Late stage assay provider counterscreen for P450 inhibition. LC-MS/MS-based assay to determine inhibitory activity of compounds against purified recombinantly expressed CYP3A4 and CYP3A5
Name: Late stage assay provider counterscreen for P450 inhibition. LC-MS/MS-based assay to determine inhibitory activity of compounds against purified recombinantly expressed CYP3A4 and CYP3A5. ..more
Depositor Specified Assays
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Patrick Griffin, TSRI
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: U54 MH084512
Grant Proposal PI: Patrick Griffin, TSRI
External Assay ID: Recombinant_CYP_INH_SAR_MDCSRUN
Name: Late stage assay provider counterscreen for P450 inhibition. LC-MS/MS-based assay to determine inhibitory activity of compounds against purified recombinantly expressed CYP3A4 and CYP3A5.
The goal of this project is to identify modulators (inverse agonists) of the orphan nuclear receptor LRH-1, which has been implicated in cancer by enhancing proliferation and cell cycle progression and metabolic disorders through its regulation of genes involved cholesterol and bile acid homeostasis. Some compounds were found to be selective CYP3A4 vs CYP3A5 inhibitors and additional SAR was done to expand the selectivity wihtout regard to LHR-1 activity (1-2).
1. Li X, Song X, Kamenecka TM, Cameron MD. Discovery of a Highly Selective CYP3A4 Inhibitor Suitable for Reaction Phenotyping Studies and Differentiation of CYP3A4 and CYP3A5. Drug Metab Dispos. 2012 Sep;40(9):1803-9.
2.Song X, Li X, Ruiz CH, Yin Y, Feng Y, Kamenecka TM, Cameron MD. "Imidazopyridines as selective CYP3A4 inhibitors." Bioorg Med Chem Lett. 2012 Feb 15;22(4):1611-4.
CYP3A4, CYP3A5, cytochrome P450
§ Panel component ID.
The purpose of this assay is to determine inhibitory activity of compounds against purified
recombinantly expressed CYP3A4 and CYP3A5.
This assay monitors CYP3A4 and CYP3A5 activity using recombinantly expressed P450 purchased from BD-Biosciences. Activity was kinetic based following the hydroxylation of midazolam and testosterone via LC-MS/MS assays.
Incubations utilizing recombinantly expressed P450 (BD Supersomes, BD Biosciences, Woburn, MA) were conducted similarly to what is described for HLM. Enzyme concentration for CYP3A4 and CYP3A5 incubations were 10 nM (2 pmole enzyme in 0.2 ml). Substrate concentrations were approximately equal to their respective Km. All incubations were prepared in 0.1 M potassium phosphate buffer, pH 7.4, and incubated at 37 C with shaking. Testosterone and midazolam are referred to as CYP3A substrates as they are metabolized in HLM by both CYP3A4 and CYP3A5. Analysis was by LC-MS/MS using an API4000 mass spectrometer (Applied Biosystems, Foster City, CA) interfaced with an Agilent 1200 HPLC (Agilent Technologies, Palo Alto, CA). In most cases chromatographic separation was achieved by using a Phenomenex Synergi Fusion RP C18 column (2.0 x 50 mm, 4 um) with a mobile phase consisted of 0.1% aqueous formic acid (solvent-A) and acetonitrile with 0.1% formic acid (solvent-B) run at a constant flow rate of 0.375 ml/min. A 2.5 minute HPLC method was used with % B equal to 2% at t=0 min, 80% at t=1.35-1.6 min, and 2% at t=1.61-2.5 min (all gradients were linear).
The percent inhibition for each compound was calculated as follows:
Data was curve fit using GraphPad Prism. Unless otherwise noted the built-in one-site competition model was used where the following equation was used:
Y = Bottom + ( Top - Bottom ) / ( 1+10^( X - LogIC50 ) )
X is the log(Concentration)
Y is the percent inhibition
Bottom and Top refer to the minimum and maximum for the curves. In most cases the values were constrained between 0 and 100%.
Values are mean of two or more experiments using midazolam hydroxylation as a measure or CYP3A4/5 activity. The error in these values is within +/- 30% of the mean.
For compounds that inhibited the target at less than 100% at the highest concentration tested (60 uM), the percent inhibition at 60 uM is reported.
Fold selectivity for CYP3A4/CYP3A5 is reported. In cases where a percent inhibition rather than an IC50 is reported, 60 uM is used for the selectivity calculation.
This assay was performed in the assay provider's lab. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis.
* Activity Concentration. ** Test Concentration. § Panel component ID.