Nile Red is a fluorescent allosteric substrate of cytochrome P450 3A4.
Cytochrome P450 3A4 (CYP3A4) plays a critical role in the metabolism of many drugs. CYP3A4 exhibits extraordinary substrate promiscuity and unusual allosteric kinetics. In addition, many CYPs catalyze sequential oxidations on a single substrate, but in most cases, mechanistic details of these processes are not well-established. As a result, in vivo clearance of many drugs and their metabolites is difficult to predict on the basis of the complex in vitro kinetics, and new in vitro probes are required to understand these behaviors. The near-IR fluorescent probe Nile Red, which has strong solvatochromic behavior, was investigated as a probe of allostery and sequential metabolism with CYP3A4. Nile Red binds with apparent Kd values of 0.05 and 2.3 muM, based on a sigmoidal dependence of heme spin state on Nile Red concentration, where the first equivalent of Nile Red increased the high-spin fraction by only 13% of the total change at saturation. Mass spectrometry analysis indicates that Nile Red is metabolized sequentially by CYP3A4 to the N-monoethyl and N-desethyl products, confirming that the immediate vicinity of the heme iron is one binding site. In the presence of CYP3A4, steady-state fluorescence emission and excitation spectra, as well as excited-state lifetimes at varying Nile Red concentrations, indicate a high-affinity site that modulates the fluorescent properties of Nile Red. The Nile Red binding site is competitively eliminated by itraconazole, which is a high-affinity ligand known to coordinate to the heme iron. Together, the data suggest that Nile Red binds to the active site with high affinity ( approximately 50 nM), where it is desolvated in a low-dielectric environment. In addition, Nile Red is sequentially oxidized at rates comparable to or faster than those of other in vitro probes, which emphasizes its utility in the further examination of this important kinetic phenomenon in vitro.
Lampe
Jed N
JN
Department of Medicinal Chemistry, Box 357610, University of Washington, Seattle, Washington 98195-7610, USA.
Fernandez
Cristina
C
Nath
Abhinav
A
Atkins
William M
WM
D000494:Allosteric Regulation; D002851:Chromatography, High Pressure Liquid; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D007700:Kinetics; D010078:Oxazines / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013997:Time Factors
D010078:Oxazines; D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C044808:nile red