Drug binding characterization of CYPs utilizing CW and pulsed EPR

Abstract

Electron paramagnetic resonance (EPR) methods have been used to study drug and ligand interactions with a super family of monooxygenase enzymes known as Cytochrome P450 (CYP). We examined the active-site of four different isoforms of CYPs before and after the addition of drug using EPR. Two of the CYPs studied, CYP3A4 and CYP2C9, play a major role in drug metabolism and the other two, CYP51B1 and CYP125A1, are attractive as therapeutic targets for the pathogen Mycobacterium tuberculosis. EPR has shown to be a quick and highly resolved method, in comparison to current methods such as crystal structure analysis and UV/Vis optical difference spectroscopy, to study CYPs ferric heme active-site before and after drugs bind, which could be very valuable in drug design. Chapter 2 examines pulsed EPR methods for studying the active-site in CYPs. The explanation of practical aspects of experimentation along with data processing provides the EPR background for studying ferric heme-containing enzymes enabling a researcher to extract highly resolved active-site information. The experimental EPR methods described in Chapter 2 are the methods used in Chapter 3 and 4. Chapter 3 examines the resting state active-site of all four isoforms which is structurally described as a single water molecule bound to the distal position of the ferric heme. CW EPR spectroscopy of the isoforms all gave different g-values and MCD showed that water ligands bind at different strengths depending on the CYP isoform which sheds light on substrate specificity in each isoform. An attempt was made at predicting nIR MCD transitions with the EPR parameters but results were unclear. Chapter 4 studied CYP2C9 and CYP125A1 in complex with drugs that had the same binding mode but different optical difference spectra. We showed that the low-spin complex between a drug-metabolizing CYP2C9 variant in complex with a drug PPT retains the water ligand seen in the resting state. Hydrogens from the axial water ligand are observed by pulsed EPR spectroscopy for both drug-free and drug-bound species showing that the drug does not displace the water ligand seen in the resting state. An 15N-label incorporated into PPT is .444 nm from the heme iron indicating that PPT is in the active-site. CYP125A1 gave the same EPR signatures seen for CYP2C9 and PPT along with an X-ray crystal structure of CYP125A1 in complex with LP10 showing a water-bridged complex. The same binding mode was seen in both complexes but optical difference spectra of CYP2C9 and PPT resemble ‘classic’ type II behavior while those of CYP125A1 and LP10 have reverse type I behavior, again providing direct evidence that optical difference spectra are not reliable for characterization of drug binding mode.