these inhibi- tors are classified as serine protease inhibitors. One of the well known serine protease inhibitory agents, the Bowman–Birk inhibitor, found in seeds of legumes and other plants, belongs to the Bowman–Birk inhibitor familyofsmallproteinswitha M r rangeof6000–8000. 2 These proteins inhibit trypsin, chymotrypsin, or elas- tase, depending on the configuration of the reactive site loop within the inhibitor. Recently, a 14-amino acid peptide, termed sunflower trypsin inhibitor (SFTI-1) was isolated from sunflower seeds. 3 SFTI-1 inhibited b-trypsin with an impressive subnanomolar K i of 0.1nM, and it inhibited cathepsin G with a comparable K i . 3 SFTI-1 has considerable selectivity; for example, it proved to be 74-fold less inhibitory for chymotrypsin, and was found to be 3 orders of magnitude less inhibitory for elastase and thrombin.Incontrast,ithadnoeffectonFactorXa. 3 Thenaturalproduct,SFTI-1waspartiallycharacterized by classical techniques, and its structure confirmed, based on the electron density map of the inhibitor co- crystallized with bovine-b-trypsin. 3 SFTI-1isoneofthe smallest, naturally occurring plant protein inhibitors reported to date, with a molecular mass of 1513. It has considerably enhanced potency relative to other pep- tides of similar length. Its backbone-cyclized peptide structure is additionally stabilized by a cystine disulfide bond. We report here the facile synthesis of SFTI-1. This methodology will also provide for the synthesis of various analogues, with altered inhibitory profiles, rela- tivetovariousserineproteasesofinterest. One such serine protease of interest is known as matriptase (or MT-SP1), a member of the emerging class of type II transmembrane serine proteases. 4 6 The mousehomologueofmatriptasehasalsobeendescribed and is termed epithin. 7 Matriptase/epithin is of con- siderable interest for the development and pathogenesis of epithelial tissues. Although matriptase is initially synthesized by multiple types of epithelial cells as a transmembrane serine protease, it was isolated origin- ally from human milk in its activated form complexed with its cognate Kunitz type of serine inhibitor (KSPI), the hepatocyte growth factor activator inhibitor (HAI- 1). Additionally, matriptase was isolated from human breast cancer cells in culture. 4,5,8 Matriptase may func- tion to degrade the extracellular matrix, as well as sev- eral cellular regulatory proteins; specifically, it may activatehepatocytegrowthfactor(HGF)bycleavingits inactive proform, it may activate urokinase by cleaving itszymogen,anditmaycleaveandactivatetheprotease 0960-894X/01/$ - see front matter. Published by Elsevier Science Ltd. PII:S0960-894X(01)00493-0 Bioorganic & Medicinal Chemistry Letters 11 (2001) 2515–2519 *Corresponding author. Tel.: +1-301-846-5904; fax: +1-301-846- 6033; e-mail: proll@helix.nih.gov