Recently considerable effort has been invested

Recently, considerable effort has been invested in developing FDPS inhibitors based on alternative scaffolds (including nonbisphosphonates) that allow other tissue types and parasite-derived forms of FDPS , to be targeted. There are several limits in analyzing inhibitors of FDPS activity: the most widely used assays are discontinuous, time and labor intensive, and involve the use of radiolabeled substrates or only measure the release of pyrophosphate , and not FPP production and can hence be susceptible to artifacts. The most common FDPS assay involves using radiolabeled IPP in the enzymatic reaction followed by separation of residual IPP from FPP and subsequent quantification of radioactivity via liquid scintillation counting. Clearly, such a labor-intensive process is an impediment to efficient high throughput screening. As an alternative, a continuous spectrophotometric assay for FDPS has been developed , . That assay uses a series of coupled enzymatic reactions where a pyrophosphatase initially converts the diphosphate product, generated in the FDPS reaction, to inorganic phosphate. The resulting phosphate is then used by a purine nucleoside phosphorylase to convert 2-amino-6-mercapto-7-methylpurine riboside to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine; the purine product of that last reaction is UV active and its production can be monitored at 360nm (=11,000Mcm). While the resulting assay is continuous, its limit of detection is approximately 1nmol of product. Additionally, because the assay monitors phosphate production, it is prone to artifacts due to background phosphate production when performed using biological extracts. Finally because this assay involves the use of two coupling GDC-0994 to measure FDPS activity, there is increased likelihood for the assay to yield false positives in inhibitor screening since inhibition of either of the coupling enzymes will result in apparent reductions in FDPS activity. Protein farnesyltransferase (PFTase) catalyzes the posttranslational attachment of a sesquiterpenoid farnesyl moiety onto the C-terminal ends of proteins which contain a four amino acid consensus sequence commonly referred to as a “CAAX box” where C is cysteine, “A” refers to aliphatic amino acids, and “X” is a directing residue, typically serine or methionine . PFTase catalyzes the reaction using FPP as a substrate and links it to the target protein via a thioether bond. Prenylation plays a role in targeting proteins to the plasma membrane and is critical for enabling key protein–protein interactions to occur . It has been implicated as a possible therapeutic target for cancer treatment because mutated forms of RAS are present in many human cancers, but in order for these oncogenic variants to be active, they must be prenylated . In 1992, Pompliano and co-workers developed a continuous spectrofluorimetric assay for monitoring the activity of PFTase that has been extensively used by a number of groups , . This assay uses FPP and a CAAX box-containing peptide functionalized with an N-terminal dansyl glycine residue. In this continuous assay, farnesylation of the peptide results in an increase in dansyl group fluorescence (an environmentally sensitive fluorophore) due to the increase in hydrophobicity that occurs in the peptide upon prenylation. Since the smaller isoprenoid diphosphates DMAPP and GPP are relatively poor substrates for PFTase, we reasoned that the production of FPP by FDPS could be coupled with its subsequent incorporation into a peptide substrate catalyzed by PFTase. Several groups have developed coupled assays to detect FPP based on this sequence of enzymatic reactions, but in all cases to date, the assays have been discontinuous in nature , . The amounts of prenylated products were determined via HPLC separation and peak integration. We hypothesized that by using a dansylated peptide as noted above, a continuous increase in fluorescence would occur as FPP is generated by FDPS and transferred to the peptide by PFTase (). In this report we describe this new assay and demonstrate its utility by employing it to measure the IC values for several inhibitors of FDPS.