IDENTIFICATION OF NOVEL SSAO/VAP-1 INHIBITORS BY HIGH THROUGHPUT SCREENING

E Cuadrado1, C Carpéné2, J Mercader2, MI Loza1, MI Cadavid1, J Brea1. 1University of Santiago de Compostela, Department of Pharmacology- 15782, Spain, 2INSERMN, I2MC U1048, France

Semicarbazide-sensitive amine oxidase (SSAO) is a cell-surface enzyme found in adipocytes and vessels. This enzyme is identical to Vascular Adhesion Protein-1 (VAP-1), an adhesion molecule mainly found in endothelial cells of inflammed regions. Recently, novel SSAO/VAP-1 inhibitors were tested and patented for their anti-inflammatory properties (Dunkel et al., Curr Med Chem 2008; 15:1827-39), while the prototypical inhibitor, semicarbazide was reported to limit fat accumulation in rodents (Mercader et al. J Obes 2011; 2011:475786). Our aim was to identify novel compounds that could inhibit SSAO in human fat cells more efficiently than semicarbazide, which is currently suspected to be genotoxic.

We developed a miniaturized fluorescent method that detected the hydrogen peroxide generated by human aypose tissue preparations during amine oxidation. By using this methodology, a High Throughput Screening campaign of the Prestwick chemical library was carried out in 96-well plates. All the compounds were tested as inhibitors of 50 µM benzylamine oxidation at a final concentration of 10 µM. In these conditions, 14 compounds were classified as hits, being phenelzine the most active one (101.8 ± 4.6 % inhibition, when compared to 1 mM semicarbazide used as 100 % inhibition reference). Such strong inhibition of phenelzine of human SSAO by low doses, when compared to semicarbazide, was observed by first time in humans and is in complete agreement with the previously observed inhibitions of bovine (Lizcano et al., Biochem Pharmacol 1996;52:187-95) or rat (Carpéné et al., Pharmacol Res 2008; 57:426-34) SSAO activities by this hydrazine derivative.

These results are an interesting starting point to decipher whether: 1) the SSAO inhibition may play a role in the clinical usefulness of phenelzine in treating depressive patients; 2) whether other pathologies may benefit from the SSAO inhibitory properties of this drug.

Moreover, phenelzine and the diverse other chemical entities which partially impaired benzylamine oxidation became an starting point for hit to lead process at novel SSAO inhibitors discovery for obesity treatment.

Grants: This work was supported by SUDOE INTERREG IVB (SOE1/P1/E178).