title: 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.
authors.affiliation: Department of Medicinal Chemistry, Box 357610, and Department of Chemistry, Box 351700, University of Washington , Seattle, Washington 98195, United States.
authors.forename: Kip P
authors.initials: KP
authors.lastname: Conner
authors.forename: Alex A
authors.initials: AA
authors.lastname: Cruce
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Krzyaniak
authors.forename: Alina M
authors.initials: AM
authors.lastname: Schimpf
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Frank
authors.forename: Paul
authors.initials: P
authors.lastname: Ortiz de Montellano
authors.forename: William M
authors.initials: WM
authors.lastname: Atkins
authors.forename: Michael K
authors.initials: MK
authors.lastname: Bowman
mesh_terms: D000631:Aminopyridines / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D065729:Cytochrome P-450 CYP2C9 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D036145:Ginsenosides / Q000737:chemistry*; D006418:Heme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000737:chemistry*; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012502:Sapogenins / Q000737:chemistry*; D014867:Water / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000631:Aminopyridines; D001426:Bacterial Proteins; D036145:Ginsenosides; D007211:Indoles; D012502:Sapogenins; C552044:alpha-((4-methylcyclohexyl)carbonylamino)-N-4-pyridinyl-1H-indole-3-propanamide; C507965:ginsenoside 20S-protopanaxatriol; D014867:Water; D006418:Heme; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9