Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP enzyme in the liver and metabolizes approximately 50% of the drugs, including anti-retrovirals. Although CYP3A4 induction by ethanol and impact of CYP3A4 on drug metabolism and toxicity is known, CYP3A4-ethanol physical interaction and its impact on drug binding, inhibition, or metabolism is not known. Therefore, we studied the effect of ethanol on binding and inhibition of CYP3A4 with a representative protease inhibitor, nelfinavir, followed by the effect of alcohol on nelfinavir metabolism. Our initial results showed that methanol, ethanol, isopropanol, isobutanol, and isoamyl alcohol bind in the active site of CYP3A4 and exhibit type I spectra. Among these alcohol compounds, ethanol showed the lowest KD (5.9 +/- 0.34 mM), suggesting its strong binding affinity with CYP3A4. Ethanol (20 mM) decreased the KD of nelfinavir by >5-fold (0.041 +/- 0.007 vs. 0.227 +/- 0.038 mu M). Similarly, 20 mM ethanol decreased the 1050 of nelfinavir by >3-fold (2.6 +/- 0.5 vs. 8.3 +/- 3.1 mu M). These results suggest that ethanol facilitates binding of nelfinavir with CYP3A4. Furthermore, we performed nelfinavir metabolism using LCMS. Although ethanol did not alter k(cat), it decreased the Km of nelfinavir, suggesting a decrease in catalytic efficiency (k(cat)/K-m). This is an important finding because alcoholism is prevalent in HIV-1-infected persons and alcohol is shown to decrease the response to antiretroviral therapy. Published by Elsevier Inc.