A novel, "double-clamp" binding mode for human heme oxygenase-1 inhibition

Abstract

The development of heme oxygenase (HO) inhibitors is critical in dissecting and understanding the HO system and for potential therapeutic applications. We have established a program to design and optimize HO inhibitors using structure-activity relationships in conjunction with X-ray crystallographic analyses. One of our previous complex crystal structures revealed a putative secondary hydrophobic binding pocket which could be exploited for a new design strategy by introducing a functional group that would fit into this potential site. To test this hypothesis and gain further insights into the structural basis of inhibitor binding, we have synthesized and characterized 1-(1H-imidazol-1-yl)-4,4-diphenyl-2-butanone (QC-308). Using a carbon monoxide (CO) formation assay on rat spleen microsomes, the compound was found to be ∼15 times more potent (IC(50) = 0.27±0.07 µM) than its monophenyl analogue, which is already a potent compound in its own right (QC-65; IC(50) = 4.0±1.8 µM). The crystal structure of hHO-1 with QC-308 revealed that the second phenyl group in the western region of the compound is indeed accommodated by a definitive secondary proximal hydrophobic pocket. Thus, the two phenyl moieties are each stabilized by distinct hydrophobic pockets. This "double-clamp" binding offers additional inhibitor stabilization and provides a new route for improvement of human heme oxygenase inhibitors.

Figure 1. The oxidative degradation of heme in the carbon monoxide/heme oxygenase (CO/HO) pathway.

Figure 2. Structures of azalanstat and QC-xx compounds used for crystallization studies.

Figure 3. Structure of 1-(1H-imidazol-1-yl)-4,4-diphenyl-2-butanone (QC-308).

QC-65 lacks the second phenyl moiety.

Figure 4. Inhibition of HO-1 activity by QC-308.

Enzyme activity was determined by measuring the CO produced in 15 min from 50 µM methemalbumin using 0.5 mg/mL rat spleen microsomes. The IC₅₀ was determined by nonlinear regression using GraphPad Prism version 4. Curves represent two independent trials, with each performed in duplicate.

Figure 5. Spectral analysis of QC-308 binding to hHO-1.

(A) Heme-conjugated hHO-1 (10 µM) in 20 mM potassium phosphate (pH 7.4) was incubated with increasing concentrations of QC-308 at room temperature. Absorbances were measured over a range of 300-700 nm at intervals of 1 nm, and values were corrected for buffer (20 mM potassium phosphate, pH 7.4). The assays were performed in duplicate and the values averaged. The Soret peak gradually shifted from 404 to 410 nm with increasing concentrations of QC-308. Secondary peaks centered at 535 and 560 nm were amplified with increasing concentrations of inhibitor, while a third minor peak at 630 nm decreased until no longer detectable at high inhibitor concentrations (B) Heme degradation rates in the presence of QC-308. Heme degradation was subsequently initiated by the addition of 1 mM l-ascorbic acid and allowed to proceed for 90 min at room temperature. Absorbances were measured at 404, 406, 408 and 410 nm at 1 min intervals and normalized to the initial absorbance (t = 0) for the respective condition. Graph is representative of one replicate for each condition at the wavelength of its Soret peak (indicated in legend). (C) Initial rates were determined for each condition over a period of 1 min (from t = 2–3 min) at the wavelength corresponding to its respective Soret peak as indicated in the legend in (B). For each replicate, values were normalized to the respective control condition, and subsequently averaged. Parallel reactions were also performed for heme-conjugated hHO-1 in the absence of both inhibitor and electron donor (l-ascorbic acid) as a negative control (i.e., no oxidative degradation). (D) Spectral analysis following heme degradation. Absorbances were measured and analyzed as described in (A). Heme degradation corresponded to the disappearance of the Soret peak. Increasing concentrations of inhibitor resulted in increased attenuation of the loss of the Soret peak as well as of the secondary peaks at 535 and 560 nm but the appearance of a peak at 699 nm. Inset depicts the fraction of heme still undegraded after the 90 min reaction, relative to that present at t = 0 for each condition.

Figure 6. Crystal structure of heme–conjugated hHO-1 in complex with QC-308 at 2.85 Å resolution.

(A) Ribbon diagram of the inhibitor binding site. Heme (orange) and QC-308 (yellow) are depicted as stick models. An omit map (F_o-F_c) contoured at 2σ is superimposed. Dashed lines indicate coordination of imidazole nitrogens of QC-308 and His25 with the heme Fe. Residues involved in inhibitor binding are indicated. (B) Electrostatic surface potentials revealing the presence of two distal hydrophobic pockets (1° HP and 2° HP) which accommodate the two phenyl groups of QC-308: a “double-clamp”. Dashes indicate coordination of the imidazole group with the heme Fe. Blue and red colours indicate positive and negative electrostatic potentials, respectively, as calculated using PyMOL .

Figure 7. Inhibition of HO-2 activity by QC-308.

Enzyme activity was determined by measuring the CO produced in 15 min from 50 µM methemalbumin using 0.5 mg/mL rat brain microsomes. Calculations were performed as described for Figure 4.

Figure 8. Spectral analysis of QC-308 binding to hHO-2.

Analyses were done in parallel to those in Figure 5.

Figure 9. Ribbon diagram showing the structural alignment of hHO-2 (cyan) with the hHO-1–QC-308 complex (green).

Residues involved in QC-308 binding are depicted as stick models, as are heme and QC-308 (yellow). The two structures are virtually identical in this catalytic core. Residues of hHO-2 which differ amongst the contact residues of the hHO-1 inhibitor binding site are depicted in red. Structural alignments were performed using “Superpose” in CCP4 , .