Late stage assay provider counterscreen for P450 inhibition based on the formation of Vincristine M1 formation in Genotyped HLM
Name: Late stage assay provider counterscreen for P450 inhibition based on the formation of Vincristine M1 formation in Genotyped HLM. ..more
BioActive Compound: 1
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: M1_Formation_in_Genotyped_HLM_SAR_MDCSRUN
Name: Late stage assay provider counterscreen for P450 inhibition based on the formation of Vincristine M1 formation in Genotyped HLM.
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 CYP 3A5 inhibitors and additional SAR was done to expand the selectivity wihtout regard to LHR-1 activity (1-7).
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.
3. Dennison JB, Jones DR, Renbarger JL, and Hall SD (2007) Effect of CYP3A5 expression on vincristine metabolism with human liver microsomes. J Pharmacol Exp Ther 321:553-563.
4. Dennison JB, Kulanthaivel P, Barbuch RJ, Renbarger JL, Ehlhardt WJ, and Hall SD (2006) Selective metabolism of vincristine in vitro by CYP3A5. Drug Metab Dispos 34:1317-1327.
5. Dennison JB, Mohutsky MA, Barbuch RJ, Wrighton SA, and Hall SD (2008a) Apparent high CYP3A5 expression is required for significant metabolism of vincristine by human cryopreserved hepatocytes. J Pharmacol Exp Ther 327:248-257.
6. Dennison JB, Renbarger JL, Walterhouse DO, Jones DR, and Hall SD (2008b) Quantification of vincristine and its major metabolite in human plasma by high-performance liquid chromatography/tandem mass spectrometry. Ther Drug Monit 30:357-364.
7. Egbelakin A, Ferguson MJ, MacGill EA, Lehmann AS, Topletz AR, Quinney SK, Li L, McCammack KC, Hall SD, and Renbarger JL (2011) Increased risk of vincristine neurotoxicity associated with low CYP3A5 expression genotype in children with acute lymphoblastic leukemia. Pediatr Blood Cancer 56:361-367.
CYP3A4, CYP3A5, cytochrome P450
The purpose of this assay is to determine if CYP3A5 activity could be "isolated" in human liver microsomes that contained both CYP3A4 and CYP3A5. Midazolam hydroxylation was used as a measure of total CYP3A4+3A5 activity and Vincristine M1 formation was used as an approximate marker of CYP3A5 activity as it is moderately selective for CYP3A5 (between 5-10-times greater activity with CYP3A5). Selective inhibitors were able to inhibit the CYP3A4 contribution to midazolam hydroxylation while allowing CYP3A5 to remain active. The correlation of midazolam hydroxylation and Vincristine M1 formation improved with selective inhibitors.
This assay preferentially monitors CYP3A5 activity by fololowing the generation of the moderately selective Vincristine M1 metabolite. Approximately 20 human liver donors of known CYP3A5 genotype were compared. The donors were comprised of CYP3A5 *1/*1, *1/*3, and *3/*3 genotypes which have high, medium, and low CYP3A5 expression.
Vincristine M1 formation in genotyped individual donor microsomes was evaluated with slight modification of established methods (3,6). Incubations containing 20 uM vincristine, 0.1 mg HLM protein/ml and 1 mM NADPH prepared in 100 mM potassium phosphate buffer, pH 7.4, were stopped after 15 min by the addition of an equal volume of acetonitrile containing 1 uM vinblastine as an internal standard. Analysis of vincristine M1 was by LC-MS/MS using a RP-amide column (Ascentis Express from Supelco, 2.7 um, 2.1*100 mm) at 300 uL/min with a 9 minute linear gradient elution from 85% A (water + 0.1% formic acid) to 80% B (acetonitrile + 0.1% formic acid).
P450 activity was assayed by midazolam hydroxylation was conducted with 5 uM midazolam in 0.1 M potassium phosphate buffer, pH 7.4, and incubated at 37 C with shaking. 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).
Correlation analysis was done in GraphPad Prism with two-tailed P value determination using the Pearson method which assumes the data are from Gaussian populations.
PubChem Activity Outcome and Score:
Compounds with correlation coefficient greater than or equal to 0.9 were considered active. Compounds with correlation coefficient less than 0.9 were considered inactive.
The PubChem Activity Score is assigned a value of 100 for active compounds, and 0 for inactive compounds.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
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.
** Test Concentration.
Data Table (Concise)