A rapid and simple HPLC-UV method for the determination of inhibition characteristics of farnesyl transferase inhibitors.
Ras proteins play an important role in the development of cancer. Farnesyl transferase inhibitors (FTIs) block the first obligatory post-translational step for activation, prenylation, of Ras proteins. To find new potent FTIs, rapid enzyme activity assays are required to reduce FTI development time. Most assays to date are based on radioactive labelled substrates. We developed a new, in vitro, farnesyl transferase assay based on gradient chromatography coupled to UV detection. Unfarnesylated and farnesylated H-Ras proteins were resolved on a C18 wide-pore HPLC column and their concentrations were determined with use of a calibration curve of unfarnesylated H-Ras. The assay was used to investigate inhibition characteristics of FTIs. The IC50 values of the FTIs L778,123 and SCH66336 were 4.2 nm and 78 microm, respectively. This assay could support the screening and development of FTIs to obtain rapid insights into their inhibitory properties.
Appels
Natalie M G M
NM
Department of Pharmacy and Pharmacology, Slotervaart Hospital/The Netherlands Cancer Institute, Louwesweg 6, 1066 EC Amsterdam, The Netherlands. apnap@slz.nl
Tung
Kien-On
KO
Rosing
Hilde
H
Schellens
Jan H M
JH
Beijnen
Jos H
JH
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D051231:Farnesyltranstransferase / Q000037:antagonists & inhibitors*; D006863:Hydrogen-Ion Concentration; D013056:Spectrophotometry, Ultraviolet; D018631:ras Proteins / Q000302:isolation & purification / Q000378:metabolism
D004791:Enzyme Inhibitors; D051231:Farnesyltranstransferase; D018631:ras Proteins