Novel farnesol and geranylgeraniol analogues: A potential new class of anticancer agents directed against protein prenylation.

Protein farnesyltransferase (FTase), the enzyme responsible for protein farnesylation, has become a key target for the rational design of cancer chemotherapeutic agents. Herein it is shown that certain novel prenyl diphosphate analogues are potent inhibitors of mammalian FTase. Furthermore, the alcohol precursors of two of these compounds are able to block anchorage-independent growth of ras-transformed cells. While 3-allylfarnesol inhibits protein farnesylation, 3-vinylfarnesol instead leads to abnormal prenylation of proteins with the 3-vinylfarnesyl group. In a similar manner, 3-allylgeranylgeraniol acts as a highly specific inhibitor of protein geranylgeranylation, while 3-vinylgeranylgeraniol restores protein geranylgeranylation in cells. This study indicates that certain prenyl alcohol analogues can act as prenyltransferase inhibitors in situ, via a novel prodrug mechanism. These analogues may prove to be valuable tools for investigating the therapeutic consequences of inhibiting geranylgeranylation relative to farnesylation. Furthermore, the 3-vinyl alcohol analogues can inhibit transformed cell growth through a mechanism not involving prenyltransferase inhibition.

Affinity labeling of the active site and the reactive sulfhydryl associated with activation of rat liver phenylalanine hydroxylase.

A pterin analogue, 5-[(3-azido-6-nitrobenzylidene)amino]-2,6-diamino-4-pyrimidinone (ANBADP), was synthesized as a probe of the pterin binding site of phenylalanine hydroxylase. The photoaffinity label has been found to be a competitive inhibitor of the enzyme with respect to 6,7-dimethyltetrahydropterin, having a Ki of 8.8 +/- 1.1 microM. The irreversible labeling of phenylalanine hydroxylase by the photoaffinity label upon irradiation is both concentration and time dependent. Phenylalanine hydroxylase is covalently labeled with a stoichiometry of 0.87 +/- 0.08 mol of label/enzyme subunit. 5-Deaza-6-methyltetrahydropterin protects against inactivation and both 5-deaza-6-methyltetrahydropterin and 6-methyltetrahydropterin protect against covalent labeling, indicating that labeling occurs at the pterin binding site. Three tryptic peptides were isolated from [3H]ANBADP-photolabeled enzyme and sequenced. All peptides indicated the sequence Thr-Leu-Lys-Ala-Leu-Tyr-Lys (residues 192-198). The residues labeled with [3H]ANBADP were Lys198 and Lys194, with the majority of the radioactivity being associated with Lys198. The reactive sulfhydryl of phenylalanine hydroxylase associated with activation of the enzyme was also identified by labeling with the chromophoric label 5-(iodoacetamido)fluorescein [Parniak, M. A., & Kaufman, S. (1981) J. Biol. Chem. 256, 6876]. Labeling of the enzyme resulted in 1 mol of fluorescein bound per phenylalanine hydroxylase subunit and a concomitant activation of phenylalanine hydroxylase to 82% of the activity found with phenylalanine-activated enzyme. Tryptic and chymotryptic peptides were isolated from fluorescein-labeled enzyme and sequenced. The modified residue was identified as Cys236.

Dolichylpyrophosphate oligosaccharides: large-scale isolation and evaluation as oligosaccharyltransferase substrates.

Oligosaccharyltransferase (OST) catalyzes the transfer of a branched oligosaccharide from a dolichylpyrophosphate oligosaccharide (Dol-PP-OS) to the asparagine of a nascent polypeptide chain in vivo and peptide substrates in vitro. Here we report the isolation and purification of Dol-PP-OS from bovine pancreas and thyroid. Steady-state kinetic parameters comparing the two Dol-PP-OS to a shorter dolichylpyrophosphate disaccharide (DolPP-DS) previously synthesized in our laboratory are reported. These were determined for Dol-PP-OS, Dol-PP-DS, and the tripeptide Bz-Asn-Leu-Thr-NH2 with solubilized OST and, for the first time, saturation kinetics were observed for all substrates. The kinetic data provide a basis for analyzing quantitatively the individual contributions of oligosaccharide donor and peptide acceptor substrates to OST-catalyzed glycosylation.

Expression of rat liver phenylalanine hydroxylase in insect cells and site-directed mutagenesis of putative non-heme iron-binding sites.

Rat liver phenylalanine hydroxylase was expressed in both Escherichia coli and the Spodoptera frugiperda insect cell line, Sf9. Recombinant enzyme from E. coli was inactive and contained less than 0.1 iron atom/subunit. In contrast, recombinant enzyme expressed in Sf9 cells using a baculovirus vector was active and identical in several properties to phenylalanine hydroxylase from rat liver: the Km for 6-methyltetrahydropterin was 39 microM (compared with 35 microM for the rat liver enzyme), 1 atom of iron was "associated" per enzyme subunit, and electron paramagnetic resonance spectra showed that iron was distributed within two distinct environments. Putative iron-binding sites of phenylalanine hydroxylase were studied by mutating either histidine 284 or 289 to serine and expressing these mutant enzymes (PAH-H284S and PAH-H289S) in Sf9 cells. Mutants were expressed at levels similar to wild-type PAH, but contained < or = 0.1 iron/subunit and were inactive. Thus, both His284 and His289 apparently are required for iron binding and hydroxylation activity.

Glycosylation of the overlapping sequons in yeast external invertase: effect of amino acid variation on site selectivity in vivo and in vitro.

Yeast invertase contains 14 sequons, all of which are glycosylated to varying degrees except for sequon 5 which is marginally glycosylated, if at all. This sequon overlaps with sequon 4 in a sequence consisting of Asn92-Asn93-Thr94-Ser95(Reddy et al., 1988, J. Biol. Chem., 263, 6978-6985). To determine whether glycosylation at Asn93is sterically hindered by the oligosaccharide on Asn92, the latter amino acid was converted to a glutamine residue by site-directed mutagenesis of the SUC2 gene in a plasmid vector which was expressed in Saccharomyces cerevisiae. A glycopeptide encompassing sequons 3 through 6 was purified from a tryptic digest of the mutagenized invertase and sequenced by Edman degradation, which revealed that Asn93 of sequon 5 contained very little, if any, carbohydrate, despite the elimination of sequon 4. When Ser and Thr were inverted to yield Asn-Asn-Ser-Thr carbohydrate was associated primarily with the second sequon, in agreement with numerous studies indicating that Asn-X-Thr is preferred to Asn-X-Ser as an oligosaccharide acceptor. However, when the invertase overlapping sequons were converted to Asn-Asn-Ser-Ser, both sequons were clearly glycosylated, with the latter sequon predominating. These findings rule out steric hindrance as a factor involved in preventing the glycosylation of sequon 5 in invertase. Comparable results were obtained using an in vitro system with sequon-containing tri- and tetrapeptides acceptors, in addition to larger oligosaccharide acceptors.

Novel limonene phosphonate and farnesyl diphosphate analogues: design, synthesis, and evaluation as potential protein-farnesyl transferase inhibitors.

Limonene and its metabolite perillyl alcohol are naturally-occurring isoprenoids that block the growth of cancer cells both in vitro and in vivo. This cytostatic effect appears to be due, at least in part, to the fact that these compounds are weak yet selective and non-toxic inhibitors of protein prenylation. Protein-farnesyl transferase (FTase), the enzyme responsible for protein farnesylation, has become a key target for the rational design of cancer chemotherapeutic agents. Therefore, several alpha-hydroxyphosphonate derivatives of limonene were designed and synthesized as potentially more potent FTase inhibitors. A noteworthy feature of the synthesis was the use of trimethylsilyl triflate as a mild, neutral deprotection method for the preparation of sensitive phosphonates from the corresponding tert-butyl phosphonate esters. Evaluation of these compounds demonstrates that they are exceptionally poor FTase inhibitors in vitro (IC50 > or = 3 mM) and they have no effect on protein farnesylation in cells. In contrast, farnesyl phosphonyl(methyl)phosphinate, a diphosphate-modified derivative of the natural substrate farnesyl diphosphate, is a very potent FTase inhibitor in vitro (Ki=23 nM).