Abstract
Some trans-prenyltransferases, such as long-chain C40 octaprenyl diphosphate synthase (OPPS), short-chain C15 farnesyl diphosphate synthase (FPPS), and C20 geranylgeranyl diphosphate synthase (GGPPS), are important drug targets. These enzymes catalyze chain elongation of FPP or geranyl diphosphate (GPP) through condensation reactions with isopentenyl diphosphate (IPP), forming designated numbers of trans-double bonds in the final products. To facilitate drug discovery, we report here a sensitive and reliable fluorescence-based assay for monitoring their activities in real time. MANT-O-GPP, a fluorescent analogue of FPP, was used as an alternative substrate and converted by the wild-type OPPS and the engineered FPPS and GGPPS into sufficiently long products with enhanced fluorescence intensities. This fluorescence probe was used to reveal the inhibitory mechanism of zoledronate, a bisphosphonate drug that targets human FPPS and possibly GGPPS.
MeSH terms
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Alkyl and Aryl Transferases / antagonists & inhibitors
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Alkyl and Aryl Transferases / chemistry
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Alkyl and Aryl Transferases / genetics
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Amino Acid Substitution
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Dimethylallyltranstransferase / antagonists & inhibitors*
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Dimethylallyltranstransferase / chemistry*
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Dimethylallyltranstransferase / genetics
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Diphosphonates / pharmacology
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Drug Discovery
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Enzyme Inhibitors / chemistry
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Enzyme Inhibitors / pharmacology
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Farnesyltranstransferase / antagonists & inhibitors
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Farnesyltranstransferase / chemistry
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Farnesyltranstransferase / genetics
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Fluorescent Dyes / chemistry*
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Geranyltranstransferase / antagonists & inhibitors
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Geranyltranstransferase / chemistry
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Geranyltranstransferase / genetics
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Humans
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Imidazoles / pharmacology
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Kinetics
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Models, Molecular
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Molecular Probe Techniques
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Molecular Probes / chemistry*
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Mutagenesis, Site-Directed
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Polyisoprenyl Phosphates / chemistry*
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Protein Conformation
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Saccharomyces cerevisiae Proteins / antagonists & inhibitors
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Sesquiterpenes / chemistry*
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Substrate Specificity
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Zoledronic Acid
Substances
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Diphosphonates
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Enzyme Inhibitors
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Fluorescent Dyes
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Imidazoles
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Molecular Probes
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Polyisoprenyl Phosphates
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Recombinant Proteins
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Saccharomyces cerevisiae Proteins
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Sesquiterpenes
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Zoledronic Acid
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farnesyl pyrophosphate
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Alkyl and Aryl Transferases
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octaprenyl pyrophosphate synthetase
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Dimethylallyltranstransferase
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Geranyltranstransferase
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Farnesyltranstransferase