Human Hepatic Microsome Stability
This assay was established to measure the extent of potential hepatic metabolism of compounds. Liver microsomes consist mainly of endoplasmatic reticulum, contain many drug-metabolizing enzymes including cytochrome P450s (CYPs), flavin monooxygenases, carboxylesterases, and epoxide hydrolase. ..more
BioActive Compound: 1
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, Lake Nona, FL)
Network: NIH Molecular Libraries Screening Centers Network (MLSCN)
Grant Proposal Number:
Assay Provider: Dr. Layton Smith, Sanford-Burnham Medical Research Institute
This assay was established to measure the extent of potential hepatic metabolism of compounds. Liver microsomes consist mainly of endoplasmatic reticulum, contain many drug-metabolizing enzymes including cytochrome P450s (CYPs), flavin monooxygenases, carboxylesterases, and epoxide hydrolase.
Metabolic stability of the compound is measured using LC/MS/MS detection of parent compound, and is reported as percent compound remaining at the end of the experiment.
This assay was performed on hits and hit analogs to profile their microsome stability, as part of MLPCN activities at the Sanford-Burnham Medical Research Institute. Compounds are either acquired from commercial sources or synthesized internally.
1) Di L , Kerns EH, Hong Y, Kleintop TA, McConnell OJ, Huryn DM, Optimization of a higher throughput microsomal stability assay. J Biomol Screen 2003, 8:453-62
2) Clarke, S.E. and Jeffrey, P. Utility of metabolic stability screening: comparison of in vitro and in vivo clearance. Xenobiotica 2001, 31:591
3) Kariv, I Rourick, RA, Kassel DB, and Chung TD. Improvement of "hit-to-lead" optimization by integration of in vitro HTS experimental models of early determination of pharmacokinetic properties. Comb Chem High Throughput Screen 2002, 5: 459-472
1) Human liver microsomes ((XenoTech, final concentration in assay: 0.5mg/mL)
2) NADPH Regenerating System, Solutions A and B (BD Biosciences), final concentrations in assay:
- NADP+ (1.55mM)
- glucose-6-phosphate (1.33 mM)
- magnesium chloride hexahydrate (1.33 mM)
- Glucose-6-phosphate dehydrogenase 0.4 U/mL
3) 96-well microplates and 96-deep well plates
4) 100% acetonitrile stop solution containing internal standards
5) Control compounds: Verapamil-HCl and Testosterone, final concentration in assay: 20 uM and 50 uM, respectively
6) Test compounds, final concentration in assay: 1 uM
7) The pH of the reactions was kept at ~ 7.4 with potassium phosphate buffer (BD Biosciences)
8) The Tecan Freedom Evo 150 automated liquid handling instrument (Tecan US) was used in this assay.
1) Compound stock solutions were initially prepared in 100% DMSO and subsequently diluted in acetonitrile for the assay (final organic concentration in each incubate 1.2%).
2) The reactions were started after adding NADPH and time 0 min aliquots were promptly collected and mixed with acetonitrile (spiked with internal standards) to quench the reactions.
3) The remainder of the reaction volume was incubated at 37 oC with shaking for 60min.
4) Additional aliquots were collected and the reaction was stopped by adding acetonitrile (spiked with internal standards).
5) The amount of compound in the supernatant was determined by LC/MS/MS (API 4000 Q-Trap, Applied Biosystems).
6) The percent of parent compound remaining was calculated by the following formula:
%parent compound remaining = (concentration at 60 min/concentration at 0 min)* 100
7) All reactions were run in triplicate, except negative controls (no NADPH) which were performed as single reactions.
8) Results are the mean of each reaction triplicate, normalized to the internal standard, and expressed as a percent compound remaining after 60 min.
Compounds that have <90% remaining after 60 minutes are considered active.
Scoring for single-concentration screening:
Activity scoring rules developed at Sanford-Burnham Center for Chemical Genomics employs a 3-tiered system:
1) The first tier (0-40 range) is reserved for primary or single-concentration screening data and is not applicable to this assay.
2) The second tier (41-80 range) is reserved for dose-response confirmation data of the primary hits that are cherry picked from the HTS mother plates and is not applicable to this assay.
3) The third tier (81-100 range) is reserved for dry-powder compounds that represent purchased and resynthesized positives and their analogues.
Inactive compounds are assigned a score of 81.
Active compounds have a score between 82 and 100.
82 + (18 - (18*(%compound remaining/90))
** Test Concentration.
Data Table (Concise)