Caratterizzazione preclinica in vitro di nuovi inibitori non peptidomimetici della glutatione trasferasi P1-1

Glutathione transferases (formerly glutathione S-transferases, GSTs) are a superfamily of enzymes involved in the glutathione (GSH)-dependend detoxification of a wide range of chemicals, including drugs. Moreover, certain members of this superfamily interact with and modulate the activity of protein kinases involved in key cellular processes, including proliferation and apoptosis [Ruzza et al., 2009]. Among them, GSTP1-1 is frequently overexpressed in a variety of human tumors, where it contributes to conferring resistance to different anticancer agents. In light of these observations, several GST inhibitors or GST-activated prodrugs have been investigated throughout the years; however, none of them has been approved for use as antitumor drug [Ruzza et al., 2009; Sau et al., 2010]. 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) and its structural analogue MC3181 are promising anticancer agents with potent inhibitory activity towards GSTP1-1 [Ricci et al., 2005; Pasello et al., 2011; De Luca et al., 2014]. A first aim of this work was to evaluate the metabolic fate of these compounds in humans and in laboratory animal species. The metabolic stability of NBDHEX and MC3181 was assessed by high performance liquid chromatography (HPLC) or LC coupled to diode array detection and tandem mass spectrometry (LC-DAD-MS/MS), upon incubation of each drug with human, mouse or rat liver microsomes or cytosol as enzyme source. The reactions investigated were UDP-glucuronic acid (UDPGA)-dependent glucuronidation, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent microsomal oxidation, and oxidized nicotinamide adenine dinucleotide (NAD+)-dependent cytosolic oxidation. Both NBDHEX and MC3181 underwent glucuronidation and microsomal NADPH-dependent oxidation in all of the investigated species. Moreover, NBDHEX, but not MC3181, underwent alcohol dehydrogenase-dependent oxidation in both human, rat and mouse cytosol. The identity of NBDHEX glucuronide was confirmed by nuclear magnetic resonance analyses. Finally, interspecies differences were identified in the glucuronidation of both compounds and in the cytosolic oxidation of NBDHEX, while sex-related differences were observed in the rate of glucuronidation of MC3181 as well as in the rate of cytosolic oxidation of NBDHEX. Sulfasalazine (sulfasalazopyridine; SASP), a drug currently used to treat rheumatic and inflammatory bowel diseases, is a non-substrate inhibitor of various human GSTs, including GSTP1-1 [Ahmad et al., 1992]. Despite this, its poor oral bioavailability and metabolic instability (which is mainly linked to the presence of an azo group in its structure) hinder its use as an anticancer agent. In this work, 30 SASP analogues containing an imidazole ring in substitution of the azo group of SASP (i.e. salycylbenzoimidazole derivatives, EML), have been investigated as potential inhibitors of human GSTP1-1. Further structural modifications involved the sulfonamide as well as the aminosalicylic moiety of the drug. Seven out of 30 of the salycylbenzoimidazole derivatives studied (i.e. EML340, EML277, EML259, EML337, EML357, EML279, and EML338) inhibited human placental GST (mostly GSTP1-1) conjugation activity more efficiently than the parent compound, SASP. Thus, the azo group seems to be not essential for inhibition of the enzyme activity. Further enzyme inhibition assays carried out using human recombinant GST A1-1, M1-1 and P1-1, showed that EML337 displays a certain grade of selectivity for GSTP1-1, while EML340 and EML277 were highly selective towards GSTM1-1. Finally, preliminary in vitro cytotoxicity assays indicate that the methyl esters EML259, EML337 and EML339 can interfere with the growth of A375 and SK-MEL 23 human melanoma cell lines.