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- Methods and Material
- Results and Discussion
- Conclusions and Future Directions
Imidazole-based compounds previously synthesized in our laboratory were selected and reconsidered as inhibitors of heme oxygenase-1 obtained from the microsomal fractions of rat spleens. Most of tested compounds were good inhibitors with IC50 values in the low micromolar range. Compounds were also assayed on membrane-free full-length recombinant human heme oxygenase-1; all tested compounds were unable to interact with human heme oxygenase-1 at 100 μm concentrations with the exception of compounds 11 and 13 that inhibited the enzyme of 54% and 20%, respectively. The binding of the most active compound 11 with heme or heme-conjugated human heme oxygenase-1 was also examined by spectral analyses. When heme was not conjugated to human heme oxygenase-1, compound 11 caused changes in the heme spectrum only at concentration 50-fold (100 μm) higher than that required to inhibit rat heme oxygenase-1; when heme was conjugated to human heme oxygenase-1, compound 11 was able to form a heme-compound 11 complex also at low micromolar concentrations. To obtain information on the binding mode of the tested compounds with enzyme, docking studies and pharmacophore analysis were performed. Template docking results were in agreement with experimental inhibition data and with a structure-based pharmacophoric model. These data may be exploitable to design new OH-1 inhibitors.
Heme oxygenase (HO) is a microsomal enzyme catalyzing the first, rate-limiting step in degradation of heme, yielding equimolar quantities of carbon monoxide (CO), Fe2+, and biliverdin (1). Finally, biliverdin is converted by biliverdin reductase to bilirubin (2), which can be oxidized by cytochrome P450 (CYP450) enzymes (3). Three distinct mammalian HO isoforms (HO-1, HO-2, and HO-3) have been identified, which are the products of different genes (4). HO-1, the inducible 32-kDa isoform, is highly expressed in the liver and spleen, but can be also detected in many other tissues. HO-2 is a constitutively expressed 36-kDa protein, present in high levels in the brain, testes, or endothelial cells. HO-3 was postulated as a 33-kDa protein expressed in different organs, very similar to HO-2, but with much lower catalytic activity (5). The HO system has been demonstrated to have a variety of cellular regulatory actions including anti-inflammatory, anti-apoptotic, anti-proliferative, and vasodilator effects, owing to contributing and complementary effects of each of the metabolites produced (6–9).
Interestingly, expression of HO-1 is usually increased in tumors, compared with surrounding healthy tissues (10–13). It has been reported that the growth of a number of tumors is dependent on HO-1 activity (14). These results support the idea that HO-1 may be a potential target in antitumor therapy. Thus, pharmacologic inhibition of HO-1 has been suggested as a new therapeutic option and potential sensitizer to chemotherapy, radiotherapy, or photodynamic therapy for several tumors (15–18). The efficacy of such treatments has been proven in animal models. Thus, administration of metalloporphyrins such as tin protoporphyrin (SnPP) or zinc protoporphyrin (ZnPPIX) (19,20) significantly suppressed the growth of hepatoma in rats (21), and sarcoma (22) or lung cancer in mice (23). However, owing to the close structural similarity between heme and the metalloporphyrin-HO inhibitors, specificity becomes a problem. In several systems, proteins bind heme and employ it for functions such as regulation of enzyme activity (i.e., of soluble guanylyl cyclase sGC) or as the key component of the active site of an enzyme (i.e., nitric oxide synthase NOS and CYP450). Accordingly, the use of metalloporphyrins to ascribe various physiological roles to the CO/HO system has been the subject of some criticism because they have been shown to affect the activity of both NOS and sGC (24,25). Thus, programs in various laboratories were concerned with the design of HO inhibitors that are not based on the porphyrin nucleus and have the goal of obtaining more selective HO inhibitors.
Vlanakis et al. (26–28) and Roman et al. (29,30) describe HO inhibitory activity of a series of imidazole–dioxolanes designed basing on the structure of azalanstat (1, Figure 1), the first non-porphyrin inhibitor of HO (26). Appropriate synthetic modifications to the structure of 1 lead to the development of imidazole-based analogues having enhanced inhibitory potency for HO-1 over other heme-dependent enzymes (such as sGC and NOS) (31), and an isozyme-dependent HO selectivity (26,27). Representative compounds 1–3 emerged from these studies are depicted in Figure 1.
Rahman et al. (32,33) continued to investigate on azole-based HO-1 inhibitors, which act in a non-competitive manner with respect to heme. These studies provided valuable information about the mode of binding of these compounds to HO-1 causing inhibition of heme oxidation. Furthermore, they elucidated the main structural features required for the binding to HO-1. The mechanism through these compounds inhibit heme oxidation is the disruption of an ordered hydrogen-bond network involving Asp140 and, ultimately, displacement of the distal water ligand deemed to be critical for catalysis. Structural characterization by X-ray crystallography of these azole-based inhibitors in complex with HO-1 (some of them are listed in Figure 1) shows that the main features for binding include coordination with the heme iron of the N-3 nitrogen present in the imidazolyl moiety of the inhibitors, and stabilization by an interaction between hydrophobic groups of the inhibitors and a distal hydrophobic pocket in the heme-binding pocket. It should be noted that all of the structural analyses of inhibitors in complex with HO-1 presented in the above cited studies have been performed using soluble, truncated versions of the protein. The full-length human heme oxygenase (hHO-1) has been recently expressed and purified (34). Characterization of the full-length hHO-1 demonstrated a 2,3-fold greater activity relative to that of the truncated, soluble form, which was increased even further in the presence of lipids. Further analyses suggested an important role of the C-terminal hydrophobic tail in membrane incorporation as well as formation of a high-affinity complex with CYP450 reductase (34–36). Then, the chance that this domain could significatively influence hHO-1 conformation should be taken into account for future design of novel inhibitors.
In recent years, our research group has conducted extensive investigations on a number of imidazole-based compounds designed as NOS inhibitors (37–42). These molecules present an imidazole nucleus separated from a hydrophobic aryl moiety by different spacers such as an ethanone, an alkylene, an ethanol, or an alkoxy chain of different length, giving compounds of general formulae 4–7 (Figure 2). The majority of these compounds possesses the two key features required for the binding to HO-1 (i.e., the N-3 imidazole nitrogen and a hydrophobic moiety); consequently, they might constitute a collection of imidazole-based compounds useful to select novel inhibitors of this enzyme.
A virtual screening for the above-mentioned imidazole collection using the X-ray structure (PDB_ENTRY = 3CZY) of the HO-1 co-crystallized with 1-(adamantan-1-yl)-2-(1H-imidazol-1-yl)ethanone AD8_901 (3, Figure 1) was undertaken to select new potential inhibitors. From this screening, a group of compounds showed interesting binding energies; six of them (8–13, Figure 3), further characterized by low or no effect on NOS (37,40,42) and CYP450 activities (data not shown), were chosen for biological and molecular modeling studies. Thus, compounds 8–13 were tested in vitro to evaluate their inhibitory activity on HO-1. Enzyme inhibition assays were performed on HO-1 obtained from rat spleen as the microsomal fraction and on full-length recombinant hHO-1. The binding of the tested compounds to heme-conjugated hHO-1 was additionally examined by spectral analyses. Moreover, to rationalize the binding mode, compounds 8–13 were the object of molecular docking studies and of the generation of a pharmacophoric model by the use of ligand scout software (Inte:Ligand Software-Entwicklungs und Consulting GmbH, Vienna, Austria).
Figure 3. Chemical structures of tested compounds 8–13: 8: 1-[6-(4-Bromophenoxy)exyl]-1H-imidazole; 9: 1-(2-Phenoxyethyl)-1H-imidazole; 10: 2-(1H-Imidazol-1-yl)-1-(4-nitrophenyl)ethanol; 11: 1-[4-(3-Bromophenoxy)butyl]-1H-imidazole; 12: 1-(4-Bromophenyl)-2-(1H-imidazol-1-yl)ethanone; 13: 1-(4-Bromophenyl)-2-[2-(1-methylethyl)-1H-imidazol-1-yl]ethanone.
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Conclusions and Future Directions
- Top of page
- Methods and Material
- Results and Discussion
- Conclusions and Future Directions
HO-1 inhibition of a set of imidazole-based compounds (8–13), which contain the imidazole as a common core but differ in side-chain substitution, was reported. Obtained data revealed general good inhibition of HO-1 derived from rat spleen microsomal fractions. Compound 11, which possesses a 3-bromophenoxybutyl chain, was found as a quite potent inhibitor (IC50 = 2.1 μm). Moreover, compound 11 was able to form a complex with heme bound to recombinant hHO-1 at low micromolar concentrations. This could justify its selectivity versus HO-1 rather than other heme enzymes such as NOS or CYP450. Docking studies proved the existence of diverse binding modes for various compounds: the imidazole interaction with the heme iron is likely to be a fundamental determinant of the difference in the binding. Docking results were in good agreement both with experimental data and the built structure-based pharmacophoric model, which might be useful to design novel potential OH-1 inhibitors. Future studies aiming to better define the SARs, also by scaffold hopping to replace the multitasking imidazole ring, are in progress. Our results support the potential role of the subject compounds as pharmacological tools to elucidate the physiological functions of HO-1; selective compounds might be also taken into account for therapeutic applications.