Isocyanides inhibit human heme oxygenases at the verdoheme stage.
Heme oxygenases (HO) catalyze the oxidative cleavage of heme to generate biliverdin, CO, and free iron. In humans, heme oxygenase-1 (hHO-1) is overexpressed in tumor tissues, where it helps to protect cancer cells from anticancer agents, while HOs in fungal pathogens, such as Candida albicans, function as the primary means of iron acquisition. Thus, HO can be considered a potential therapeutic target for certain diseases. In this study, we have examined the equilibrium binding of three isocyanides, isopropyl, n-butyl, and benzyl, to the two major human HO isoforms (hHO-1 and hHO-2), Candida albicans HO (CaHmx1), and human cytochrome P450 CYP3A4 using electronic absorption spectroscopy. Isocyanides coordinate to both ferric and ferrous HO-bound heme, with tighter binding by the more hydrophobic isocyanides and 200-300-fold tighter binding to the ferrous form. Benzyl isocyanide was the strongest ligand to ferrous heme in all the enzymes. Because the dissociation constants (KD) of the ligands for ferrous heme-hHO-1 were below the limit of accuracy for equilibrium titrations, stopped-flow kinetic experiments were used to measure the binding parameters of the isocyanides to ferrous hHO-1. Steady-state activity assays showed that benzyl isocyanide was the most potent uncompetitive inhibitor with respect to heme with a KI = 0.15 microM for hHO-1. Importantly, single turnover assays revealed that the reaction was completely stopped by coordination of the isocyanide to the verdoheme intermediate rather than to the ferric heme complex. Much tighter binding of the inhibitor to the verdoheme intermediate differentiates it from inhibition of, for example, CYP3A4 and offers a possible route to more selective inhibitor design.
Evans
John P
JP
Department of Pharmaceutical Chemistry, University of California, 600 16th Street, San Francisco, California 94158-2517, USA.
Kandel
Sylvie
S
Ortiz de Montellano
Paul R
PR
D000097:Acetonitriles / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003486:Cyanides / Q000737:chemistry* / Q000378:metabolism; D005290:Ferric Compounds / Q000378:metabolism; D005296:Ferrous Compounds / Q000378:metabolism; D006418:Heme / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing) / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D051547:Heme Oxygenase-1 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D009570:Nitriles / Q000737:chemistry* / Q000378:metabolism; D051381:Rats
D000097:Acetonitriles; D003486:Cyanides; D005290:Ferric Compounds; D005296:Ferrous Compounds; D009570:Nitriles; C099547:verdoheme; C006725:benzyl cyanide; C002990:butyl isocyanide; D006418:Heme; C039089:isopropyl isocyanide; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; C105245:heme oxygenase-2
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