X-ray Crystallographic and Biochemical Characterization of the Inhibitory Action of an Imidazole−Dioxolane Compound on Heme Oxygenase^,

Abstract

Heme oxygenase (HO) catalyzes the regiospecific cleavage of the porphyrin ring of heme using reducing equivalents and O2 to produce biliverdin, iron, and CO. Because CO has a cytoprotective effect through the p38-MAPK pathway, HO is a potential therapeutic target in cancer. In fact, inhibition of the HO isoform HO-1 reduces Kaposi sarcoma tumor growth. Imidazole-dioxolane compounds have recently attracted attention because they have been reported to specifically inhibit HO-1, but not HO-2, unlike Cr-containing protoporphyrin IX, a classical inhibitor of HO, that inhibits not only both HO isoforms but also other hemoproteins. The inhibitory mechanism of imidazole-dioxolane compounds, however, has not yet been characterized. Here, we determine the crystal structure of the ternary complex of rat HO-1, heme, and an imidazole-dioxolane compound, 2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane. This compound bound on the distal side of the heme iron, where the imidazole and 4-chlorophenyl groups were bound to the heme iron and the hydrophobic cavity in HO, respectively. Binding of the bulky inhibitor in the narrow distal pocket shifted the distal helix to open the distal site and moved both the heme and the proximal helix. Furthermore, the biochemical characterization revealed that the catalytic reactions of both HO-1 and HO-2 were completely stopped after the formation of verdoheme in the presence of the imidazole-dioxolane compound. This result should be mainly due to the lower reactivity of the inhibitor-bound verdoheme with O2 compared to the reactivity of the inhibitor-bound heme with O2.

Structural insights into human heme oxygenase-1 inhibition by potent and selective azole-based compounds

“…The potency of this compound was very similar to that of QC-1 (6 + 1 mM) for HO-1, but removal of the bulky 4-aminophenylthiol groups in the northeastern region resulted in decreased activity towards HO-2 in comparison with QC-1 whose IC 50 was 28 + 18 mM. This compound was also the first to be crystallized, as reported by Sugishima et al [76], in complex with rat HO-1 (rHO- 77]. The similarity in inhibitory effect was quite interesting, given the structural differences between these two compounds.…”

“…Comparison of the structures of the two inhibitor -HO-1 complexes revealed a common binding mode for these seemingly structurally distinct, imidazole-based compounds (figure 4a,b) [76,77]. While it appears that the inhibitors bind within the heme-binding pocket, they do not displace and rather require the heme moiety for binding, which explains the non-competitive nature of the inhibition as determined through kinetic analyses.…”

“…The only commonality was the eastern imidazole moiety. Interestingly, spectral analyses of both inhibitor-enzyme complexes also showed a red-shift in the Soret peak implying that, while the heme was still conjugated to the enzyme, its environment had been modified [76,77].…”

Pharmacological and Clinical Aspects of Heme Oxygenase

“…Zinc-protoporphyrin was developed to treat cancer (Fang et al, 2004b). Recently, imidazole-dioxolane compounds have been shown to act as inhibitors of HO activity (Vlahakis et al, 2006;Sugishima et al, 2007) and shown to be effective as inhibitors of specific HO isoenzymes as well as HO activity (Kinobe et al, 2007).…”

The Effects of Azole-Based Heme Oxygenase Inhibitors on Rat Cytochromes P450 2E1 and 3A1/2 and Human Cytochromes P450 3A4 and 2D6

“…The tetrazoles exhibited less effect than the 1,2,4-triazoles, which in turn exerted less effect than their imidazole-containing counterparts. Previous work detailing the cocrystallization of HO-1 with QC-15 (Sugishima et al, 2007;Rahman et al, 2008), QC-82 (Rahman et al, 2008), and QC-86 bound (Roman et al, 2010) demonstrated that the imidazole and 1,2,4-triazole pharmacophore coordinated with the heme iron through the N3 atom in the azole ring. It is possible that the heme in the P450 active site coordinates with the HO inhibitors in a similar fashion and that, with each additional nitrogen in the five-membered ring, the displaced charge on each nitrogen in the three and/or four position results in a compound that is less tightly bound to the heme in the P450 active site and could thus be more easily displaced by the substrate, leading to decreased P450 inhibition.…”