Protein prenyltransferases catalyze the attachment of C15 (farnesyl) and C20 (geranylgeranyl) groups to proteins at specific sequences localized at or near the C-termini of specific proteins. Determination of the specific protein prenyltransferase substrates affected by the inhibition of these enzymes is critical for enhancing knowledge of the mechanism of such potential drugs. Here, we investigate the utility of alkyne-containing isoprenoid analogs for chemical proteomics experiments by showing that these compounds readily penetrate mammalian cells in culture and become incorporated into proteins that are normally prenylated. Derivatization via Cu(I) catalyzed click reaction with a fluorescent azide reagent allows the proteins to be visualized and their relative levels to be analyzed. Simultaneous treatment of cells with these probes and inhibitors of prenylation reveals decreases in the levels of some but not all of the labeled proteins. Two-dimensional electrophoretic separation of these labeled proteins followed by mass spectrometric analysis allowed several labeled proteins to be unambiguously identified. Docking experiments and density functional theory calculations suggest that the substrate specificity of protein farnesyl transferase may vary depending on whether azide-or alkyne-based isoprenoid analogs is employed. These results demonstrate the utility of alkynecontaining analogs for chemical proteomic applications. Protein prenyltransferases catalyze the attachment of C15 (farnesyl) and C20 (geranylgeranyl) groups to proteins at specific sequences localized at or near the C-termini of certain proteins via the reaction shown in Figure 1. Protein farnesyltransferase (PFTase) and protein geranylgeranyltransferase type 1 (PGGTase-I) alkylate simple tetrapeptide (CAAX box) substrates, while protein geranylgeranyltransferase type 2 (PGGTase-II) modifies more cryptic sequences (1). The inhibition of protein farnesylation has been a target for disease intervention for the past two decades, and protein farnesyltransferase inhibitors (FTIs) have been evaluated as therapeutic agents for several medical problems. These include a number of forms of cancer, malaria, and related protozoan infections, and certain progerias; protein geranylgeranyltransferase inhibitors (GGTIs) are also in development (2,3). Despite copious amounts of research, much still remains unclear about protein prenylation and its inhibition. For example, the driving force behind FTI development for cancer therapy has focused on the oncogenic Ras proteins, because they must be farnesylated to be active (4). During preclinical studies with these inhibitors, antiproliferative and pro-apoptotic activity were observed in cases where oncogenic Ras was not present, suggesting that other downstream effectors contribute to the anticancer activity of FTIs (5). While decreases in the levels of a number of prenylated proteins have been shown to occur upon treatment with FTIs, direct evidence that these species, and not other undiscovered prenylated protei...

A number of biochemical processes rely on isoprenoids, including the post-translational modification of signaling proteins and the biosynthesis of a wide array of compounds. Photoactivatable analogues have been developed to study isoprenoid utilizing enzymes such as the isoprenoid synthases and prenyltransferases. While these initial analogues proved to be excellent structural analogues with good cross-linking capability, they lack the stability needed when the goals include isolation of cross-linked species, tryptic digestion, and subsequent peptide sequencing. Here, the synthesis of a benzophenone-based farnesyl diphosphate analogue containing a stable phosphonophosphate group is described. Inhibition kinetics, photolabeling experiments, as well as X-ray crystallographic analysis with a protein prenyltransferase are described, verifying this compound as a good isoprenoid mimetic. In addition, the utility of this new analogue was explored by using it to photoaffinity label crude protein extracts obtained from Hevea brasiliensis latex. Those experiments suggest that a small protein, rubber elongation factor, interacts directly with farnesyl diphosphate during rubber biosynthesis. These results indicate that this benzophenone-based isoprenoid analogue will be useful for identifying enzymes that utilize farnesyl diphosphate as a substrate.

The Similarity Ensemble Approach (SEAa) relates proteins based on the set-wise chemical similarity among their ligands. It can be used to rapidly search large compound databases and to build crosstarget similarity maps. The emerging maps relate targets in ways that reveal relationships one might not recognize based on sequence or structural similarities alone. SEA has previously revealed cross talk between drugs acting primarily on G-protein coupled receptors (GPCRs). Here we used SEA to look for potential off-target inhibition of the enzyme protein farnesyltransferase (PFTase) by commercially available drugs. The inhibition of PFTase has profound consequences for oncogenesis, as well as a number of other diseases. In the present study, two commercial drugs, Loratadine and Miconazole, were identified as potential ligands for PFTase and subsequently confirmed as such experimentally. These results point towards the applicability of SEA for the prediction of not only GPCR-GPCR drug cross talk, but also GPCR-enzyme and enzyme-enzyme drug cross talk.

Originally designed to block the prenylation of oncogenic Ras, inhibitors of protein farnesyltransferase currently in pre-clinical and clinical trials are showing efficacy in cancers with normal Ras. Blocking protein prenylation has also shown promise in the treatment of malaria, Chagas disease, and progeria syndrome. A better understanding of the mechanism, targets and in vivo consequences of protein prenylation are needed to elucidate the mode of action of current PFTase inhibitors and to create more potent and selective compounds. Caged enzyme substrates are useful tools for understanding enzyme mechanism and biological function. Reported here is the synthesis and characterization of caged substrates of PFTase. The caged isoprenoid diphosphates are poor substrates prior to photolysis. The caged CAAX peptide is a true catalytically caged substrate of PFTase in that it is to not a substrate, yet is able to bind to the enzyme as established by inhibition studies and x-ray crystallography. Irradiation of the caged molecules with 350 nm light readily releases their cognate substrate, and their photolysis products are benign. These properties highlight the utility of those analogues towards a variety of in vitro and in vivo applications.

Protein farnesyltransferase (PFTase) catalyzes the attachment of a geranylazide moiety to a peptide substrate, N-dansyl-GCVIA. Because geranylazide is actually a mixture of isomeric, interconverting primary and secondary azides, incorporation of this isoprenoid into peptides can potentially result in a corresponding mixture of prenylated peptides. Here, we first examined the reactivity of geranyl azide in a model Staudinger reaction and determined that a mixture of products is formed. We then describe the synthesis of 6,7-dihydrogeranylazide diphosphate and demonstrate that this compound allows exclusive incorporation of a primary azide into a peptide. The resulting azide-containing peptide was derivatized with a triphenylphosphinebased reagent to generate an O-alkyl imidate-linked product. Finally, we show, using a series of model reactions, that the Staudinger ligation frequently produces small amounts of O-alkyl imidate products in addition to the major amide-linked products. Thus, the alkoxyimidates we have observed as the exclusive products in the reactions of peptides containing prenylated azides also appear to be a common type of product formed using other azidecontaining reactants, although at greatly reduced levels. This method for chemical modification of the C-terminus of a protein should be useful for a variety of applications in protein chemistry.

No abstract

Photoactive analogs of farnesyl diphosphate (FPP) are useful probes in studies of enzymes that employ this molecule as a substrate. Here, we describe the preparation and properties of two new FPP analogs that contain diazotrifluoropropanoyl photophores linked to geranyl diphosphate via amide or ester linkages. The amide-linked analog (3) was synthesized in 32 P-labeled form from geraniol in seven steps. Experiments with Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) showed that 3 is an alternative substrate for the enzyme. Photolysis experiments with [ 32 P]3 demonstrate that this compound labels the b-subunits of both farnesyltransferase and geranylgeranyltransferase (types 1 and 2). However, the amide-linked probe 3 undergoes a rearrangement to a photochemically unreactive isomeric triazolone upon long term storage making it inconvenient to use. To address this stability issue, the ester-linked analog 4 was prepared in six steps from geraniol. Computational analysis and X-ray crystallographic studies suggest that 4 binds to protein farnesyl transferase (PFTase) in a similar fashion as FPP. Compound 4 is also an alternative substrate for PFTase, and a 32 P-labeled form selectively photocrosslinks the b-subunit of ScPFTase as well as E. coli farnesyldiphosphate synthase and a germacrene-producing sesquiterpene synthase from Nostoc sp. strain PCC7120 (a cyanobacterial source). Finally, nearly exclusive labeling of ScPFTase in crude E. coli extract was observed, suggesting that [ 32 P]4 manifests significant selectivity and should hence be useful for identifying novel FPPutilizing enzymes in crude protein preparations.Key words: diazotrifluoropropanoyl, farnesyl diphosphate, germacrene synthase, photoaffinity labeling, prenyltransferase, protein prenylation, sesquiterpene synthase Abbreviations: BSA, bovine serum albumin; CPK, bovine CoreyPauling-Koltun; DATFP, diazotrifluoropropanoyl; DEAD, diethyl azodicarboxylate; DEPT, Distortionless Enhancement by Polarization Transfer; DFT, Density functional theory; DTT, dithiothreitol; EcFPPSase, E. coli farnesyl diphosphate synthase; EDTA, ethylenediaminetetraacetic acid; ESI-MS, electrospray ionization mass spectrometry; ESI-TOF, High resolution time of flight; EtOAc, Ethyl acetate; FAB-MS, fast atom bombardment mass spectrometry; FPP, farnesyl diphosphate; FT-IR, Fourier transform infrared spectrum; GGPP, geranylgeranyl diphosphate; HPLC, high performance liquid chromatography; HR-ESI-MS, High resolution electrospray ionization mass spectrometry; HR-FAB-MS, High resolution fast atom bombardment mass spectrometry; HsPGGTase I, H. sapiens protein geranylgeranyltransferase type 1; NMR, nuclear magnetic resonance; NoSTSase, Nostoc sp. strain PCC7120 sesquiterpene synthase; PFTase, protein farnesyl transferase; PPTS, pyridinium p-toluenesulfonate; QM-MM, quantum mechanics molecular mechanics; RnPGGTase II, R. norvegicus protein geranylgeranyltransferase type 2; RnPFTase, R. norvegicus protein farnesyltransferase; ScPFTase, S. cerevisiae protein farnesyltra...