Identification of peptide inhibitors of transforming growth factor beta 1 using a phage-displayed peptide library Javier Dotor a, * , Ana B. Lo ´ pez-Va ´zquez a , Juan J. Lasarte a , Pablo Sarobe a , Marta Garcı ´a-Granero a , Jose ´ I. Riezu-Boj a , Alfonso Martı ´nez b , Esperanza Feijoo ´ c , Jacinto Lo ´ pez-Sagaseta d , Jose ´ Hermida d , Jesu ´s Prieto a , Francisco Borra ´s-Cuesta a a Division of Hepatology and Gene Therapy, Center for Applied Medical Research (CIMA), University of Navarra, Avda. Pı ´o XII, 55-31008-Pamplona, Spain b CIC bioGUNE, Technological Park of Bizkaia, Bizkaia, Spain c DIGNA Biotech, Madrid, Spain d Division of Cardiovascular Sciences, Laboratory of Thrombosis and Haemostasis, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Received 28 November 2006; received in revised form 8 June 2007; accepted 18 June 2007 Abstract Pathologies such as liver fibrosis and scleroderma are characterized by harmful levels of transforming growth factor beta 1 (TGFb1). These levels could be neutralized if inhibitors of this cytokine were available. With this aim we searched for peptides with binding affinity for TGFb1 using a phage-displayed random 15-mer peptide library. Some peptides thus identified blocked activity of TGFb1 in vitro, as measured by their capacity to restore growth of Mv-1-Lu cells in presence of added TGFb1. Also, they inhibited TGFb1-dependent expression of collagen type I mRNA in liver of mice orally insulted with CCl 4 . Intraperitoneal administration of 50 lg of peptide P17 (the most active 15-mer peptide, also referred to as P17 (1–15) ) inhibited expression of collagen type I mRNA by almost 100%. Inter- estingly, titration experiments showed that P17 (1–12) (a peptide encompassing the first 12 amino acids of P17) was approximately four times more active than P17. These results suggest that both peptides, as well as others reported here, may be of therapeutic interest in processes requiring control of undesired high levels of TGFb1. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Transforming growth factor beta 1; Inhibitor peptides; Phage display; Liver; Fibrosis 1. Introduction Transforming growth factor beta (TGFb) belongs to a family of regulatory cytokines. Among them, isoforms TGFb1, TGFb2 and TGFb3 participate in a large number of biological processes such as cell proliferation [1], embry- onic development [2], immune-modulation [3], angiogenesis [4], tumorogenesis [5], and balance between cell mass and extracellular matrix [6]. Thus, enhanced levels of TGFb, play an important role in virus-induced liver fibrosis [7–9], in alcohol-induced cirrhosis [10], scarring during wound healing [11] or undesired regulation of immune responses [3]. These undesired side effects due to TGFb, has prompted several research groups to develop strategies to neutralize this cytokine. Thus, it has been reported that antisense oligonucleotides [12,13], antibodies against TGFb [14,15], as well as synthetic peptides [16–18] can effi- ciently block the activity of TGFb. Using a computer algorithm developed in our labora- tory, we reported the identification of two peptides derived from an identical region of human and rat TGFb1 type III receptor, which are able to block TGFb1 in vitro [16]. 1043-4666/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2007.06.004 * Corresponding author. Fax: +34 948 194717. E-mail address: jdotor@unav.es (J. Dotor). www.elsevier.com/locate/issn/10434666 Cytokine 39 (2007) 106–115