Send Orders of Reprints at reprints@benthamscience.org Current Medicinal Chemistry, 2012, 19, 5205-5213 5205 Non-Ribosomal Halogenated Protease Inhibitors from Cyanobacterial Isolates as Attractive Drug Targets M.E. Silva-Stenico, J. Rigonato, M.G. Leal, M.G.M.V. Vaz, A.P.D. Andreote and M.F. Fiore* University of São Paulo, Center for Nuclear Energy in Agriculture, Laboratory of Molecular Ecology of Cyanobacteria, 13400-970 Piracicaba, SP, Brazil Abstract: Cyanobacteria possess the ability to produce compounds with remarkable biological activity, and have thus attracted the attention of the pharmaceutical industry. Cyanopeptides acting as protease inhibitors have shown potential in the field of pharmacother- apy through regulation of abnormal physiological processes in the human body. Despite the already described cyanopeptide protease inhibitors, the search for new congeners is of considerable interest which may pave the way for more efficient molecules. In this study, the presence of the protease inhibitors aeruginosin and cyanopeptolin with non-, mono- and dichlorination and also genes coding for their synthetases was investigated in 90 cyanobacterial strains. Mass spectrometry analyses highlighted production of 91, 19 and 3 non-, mono- and dichlorinated congeners, respectively. The purified extract of Microcystis botrys SPC759 inhibited 61% of pepsin protease. PCR amplifications of aeruginosin and cyanopeptolin synthetase gene regions were observed in 41 and 28% of evaluated strains, respectively. The sequences obtained for the aerA-aerB (aeruginosin) and mcnC-mcnE (cyanopeptolin) gene regions grouped together with their homologues found in other cyanobacterial strains in the phylogenetic analyses with high bootstrap support. Antimicrobial activity assays performed using all intracellular extracts inhibited 31 and 26% of Gram-negative and Gram-positive pathogenic bacterial growth, respectively. The results of this study showed the production of aeruginosin and cyanopeptolin and the presence of their genes in several cyanobacterial genera for the first time besides the discovery of novel congeners. Keywords: Antimicrobial, aeruginosin, bioactive peptides, chlorination, cyanobacteria, cyanopeptolin, mass spectrometry, natural products, pepsin, protease inhibitors. INTRODUCTION Cyanobacteria are interesting microorganisms which can pro- duce a significant variety of valuable molecules with potential for pharmaceutical application. These natural products belong to sev- eral classes of substances, including enzyme, photosynthesis and Ca 2+ channel inhibitors, antibacterial, antimalarial, imunossuppres- sor, antitumoral, antiprotozoal, antiviral and cytotoxic [1-3]. Most of the cyanopeptides is synthesized by non-ribosomal pathway [4, 5] including the halogenated aeruginosins and cyanopeptolins [6]. Aeruginosins are natural linear peptides with protease inhibition activity, characterized by the presence of four subunits: N-terminal amino acid group (3-4 hydroxyphenyl lactic acid - Hpla), a hydrophobic amino acid, a central residue 2-carboxy-6-hydroxy octahydroindol (Choi) and a group with a guanidine-C terminal [7- 10]. Structural changes may occur in Hpla subunits (mono-, di- chlorination and sulphation) and Choi (sulphation, glycosylation and chlorination) Fig. (1A). Cyanopeptolins belong to a class of cyclic peptides besides being protease inhibitors. The general structure of this peptide class is characterized by a branched peptidolactone, containing seven amino acids, of which six form a ring [11]. All cyanopeptolins con- tain the residue 3-amino-6-hydroxy-2-piperidone (Ahp) and the peptide cyclization occurs by an ester bond of the -hydroxy group of threonine with the carboxy group of the terminal amino acid [12] Fig. (1B). Both aeruginosins and cyanopeptolins can be synthesized as non- mono- or di-chlorinated molecules. The genes aerJ and mcnD have been suggested to be involved in the production of chlorinated aeruginosin and cyanopeptolin, respectively [6]. The presence or absence of a chlorine atom in these peptides may modulate their inhibition activity [13]. For example, the chlorinated chlorodysinosin A has a potent effect against thrombin, while dysinosin A containing hydrogen instead of chlorine is less effective [8]. Here we present data showing the natural diversity of *Address correspondence to this author at the University of São Paulo, Center for Nuclear Energy in Agriculture, Molecular Ecology of Cyanobacteria Laboratory, 13400-970, Piracicaba-SP, Brazil; Tel: +55 19 3429 4657; Fax: +55 19 3429 4610; E-mail: fiore@cena.usp.br Fig. (1). Chemical structure of cyanopeptides protease inhibitors: A) Aeru- ginosin 98-A; B) Cyanopeptolin A. Structures adapted from Welker & von Döhren [22]. 1875-533X/12 $58.00+.00 © 2012 Bentham Science Publishers