A semi-automated method for measuring the potential for protein covalent binding in drug discovery.
BACKGROUND
Covalent protein binding of metabolically reactive intermediates of drugs has been implicated in drug toxicity including the occurrence of idiosyncratic drug toxicity. Investigators therefore would prefer to avoid developing compounds that produce significant amounts of reactive metabolites. By incubating the radiolabeled drug of interest with liver microsomes it is possible to evaluate the propensity of a drug candidate to covalently bind to proteins.


METHODS
Here we present a semi-automated method in which a Brandel cell harvester is used to collect and wash proteins that have been incubated with radiolabeled drug. This method utilizes glass fiber filter paper to capture precipitated protein, rather than the more traditional exhaustive extraction/centrifugation approach. Using model compounds (including [14C]diclofenac, [3H]imipramine, [14C]naphthalene, and [14C]L-746530) we compare the covalent binding results obtained using this method to results generated using the traditional method and we performed cross-laboratory testing of assay reproducibility.


RESULTS
It was found that results from new method correlated highly with the traditional method (R2=0.89). The cross-laboratory testing of the method showed an average interlaboratory coefficient of variation of only 18.4%.


CONCLUSIONS
This method provides comparable results to the more traditional centrifugation-based method with considerable time and labor savings.
Day
S H
SH
Medicinal Chemistry, Merck Frosst Centre for Therapeutic Research, Merck Frosst Canada and Co., 16711 Trans Canada Hwy., Kirkland, Quebec, Canada H9H 3L1. stephen_day@merck.com
Mao
A
A
White
R
R
Schulz-Utermoehl
T
T
Miller
R
R
Beconi
M G
MG
D000818:Animals; D001331:Automation / Q000379:methods*; D002250:Carbon Radioisotopes; D002498:Centrifugation / Q000379:methods; D011232:Chemical Precipitation; D004008:Diclofenac / Q000737:chemistry / Q000378:metabolism; D005374:Filtration / Q000379:methods; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism; D015394:Molecular Structure; D009281:Naphthalenes / Q000737:chemistry / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011869:Radioligand Assay / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D051381:Rats; D015203:Reproducibility of Results; D012997:Solvents / Q000737:chemistry; D014316:Tritium
D002250:Carbon Radioisotopes; D009281:Naphthalenes; D004364:Pharmaceutical Preparations; D012997:Solvents; D014316:Tritium; D004008:Diclofenac; C031721:naphthalene