Drug modulation of water-heme interactions in low-spin P450 complexes of CYP2C9d and CYP125A1

Abstract

Azoles and pyridines are commonly incorporated into small molecule inhibitor scaffolds that target cytochromes P450 (CYPs) as a strategy to increase drug binding affinity, impart isoform-dependent selectivity, and improve metabolic stability. Optical absorbance spectra of the CYP-inhibitor complex are widely used to infer whether these inhibitors are ligated directly to the heme iron as catalytically inert, low-spin (type II) complexes. Here, we show that the low-spin complex between a drug-metabolizing CYP2C9 variant and 4-(3-phenylpropyl)-1H-1,2,3-triazole (PPT) retains an axial water ligand despite exhibiting elements of "classic" type II optical behavior. Hydrogens of the axial water ligand are observed by pulsed electron paramagnetic resonance (EPR) spectroscopy for both inhibitor-free and inhibitor-bound species and show that inhibitor binding does not displace the axial water. A (15)N label incorporated into PPT is 0.444 nm from the heme iron, showing that PPT is also in the active site. The reverse type I inhibitor, LP10, of CYP125A1 from Mycobacterium tuberculosis, known from X-ray crystal structures to form a low-spin water-bridged complex, is found by EPR and by visible and near-infrared magnetic circular dichroism spectroscopy to retain the axial water ligand in the complex in solution.

Figure 1

Absorbance difference spectra (left) and binding isotherm (right) for CYP2C9d with PPT (top) and 1,2,3-TRZ (bottom). Note the lack (top left) of the positive peak observed previously in the Soret region (350–470 nm) with other nitrogenous ligands for CYP2C9. The α/β bands in the absolute absorbance spectra in the inset are similar to those found in classic type II spectra.

Figure 2

EPR spectral shifts for CYP2C9d with addition of inhibitor: red, CYP2C9d spectrum with no added drug; green, addition of 1,2,3-TRZ shifts the g_z peak to higher g values (lower field), indicative of water displacement and type II binding; blue, addition of PPT shifts the g_z peak to a lower g value (higher field).

Figure 3

CW EPR spectrum of CYP125A1 (blue) and in complex with pyridine inhibitor LP10 (red). The inset shows a noticeable sharpening and a shift of the g_z feature.

Figure 4

HYSCORE spectra (multicolor surface) and simulations of the water peaks (green contours) show consistent fits with and without the drug for CYP2C9d and CYP125A1. The parameters used to simulate the HYSCORE spectra are listed in Table 2.

Figure 5

Definition of the polar angles θ and ϕ for the hydrogens of the water relative to the g axis system of the heme. The length of the red vector connecting the heme iron and water hydrogen is R.

Figure 6

HYSCORE (left and center) and difference HYSCORE (right) spectra of the CYP2C9–(PPT) complex with [¹⁵N]PPT or [¹⁴N]PPT at g = 2.396 (top row) and g = 2.376 (bottom row). The black contour lines are simulations using parameters listed in Table 2. In the difference spectra (right), blue indicates negative intensity (decrease of some ¹⁴N peaks) and red indicates positive (appearance of ¹⁵N peaks).

Figure 7

MCD spectra (6 T) of CYP125A1 (red) and CYP125A1-(LP10) (yellow). The visible MCD spectra are shown at 298 K (top left) and 4.2 K (top right). The corresponding near-infrared spectra at the same temperature are shown in the bottom panels.