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BioAssay: AID 686941

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
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 Tested Compounds
 Tested Compounds
All(31)
 
 
Unspecified(31)
 
 
 Tested Substances
 Tested Substances
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Unspecified(31)
 
 
 Related BioAssays
 Related BioAssays
AID: 686941
Data Source: The Scripps Research Institute Molecular Screening Center (Recombinant_CYP_INH_SAR_MDCSRUN)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2013-04-16
Hold-until Date: 2013-10-29
Modify Date: 2013-10-29

Data Table ( Complete ):           All
Tested Compounds:
Depositor Specified Assays
Show more
AIDNameTypeComment
602396Luminescence-based cell-based primary high throughput screening assay to identify inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2)screeningPrimary screen (LRH-1 inhibitors in singlicate)
602418Summary of the probe development efforts to identify inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2)summarySummary (LRH-1 inhibitors)
651611Counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput assay to identify nonselective inhibitors of the Steroidogenic acute regulatory protein (StAR) promoter or luminescence assay artifactsscreeningCounterscreen (StAR promoter non-selective inhbitors or luminescence assay artifacts in quadruplicate)
651613Luminescence-based cell-based high throughput confirmation assay for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2)screeningConfirmation assay (LRH-1 inverse agonists in triplicate)
651614Counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput assay to identify inverse agonists of the Steroidogenic Factor 1 Nuclear Receptor (SF1; NR5A1)screeningCounterscreen (SF1 inverse agonists in triplicate)
651615Counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16)screeningCounterscreen (VP16 inhibitors in triplicate)
651968Counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput dose response assay to identify inverse agonists of the Steroidogenic Factor 1 Nuclear Receptor (SF1; NR5A1)confirmatoryDose response counterscreen (SF1 inverse agonists in quadruplicate)
651969Counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput dose response assay to identify nonselective inhibitors of the Steroidogenic acute regulatory protein (StAR) promoter or luminescence assay artifactsconfirmatoryDose response counterscreen (StAR promoter non-selective inhbitors or luminescence assay artifacts in quadruplicate)
651970Luminescence-based cell-based high throughput dose response assay for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2)confirmatoryDose response counterscreen (LRH-1 inverse agonists in quadruplicate)
651973Counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput dose response assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16)confirmatoryDose response counterscreen (VP16 inhibitors in quadruplicate)
651974Late stage counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput dose response assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16)confirmatoryLate stage dose response counterscreen (VP16 inhibitors in triplicate)
651975Late stage counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput dose response assay to identify nonselective inhibitors of the Steroidogenic acute regulatory protein (StAR) promoter or luminescence assay artifactsconfirmatoryLate stage dose response counterscreen (StAR promoter inhibitors or luminescence assay artifacts in triplicate)
651976Late stage luminescence-based cell-based high throughput dose response assay for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2)confirmatoryLate stage dose response (LRH-1 inverse agonists in triplicate)
651977Late stage counterscreen for inverse agonists of the liver receptor homolog-1 (LRH-1; NR5A2): Luminescence-based cell-based high throughput dose response assay to identify inverse agonists of the Steroidogenic Factor 1 Nuclear Receptor (SF1; NR5A1)confirmatoryLate stage dose response counterscreen (SF1 inverse agonists in triplicate)
Description:
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.

Description:

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).

References:

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.

Keywords:

CYP3A4, CYP3A5, cytochrome P450
Panel Information
Targets
PID§NameSubstancePanel TargetsDescriptionAdditional Information
ActiveInactive
1CYP3A4cytochrome P450, family 3, subfamily A, polypeptide 4 [Homo sapiens] [gi:51094614]
Taxonomy id: 9606
Gene id: 1576
2CYP3A5RecName: Full=Cytochrome P450 3A5; AltName: Full=CYPIIIA5; AltName: Full=Cytochrome P450 HLp2; AltName: Full=Cytochrome P450-PCN3 [gi:117157]
Taxonomy id: 9606
Gene id: 1577

§ Panel component ID.
Protocol
Assay Overview:

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.

Protocol Summary:

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 ) )

Where:

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.
Comment
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.
Result Definitions
TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1QualifierQualifier identifies if the resultant selectivity value came from dividing two IC50 values, or from using a lower estimate based on the percent inhibition for one of the targets at the highest concentration tested, 60 uM.String
2Fold Selectivity for CYP3A4/CYP3A5Selectivity for CYP3A4 over CYP3A5Floatratio
3IC50 [CYP3A4]*Value of IC50 for CYP3A4 expressed in uM1cytochrome P450, family 3, subfamily A, polypeptide 4 [Homo sapiens]FloatμM
4Inhibition at 60 uM [CYP3A4] (60μM**)Percent inhibition of CYP3A4 at the highest concentration tested, 60 uM.1Float%
5IC50 [CYP3A5]*Value of IC50 for CYP3A5 expressed in uM2RecName: Full=Cytochrome P450 3A5; AltName: Full=CYPIIIA5; AltName: Full=Cytochrome P450 HLp2; AltName: Full=Cytochrome P450-PCN3FloatμM
6Inhibition at 60 uM [CYP3A5] (60μM**)Percent inhibition of CYP3A5 at the highest concentration tested, 60 uM.2Float%

* Activity Concentration. ** Test Concentration. § Panel component ID.
Additional Information
Grant Number: U54 MH084512

Classification
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