Posttranslational modification of
proteins with farnesyl and geranylgeranyl
isoprenoids is a widespread phenomenon in eukaryotic organisms. Isoprenylation is conferred by three
protein prenyltransferases: farnesyl
transferase (FTase), geranylgeranyl
transferase type-I (
GGTase-I), and
Rab geranylgeranyltransferase (RabGGTase). Inhibitors of these
enzymes have emerged as promising therapeutic compounds for treatment of
cancer, viral and parasite originated diseases, as well as
osteoporosis. However, no generic nonradioactive
protein prenyltransferase assay has been reported to date, complicating identification of
enzyme-specific inhibitors. We have addressed this issue by developing two fluorescent analogues of farnesyl and geranylgeranyl
pyrophosphates {3,7-dimethyl-8-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-octa-2,6-diene-1}
pyrophosphate (NBD-GPP) and {3,7,11-trimethyl-12-(7-nitro-benzo[1,2,5]oxadiazo-4-ylamino)-dodeca-2,6,10-
trien-1}
pyrophosphate (
NBD-FPP), respectively. We demonstrate that these compounds can serve as efficient
lipid donors for prenyltransferases. Using these fluorescent
lipids, we have developed two simple (SDS-PAGE and bead-based) in vitro prenylation assays applicable to all prenyltransferases. Using the SDS-PAGE assay, we found that, in contrast to previous reports, the
tyrosine phosphatase PRL-3 may possibly be a dual substrate for both FTase and
GGTase-I. The on-bead prenylation assay was used to identify
prenyltransferase inhibitors that displayed nanomolar affinity for RabGGTase and FTase. Detailed analysis of the two inhibitors revealed a complex inhibition mechanism in which their association with the
peptide binding site of the
enzyme reduces the
enzyme's affinity for
lipid and
peptide substrates without competing directly with their binding. Finally, we demonstrate that the developed fluorescent
isoprenoids can directly and efficiently penetrate into mammalian cells and be incorporated in vivo into
small GTPases.