Protein Farnesyltransferase-Catalyzed Isoprenoid Transfer to Peptide Depends on Lipid Size and Shape, not Hydrophobicity (original) (raw)
Related papers
Biochemistry, 2001
Farnesylation is a posttranslational lipid modification in which a 15-carbon farnesyl isoprenoid is linked via a thioether bond to specific cysteine residues of proteins in a reaction catalyzed by protein farnesyltransferase (FTase). We synthesized the benzyloxyisoprenyl pyrophosphate (BnPP) series of transferable farnesyl pyrophosphate (FPP) analogues (1a-e) to test the length dependence of the isoprenoid substrate on the FTase-catalyzed transfer of lipid to protein substrate. Kinetic analyses show that pyrophosphates 1a-e and geranyl pyrophosphate (GPP) transfer with a lower efficiency than FPP whereas geranylgeranyl pyrophosphate (GGPP) does not transfer at all. While a correlation was found between K m and analogue hydrophobicity and length, there was no correlation between k cat and these properties. Potential binding geometries of FPP, GPP, GGPP, and analogues 1a-e were examined by modeling the molecules into the active site of the FTase crystal structure. We found that analogue 1d displaces approximately the same volume of the active site as does FPP, whereas GPP and analogues 1a-c occupy lesser volumes and 1e occupies a slightly larger volume. Modeling also indicated that GGPP adopts a different conformation than the farnesyl chain of FPP, partially occluding the space occupied by the Ca 1 a 2 X peptide in the ternary X-ray crystal structure. Within the confines of the FTase pocket, the double bonds and branched methyl groups of the geranylgeranyl chain significantly restrict the number of possible conformations relative to the more flexible lipid chain of analogues 1a-e. The modeling results also provide a molecular explanation for the observation that an aromatic ring is a good isostere for the terminal isoprene of FPP.
Journal of Peptide Research, 1999
Conformation of a novel tetrapeptide inhibitor NH2-d-Trp-d-Met-Phe(pCl)-Gla-NH2 bound to farensyl-protein transferase. Abstract: Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the C-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for membrane association and celltransforming activities of ras. Inhibitors of FPTase have been demonstrated to display antitumor activity in both tissue culture and animal models, and thus represent a potential therapeutic strategy for the treatment of human cancers. A synthetic tetrapeptide library, which included an expanded set of 68 L-, Dand noncoded amino acids, has been screened for inhibitors of FPTase activity. The tetrapeptide, NH 2 -D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH 2 was shown to be competitive with the isoprenyl cosubstrate, farnesyl diphosphate (FPP) but not with the peptide substrate, the C-terminal tetrapeptide of the Ras protein. The FPTase-bound conformation of the inhibitor, NH 2 -D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH 2 was determined by NMR spectroscopy. Distance constraints were derived from two-dimensional transferred nuclear Overhauser effect (TRNOE) experiments. Ligand competition experiments identi®ed the NOEs that originate from the activesite conformation of the inhibitor. Structures were calculated using a combination of distance geometry and restrained energy minimization. The peptide backbone is shown to adopt a reverseturn conformation most closely approximating a type II' b-turn. The resolved conformation of the inhibitor represents a distinctly different structural motif from that determined for Rascompetitive inhibitors. Knowledge of the bound conformation of this novel inhibitor provides a template and future direction for the design of new classes of FPTase antagonists. Abbreviations: FPP, farnesyl diphosphate; FPTase, farnesylprotein transferase; TRNOE, transferred nuclear Overhauser effect.
Inhibitors of farnesyl:protein transferase—A possible cancer chemotherapeutic
1996
The recent interest in inhibitors of farnesyl:protein transferase (FPTase) has resulted in a better understanding of the enzymology of this protein. Rationally designed inhibitors of prenyl transfer have emerged as potential new drug candidates because of the insight gained over bow a prenyl group is enz3'matically transferred onto a peptide thiol. This paper will explore how advances in our understanding of FPTase mediated catalysis has affected the design of FPTase inhibitors as possible cancer therapeutic agents. Without structural information of the enzyme, substrate analogues comprise the first area of drug design: these include peptidomimetics of the four C-terminal amino acids of rasP21 as well as farnesyl diphosphate analogs. In addition, phosphate anion was found to enhance the inhibitory potency of certain compounds known to be competitive with respect to farnesyl diphosphate and therefore incorporation of the phosphate anion may also provide a basis for improved inhibitor design.
Journal of Medicinal Chemistry, 1999
Crystallographic and thermodynamic studies of farnesyl protein transferase (FPT) complexed with novel tricyclic inhibitors provide insights into the observed SAR for this unique class of nonpeptidic FPT inhibitors. The crystallographic structures reveal a binding pattern conserved across the mono-, di-, and trihalogen series. In the complexes, the tricycle spans the FPT active site cavity and interacts with both protein atoms and the isoprenoid portion of bound farnesyl diphosphate. An amide carbonyl, common to the tricyclic compounds described here, participates in a water-mediated hydrogen bond to the protein backbone. Ten high-resolution crystal structures of inhibitors complexed with FPT are reported. Included are crystallographic data for FPT complexed with SCH 66336, a compound currently undergoing clinical trials as an anticancer agent (SCH 66336, 4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)-1-piperidinyl]-2-oxoethyl]-1-piperidinecarboxamide). Thermodynamic binding parameters show favorable enthalpies of complex formation and small net entropic contributions as observed for 4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-11H-benzo-[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinyl]-2-oxoethyl]pyridine N-oxide where ∆H°b ind ) -12.5 kcal/mol and T∆S°b ind ) -1.5 kcal/mol. . † Abbreviations: farnesyl protein transferase (FPT), farnesyl diphosphate (FPP), concentration of inhibitor to cause 50% inhibition (IC50), structure-activity relationship (SAR), isothermal titration calorimetry (ITC), R-hydroxyfarnesylphosphonic acid (RHFP).
Journal of Biological Chemistry, 1997
Investigation of the comparative activities of various inhibitors of farnesyl:protein transferase (FPTase) has led to the observation that the presence of phosphate or pyrophosphate ions in the assay buffer increases the potency of farnesyl diphosphate (FPP) competitive inhibitors. In addition to exploring the phenomenon of phosphate synergy, we report here the effects of various other ions including sulfate, bicarbonate, and chloride on the inhibitory ability of three FPP competitive compounds: Cbz-His-Tyr-Ser(OBn)TrpNH 2 (2), Cbz-HisTyr-(OPO 4 2؊ )-Ser(OBn)TrpNH 2 (3), and ␣-hydroxyfarnesyl phosphonic acid (4). Detailed kinetic analysis of FPTase inhibition revealed a high degree of synergy for compound 2 and each of these ions. Phosphorylation of 2 to give 3 completely eliminated any ionic synergistic effect. Moreover, these ions have an antagonistic effect on the inhibitory potency of compound 4. The anions in the absence of inhibitor exhibit non-competitive inhibition with respect to FPP. These results suggest that phosphate, pyrophosphate, bicarbonate, sulfate, and chloride ions may be binding at the active site of both free enzyme and product-bound enzyme with normal substrates. These bound complexes increase the potency of FPP competitive inhibitors and mimic an enzyme:product form of the enzyme. None of the anions studied here proved to be synergistic with respect to inhibition of geranylgeranyl transferase I. These findings provide insight into the mechanism of action of FPP competitive inhibitors for FPTase and point to enzymatic differences between FPTase and geranylgeranyl transferase I that may facilitate the design of more potent and specific inhibitors for these therapeutically relevant target enzymes.
NMR studies of novel inhibitors bound to farnesyl-protein transferase
Protein Science, 2008
Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the carboxyl-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for the membrane association and cell-transforming activities of ras. Inhibitors of FPTase have been demonstrated to inhibit ras-dependent cell transformation and thus represent a potential therapeutic strategy for the treatment of human cancers. The FPTase-bound conformation of a tetrapeptide inhibitor, CVWM, and a novel pseudopeptide inhibitor, L-739,787, have been determined by NMR spectroscopy. Distance constraints were derived from twodimensional transferred nuclear Overhauser effect experiments. Ligand competition experiments identified the NOES that originate from the active-site conformation. Structures were calculated with the combination of distance geometry and restrained energy minimization. Both peptide backbones are shown to adopt nonideal reverseturn conformations most closely approximating a type 111 P-turn. These results provide a basis for understanding the spatial arrangements necessary for inhibitor binding and selectivity and may aid in the design of therapeutic agents.
Biochemistry, 2012
Farnesylation is an important post-translational modification essential for proper localization and function of many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual isoprene moieties to examine some of the structural and electronic properties of analogue transfer to peptide catalyzed by FTase. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple turnover activity depends on the analogue structure. Analogues where the first isoprene is replaced by a benzyl group and an analogue where each isoprene is replaced by an aryl group are good substrates. In sharp contrast with the steady-state reaction, the single turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased chemical reactivity of the benzyl analogues and the increased steric bulk of other analogues. However, the single turnover rate constant for alkylation does depend on the Ca 1 a 2 X peptide sequence. These results suggest that the isoprenoid transition state conformation is preferred over the inactive E•FPP• Ca 1 a 2 X ternary complex conformation. Furthermore, these data suggest that the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the active site binding pocket and therefore might be a useful site for design of novel inhibitors.
Photoaffinity Analogues of Farnesyl Pyrophosphate Transferable by Protein Farnesyl Transferase
Journal of the American Chemical Society, 2002
Farnesylation is a posttranslational lipid modification in which a 15-carbon farnesyl isoprenoid is linked via a thioether bond to specific cysteine residues of proteins in a reaction catalyzed by protein farnesyltransferase (FTase). We synthesized analogues (3-6) of farnesyl pyrophosphate (FPP) to probe the range of modifications possible to the FPP skeleton which allow for efficient transfer by FTase. Photoaffinity analogues of FPP (5, 6) were prepared by substituting perfluorophenyl azide functional groups for the ω-terminal isoprene of FPP. Substituted anilines replace the ω-terminal isoprene in analogues 3 and 4. Compounds 3-5 were prepared by reductive amination of the appropriate anilines with 8-oxogeranyl acetate, followed by ester hydrolysis, chlorination, and pyrophosphorylation. Additional substitution of three methylenes for the-isoprene of FPP gave photoprobe 6 in nine steps. Preparation of the analogues required TiCl 4-mediated imine formation prior to NaBH(OAc)3 reduction for anilines with a pKa < 1. The azide moiety was not affected by Ph3PCl2 conversion of allylic alcohols 13-16 into corresponding chlorides 17-20. Analogues 3-6 are efficiently transferred to target N-dansyl-GCVLS peptide substrate by mammalian FTase. Comparison of analogue structures and kinetics of transfer to those of FPP reveals that ring fluorination and para substituents have little effect on the affinity of the analogue pyrophosphate for FTase and its transfer efficiency. These results are also supported with models of the analogue binding modes in the active site of FTase. The transferable azide photoprobe 5 photoinactivates FTase. Transferable analogues 5 and 6 allow the formation of appropriately posttranslationally modified photoreactive peptide probes of isoprene function.
Potent and Selective Farnesyl Transferase Inhibitors
Journal of Medicinal Chemistry, 2004
We recently described a novel series of CA 1 A 2 X peptidomimetics as farnesyl transferase inhibitors (FTIs). These compounds possess an N-(4-piperidinyl)benzamide scaffold mimicking A 1 A 2 residue. Extensive exploration of structure-activity relationships revealed that replacement of cysteine by substituted benzylimidazoles provided nanomolar FTIs with in vitro activities (18e, IC 50 ) 4.60 nM on isolated enzyme, EC 50 ) 20.0 nM for growth inhibition on a tumor cell line). The molecular docking of 18e and 19e in the active site of the enzyme provided details of key interactions with the protein and showed that the methionine or phenylalanine residue fits into the aryl binding site.