Investigation of small molecule modulators binding to P450 3A4 and histone acetiltransferase p300 enzymes by means of Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR) spectroscopies

2010 - 2012
Title: 
Investigation of small molecule modulators binding to P450 3A4 and histone acetiltransferase p300 enzymes by means of Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR) spectroscopies
Principal Investigator: 
Lucia Calucci
Project type: 
National

Abstract

CYP3A4 catalyzes the oxygen-dependent metabolism of greater than 50% of known drugs. Drug candidate interactions with this enzyme are of particular interest for new drug discovery because, first, the drug itself is a substrate for CYP3A4 and, second, the drug binding to CYP3A4 can give enzyme activation or inhibition towards another substrate, thus resulting in potentially undesirable drug-drug interactions. Since substrates that bind to this enzyme change the ferric spin state equilibrium of the heme in the active site, EPR spectroscopy is particularly suited for monitoring drug-CYP3A4 interactions. To this aim, in the present project conventional (X band) and high field (up to 12 Tesla) EPR techniques will be applied to drug-enzyme systems in solution and, possibly, in physical states mimicking the micro-arrays used for drug screening. In particular, high field EPR spectroscopy will be applied to accurately characterize structural and electronic properties of the CYP3A4 heme group as influenced by drug binding in close cooperation with computational studies. On the other hand, the binding dependent spin state equilibrium of drugs with CYP3A4 will be monitored by X band EPR titrations. Complimentary information on the structural changes induced in the heme group environment by drug binding to the enzyme will be acquired by 1H NMR experiments exploiting both chemical shift and longitudinal relaxation effects induced by the unpaired electrons on proton nuclei. Also in this case a combined approach of NMR and computational techniques will allow accurate structural information to be obtained on drug binding to CYP3A4. The EPR and NMR nvestigations will be performed on drugs chosen on the basis of in vitro enzymatic essays.
The HAT p300 is a highly potent enzyme that acetylates histones and several other proteins; its dysfunction leads to several diseases including cancer, diabetes, and asthma. Therefore small molecule modulators (activators or inhibitors) of HAT are considered as new generation therapeutics. Recently, a few successful attempts have been made to understand the mechanisms of both p300 mediated acetylation of histones and modulation of p300 activity by small molecules. However, due to their specificity, many questions remain open on the molecular interactions bringing to p300 activity modulation. In this context, our research project will contribute to the elucidation of drug binding to p300 by means of a combined approach of spin labelling EPR experiments and computational methods. In particular, the Pisa research unit will apply low temperature high field EPR experiments for a thorough characterization of the spectroscopic properties (g and hyperfine tensors components) of the nitroxide group of spin labelled drugs interacting with p300 which probe the polarity and hydrogen bonding properties of the environment. Moreover, line shape analyses of EPR spectra recorded at different frequencies (X band, high frequencies from 95 to 285 GHz) and temperatures will afford valuable information on drug dynamics (mechanism, rate, and activation energy of motions) in the enzyme binding site.