Cyprotex US, LLC Stow, Massachusetts, United States
Purpose: Cytochrome P450 46A1 (CYP46A1) is predominantly expressed in brain and catalyzes the conversion of cholesterol to 24-hydroxy cholesterol as well as the rate of cholesterol production and turnover. Like other cholesterol metabolites, 24-hydroxy cholesterol has role in the central nervous system signaling. In-Vitro and In-Vivo studies have revealed that some sterols, steroids and marketed drugs can also bind to CYP46A1. Inhibition or activation of CYP46A1 enzyme can have a therapeutic potential. Here, we describe the development of rapid, sensitive and automated CYP46A1 assay in a 384-well plate format for the detection of potential inhibitors or activators using a compound library (2321 FDA approved drugs). Based on the literature, testosterone is found to be a substrate of the CYP46A1 enzyme. Therefore, testosterone is used as a probe substrate for reliability of initial screening of cassetted compound library due to short LC-MS/MS run time compared to cholesterol. Based on the cassetted analysis results individual compounds were tested with both testosterone and cholesterol as probe substrates. The present work also provides a better understanding as to which compounds should be tested as potential inhibitors or activators for CYP46A1. Methods: Time and Protein Concentration - To determine the optimum protein concentration and incubation time for the CYP46A1 assay, a range of protein concentrations (0.1, 0.25 and 0.5 mg/mL) of CYP46A1 bactosomes were incubated across several incubation times (0, 5, 10, 30, 60, and 120 minutes). Testosterone or cholesterol were added as probe substrate to designated wells and the NADPH was added to initiate the reaction. The samples were filtered by centrifugation and analyzed using LC-MS/MS to monitor 16 beta hydroxy testosterone or 24 hydroxy cholesterol metabolite formation respectively. Determination of Km and Vmax. Using the protein concentration and incubation time determined in the previous section, the formation of testosterone or cholesterol metabolites were measured with several concentrations of probe substrate covering the expected range of Km values. Validation of the CYP46A1 assay in 384-well plate - The CYP46A1 testosterone inhibition/activation assay was performed in a high-throughput 384-well format using Bravo (liquid handler). CYP46A1 bactosomes (0.25 mg/mL) in potassium phosphate buffer containing magnesium chloride were pre-incubated for 5 minutes. Soticlestat (final concentrations of 0.5 and 5uM) was used as a positive control inhibitor and vehicle was used as a negative control. The samples were incubated for 30 minutes at 37°C. The formation of 16 beta hydroxy testosterone compared to the vehicle control will be used to calculate % inhibition as well as performance of the assay. Cassette Analysis - Test compounds (library containing 2,321 compounds) were pooled into 384-well plates (4 compounds/well) using cassette analysis for initial screening. The assay was performed as described above. Individual Testing Based on the results of the cassette analysis, 242 test compounds were defined as hits and selected for individual testing. CYP46A1 Cholesterol Inhibition/Activation assay - The same 242 test compounds and control were then tested with cholesterol as probe substrate. The samples were incubated for 30 minutes at 37°C in a CO2 incubator. The formation of 24 hydroxy cholesterol compared to the vehicle control will be used to calculate % activity and % inhibition Results: The time and protein linearity results showed that 30 min incubation time and 0.25 mg/mL CYP46A1 bactosomes were optimum to run testosterone and cholesterol assay. The control inhibitor, soticlestat, in both testosterone and cholesterol assay showed inhibition of 99% and 97% respectively. The % CV was found to be less than 2.5% for the testosterone inhibition using soticlestat at both the concentrations tested across the 384 well plate. Cassette testing detected the activation or inhibition of CYP46A1 activity in 69 wells out of two 384 well plates. This resulted in 242 compounds to be individually tested for inhibition or activation of CYP46A1 using both testosterone and cholesterol. Cassetted analysis vs individual testing of compounds in the testosterone assay for inhibition or activation data showed less than 20% CV between the results. The individual testing resulted in 15 common inhibitors and 12 common activators of CYP46A1 for the testosterone and cholesterol assays. Conclusion: The system described here not only establishes high throughput automated method using 384-well plates to predict the therapeutic inhibitors or activators of CYP46A1. The low % CV for the inhibition of testosterone using soticlestat reveals the reliability of the assay across the 384 well plate. The assay also shows testosterone could be used as the probe substrate for ease of analysis as compared to cholesterol. Cassetting was reliable to screen the compounds for the activation or inhibition of CYP46A1 activity. A possible limitation to this approach is an interaction between compounds leading to false positive or false negative results. To demonstrate the efficacy of this screen, we identified clotrimazole as an inhibitor of CYP46A1 which agrees with the previous literature. Our study results give insight into structurally diverse compounds that can inhibit or activate the CYP46A1 enzyme.
