Benzothiophene carboxamide derivatives as inhibitors of Plasmodium falciparum enoyl-ACP reductase

Benzothiophene derivatives like benzothiophene sulphonamides, biphenyls, or carboxyls have been synthesized and have found wide pharmacological usage. Here we report, bromo-benzothiophene carboxamide derivatives as potent, slow tight binding inhibitors of Plasmodium enoyl-acyl carrier protein (ACP) reductase (PfENR). 3-Bromo-N-(4-fluorobenzyl)-benzo[b]thiophene-2-carboxamide (compound 6) is the most potent inhibitor with an IC50 of 115 nM for purified PfENR. The inhibition constant (Ki) of compound 6 was 18 nM with respect to the cofactor and 91 nM with respect to crotonoyl-CoA. These inhibitors showed competitive kinetics with cofactor and uncompetitive kinetics with the substrate. Thus, these compounds hold promise for the development of potent antimalarials.     

Kinetics of slow, tight-binding inhibitors of angiotensin converting enzyme

Five phosphorus-containing inhibitors of angiotensin converting enzyme were found to exhibit slow, tight-binding kinetics by using furanacryloyl-L-phenylalanylglycylglycine as substrate at pH 7.50 and T = 25 degrees C. Two of the inhibitors, (O-ethylphospho)-Ala-Pro (2) and (O-isopropylphospho)-Ala-Pro (3), are found to follow at minimum a two-step mechanism of binding (mechanism B) to the enzyme. This mechanism consists of an initial fast formation of a weaker enzyme-inhibitor complex (Ki = 130 nM for 2 and 180 nM for 3) followed by a slow reversible isomerization to a tighter complex with measurable forward (K3) and reverse (k4) rate constants (k3 = 4.5 X 10(-2) s-1 for 2 and 5.4 X 10(-2) s-1 for 3; k4 = 9.2 X 10(-3) s-1 for 2 and 3.5 X 10(-3) s-1 for 3). For the remaining three inhibitors, phospho-Ala-Pro (1), (O-benzyl-phospho)-Ala-Pro (4), and (P-phenethylphosphono)-Ala-Pro (5), a one-step binding mechanism (mechanism A) is observed under the conditions of the experiment. The second-order rate constants k1 (M-1 s-1) for the binding of these inhibitors to converting enzyme are found to have values more than 3 orders of magnitude lower than the diffusion-controlled limit for a bimolecular reaction involving the enzyme, viz., 3.9 X 10(5) for 1, 2.2 X 10(5) for 4, and 4.8 X 10(5) for 5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible role for water dissociation in the slow binding of phosphorus-containing transition-state-analogue inhibitors of thermolysin

A number of phosphonamidate and phosphonate tripeptide analogues have been studied as transition-state-analogue inhibitors of the zinc endopeptidase thermolysin. Those with the form Cbz-GlyP(Y)Leu-X [ZGP(Y)LX, X = NH2 or amino acid, Y = NH or O linkage] are potent (Ki = 9-760 nM for X = NH, 9-660 microM for X = O) but otherwise ordinary in their binding behavior, with second-order rate constants for association (kon) greater than 10(5) M-1 s-1. Those with the form Cbz-XP(Y)-Leu-Ala [ZXP(Y)LA,XP = alpha-substituted phosphorus amino acid analogue] are similarly potent (Ki for ZFPLA = 68 pM) but slow binding (kon less than or equal to 1300 M-1 s-1). Several kinetic mechanisms for slow binding behavior are considered, including two-step processes and those that require prior isomerization of inhibitor or enzyme to a rare form. The association rates of ZFPLA and ZFP(O)LA are first order in inhibitor concentration up to 1-2 mM, indicating that any loose complex along the binding pathway must have a dissociation constant above this value. The crystallographic investigation described in the preceding paper [Holden, H. M., Tronrud, D. E., Monzingo, A. F., Weaver, L. H., & Matthews, B. W. (1987) Biochemistry (preceding paper in this issue)] identifies a specific water molecule in the active site that may hinder binding of the alpha-substituted inhibitors. The implication of this observation for a mechanism for slow binding is discussed.     

Potent and selective inhibition of zinc aminopeptidase A (EC 3.4.11.7, APA) by glutamyl aminophosphinic peptides: importance of glutamyl aminophosphinic residue in the P1 position

Through the development of a new chemical strategy, aminophosphinic peptides containing a pseudoglutamyl residue (Glu Psi(PO2-CH2)Leu-Xaa) in the N-terminal position were synthesized and evaluated as inhibitors of aminopeptidase A (APA). The most potent inhibitor developed in this study, Glu Psi(PO2-CH2)Leu-Ala, displayed a Ki value of 0.8 nM for APA, but was much less effective in blocking aminopeptidase N (APN) (Ki = 31 microM). The critical role of the glutamyl residue in this phosphinic peptide, both in potency and selectivity, is exemplified by the P1 position analogue, Ala Psi(PO2-CH2)Leu-Ala, which exhibited a Ki value of 0.9 microM toward APA but behaved as a rather potent inhibitor of APN (Ki = 25 nM). Glu Psi(PO2-CH2)Leu-Xaa peptides are poor inhibitors of angiotensin converting enzyme (Ki values higher than 1 microM). Depending on the nature of the Xaa residue, the potency of these phosphinic peptides toward neutral endopeptidase 24-11 varied from 50 nM to 3 microM. In view of the in vivo role of APA in the formation of brain angiotensin III, one of the main effector peptides of the renin angiotensin system in the central nervous system, highly potent and selective inhibitors of APA may find important therapeutic applications soon.     

Structure-activity studies of fluoroketone inhibitors of alpha-lytic protease and human leukocyte elastase

We have synthesized a series of peptidyl fluoroketones that reversibly inhibit the serine proteases human leukocyte elastase (HLE) and alpha-lytic protease (alpha-LP). Ac-ambo-AlaCF3 (1) inhibits HLE and alpha-LP with Ki's of 2.4 and 15 mM, respectively. The effects of structural variations on this parent compound on Ki and the kinetics of inhibition were studied. The acetyl group was replaced by the tripeptide Z-L-Ala-L-Ala-L-Pro to yield the tetrapeptide trifluoroketone (TFK) Z-L-Ala-L-Ala-L-Pro-ambo-AlaCF3 (2). This extension reduced Ki 3500-fold for HLE and 3000-fold for alpha-LP. Removal of a fluorine atom from a TFK decreases Ki about 15- to 30-fold with both enzymes. Replacement of one fluorine atom of 2 by a residue (-CH2-CH2-COLeuOMe) (6) which can interact with the S'1 and S'2 subsites decreased Ki 30-fold for HLE and 150-fold for alpha-LP compared to Z-L-Ala-L-Ala-L-Pro-ambo-AlaCF2H (3). The Ki of 6 for HLE is approximately equal to that of trifluoroketone 2. For alpha-LP Ki of 6 is 10-fold lower than that for the trifluoroketone 2. Inhibitors with Ki values less than 10(-7) M exhibit slow binding kinetics. By analogy to cholinesterases and chymotrypsin, it is likely that these enzymes combine with the keto form of the inhibitor to form the enzyme-inhibitor complex. Therefore, kon and Ki were corrected for the ketone concentration. The corrected kon values for the slow binding inhibitors are in most cases less than diffusion controlled, ranging between 8.2 X 10(4) and 4.68 X 10(6) M-1 s-1. An exception is Z-L-Ala-L-Ala-L-Pro-ambo-ValCF3 (8) where kon = 9 X 10(7) M-1 s-1, which is nearly diffusion controlled.     

Reaction intermediate analogues for enolase

A number of compounds that appear to be analogues of the aci form of the normal carbanion intermediate are good inhibitors of yeast enolase. These include (3-hydroxy-2-nitropropyl)phosphonate (I), the ionized (pK = 8.1) nitronate form of which in the presence of 5 mM Mg2+ has a Ki of 6 nM, (nitroethyl)phosphonate (III) (pK = 8.5; Ki of the nitronate in the presence of 5 mM Mg2+ = 1 microM), phosphonoacetohydroxamate (IV) (pK = 10.2; Ki with saturating Mg2+ for the ionized form = 15 pM), and (phosphonoethyl)nitrolate (VII) (Ki at 1 mM Mg2+ = 14 nM). The oxime of phosphonopyruvate (VI) has a pH-independent Ki of 75 microM. I, IV, VI, and VII are slow binding inhibitors. All of these compounds are trigonal at the position analogous to C-2 of 2-phosphonoglycerate and contain a phosphono group, but a negatively charged metal ligand at the position isosteric with the hydroxyl attached to C-3 of 2-phosphoglycerate (as in IV) appears to contribute more to binding than a nitro group isosteric with the carboxyl of 2-phosphoglycerate (I and III). These data support the carbanion mechanism for enolase and suggest that the 3-hydroxyl of 2-phosphoglycerate is directly coordinated to Mg2+ prior to being eliminated to give phosphoenolpyruvate.     

Structure/function relationships in the inhibition of thimet oligopeptidase by carboxyphenylpropyl-peptides.

Some novel N-[1(RS)-carboxy-3-phenylpropyl]tripeptide p-aminobenzoates have been synthesised as inhibitors of thimet oligopeptidase (EC 3.4.24.15). These compounds are considered to bind as substrate analogues with the Cpp group in S1 and the peptide portion in the S' sites. The most potent inhibitor is Cpp-Ala-Pro-Phe-pAb, which has a Ki = 7 nM. Substitution of Gly for Ala at P1' leads to weaker binding which can be ascribed to increased rotational freedom. Good substrates often have Pro at P2' and Pro is favoured over Ala at this position in the inhibitors, too. When P2' is Pro, Phe is preferred over Tyr and Trp in P3'. The p-aminobenzoate group makes an important contribution to the binding, probably by forming a salt bridge, and removal of the C-terminal negative charge results in much less potent inhibitors.     

Inhibition of cytoplasmic aspartate aminotransferase from porcine heart by R and S isomers of aminooxysuccinate and hydrazinosuccinate

D- and L-aminooxysuccinate were synthesized and evaluated as inhibitors of cytoplasmic aspartate aminotransferase (EC 2.6.1.1) from porcine heart. L-Aminooxysuccinate was shown to be a slow binding inhibitor of the pyridoxal phosphate form of the enzyme with a Ki of 160 nM and a half-life of the inhibited complex of 8 min. Kinetic analysis revealed that inhibition followed a two-step mechanism in which the last step was rate-limiting. D-Aminooxysuccinate was not inhibitory up to a concentration of 0.1 mM. These compounds were compared to D- and L-hydrazinosuccinate, which are potent slow binding inhibitors of aspartate aminotransferase with Ki values of 1.5 and 0.5 nM, respectively. Models of all four analogs were built into the active site of the closed form of the enzyme. The energy-minimized conformations of both L-isomers bound to aspartate aminotransferase show better geometry for hydrogen bond and ion pair formation than do the corresponding D-isomers. The aldimine double bond formed by the L-isomers is not coplanar with the pyridoxal phosphate ring in accordance with the spectral properties of the inhibitor complexes that are characterized by broad absorbance bands. This lack of planarity was not evident for the models of D-hydrazinosuccinate and D-aminooxysuccinate.     

Stable analogs of acyl adenylates. Inhibition of acetyl- and acyl-CoA synthetase by adenosine 5'-alkylphosphates

Adenosine 5'-alkylphosphates are potent inhibitors of acetyl- and acyl-CoA synthetase. In each case, the most effective inhibitor in the series is homologous with the tightly bound acyl adenylate intermediate. Adenosine 5'-ethylphosphate (Ki = 33 nM) is 88-fold more potent than adenosine 5'-methylphosphate (Ki = 2900 nM) as a competitive inhibitor of acetyl-CoA synthetase; the contribution of a single carbon to the observed binding energy (-11 kJ/mol) is much larger than is typically observed.     

Solubilization of Rat Brain Phencyclidine Receptors in an Active Binding Form That Is Sensitive to N-Methyl-d-Aspartate Receptor Ligands

Phencyclidine (PCP) receptors were successfully solubilized from rat forebrain membranes with 1% sodium cholate. Approximately 58% of the initial protein and 20-30% of the high-affinity PCP binding sites were solubilized. The high affinity toward PCP-like drugs, the stereo-selectivity of the sites, and the sensitivity to N-methyl-D-aspartate (NMDA) receptor ligands were preserved. Binding of the potent PCP receptor ligand N-[3H][1-(2-thienyl)cyclohexyl] piperidine ([3H]TCP) to the soluble receptors was saturable (KD = 35 nM), and PCP-like drugs inhibited [3H]TCP binding in a rank order of potency close to that observed for the membrane-bound receptors; the most potent inhibitors were TCP (Ki = 31 nM) and the anticonvulsant MK-801 (Ki = 50 nM). The NMDA receptor antagonist 2-amino-5-phosphonovaleric acid inhibited binding of [3H]TCP to the soluble receptors; glutamate or NMDA diminished this inhibition in a dose-dependent manner. Taken together, the results indicate that the soluble PCP receptor preparation contains the glutamate recognition sites and may represent a single receptor complex for PCP and NMDA, as suggested by electrophysiological data. The successful solubilization of the PCP receptors in an active binding form should now facilitate their purification.     

Kinetics of the formation and isomerization of methotrexate complexes of recombinant human dihydrofolate reductase

The kinetics of inhibitor binding to highly purified recombinant human dihydrofolate reductase (rHDHFR) have been examined. Methotrexate (MTX) binds rapidly (kon = 1.0 x 10(8) M-1 s-1) and tightly (koff/kon = 210 pM) to the preformed complex of rHDHFR with NADPH. The initial association reaction between rHDHFR.NADPH and MTX is followed by an isomerization of the resulting complex (kiso = 0.4 s-1) leading to a new conformer in which MTX is bound even more tightly (Ki = 3.4 pM). Similar results have been obtained with a major metabolite of MTX having four additional glutamate residues for which Ki = 1.4 pM. 7-HydroxyMTX, another major metabolite of MTX, is a weak inhibitor of rHDHFR (Ki = 8.9 nM), and a polyglutamate form of this metabolite is an equally weak inhibitor (Ki = 9.9 nM), so that the addition of glutamate residues to MTX or 7-hydroxyMTX has little effect on their binding. It follows that the significance of MTX polyglutamate formation relates to other roles such as increasing the cytotoxicity of MTX by prolonging intracellular retention of the drug. Another antifolate, trimethoprim, binds tightly to dihydrofolate reductases from bacterial sources, but weakly to rHDHFR in the ternary complex (KD = 0.5 microM). Although the association step is rapid (kon = 0.4 x 10(8) M-1 s-1), the dissociation rate is also rapid (koff = 15 s-1). Furthermore, there is no isomerization of the ternary complex of trimethoprim with rHDHFR, in contrast to the known isomerization of complexes of trimethoprim with bacterial dihydrofolate reductases.     

125I-Spiperone: a novel ligand for D2 dopamine receptors

125I-Spiperone binds with high affinity (KD 0.3 nM) to a single specific site (Bmax 34 pmol/g wet weight) in homogenates of rat corpus striatum. Specific binding is about 40-60 percent of total binding and is displaced stereo-specifically by butaclamol and clopenthixol. Neuroleptic drugs of various classes are potent inhibitors of 125I-spiperone binding (Ki's 1-10 nM). Selective dopamine antagonists such as sulpiride (Ki 50 nM) and dopamine agonists such as apomorphine (Ki 200 nM) are also potent inhibitors. The drug specificity of 125I-spiperone binding correlates well with that of 3H-spiperone binding, providing good evidence that 125I-spiperone labels D2 dopamine receptors in striatal membranes. 125I-Spiperone, with its high specific activity (2200 Ci/mmol) may prove to be a useful ligand in studies examining D2 dopamine receptors in soluble preparations and by autoradiography. Furthermore iodinated spiperone may be useful in radioreceptor assays of neuroleptic drug levels and, in a 123I-labeled form, for imaging of dopamine receptors, in vivo, using single photon tomography.     

Synthesis and pharmacology of pyrido[2,3-d]pyrimidinediones bearing polar substituents as adenosine receptor antagonists

Amino-substituted pyrido[2,3-d]pyrimidinediones have previously been found to bind to adenosine A1 and A2A receptors in micromolar concentrations. The present study was aimed at studying the structure-activity relationships of this class of compounds in more detail. Most of the investigated compounds were provided with polar substituents, such as ethoxycarbonyl groups and basic amino functions, in order to improve their water-solubility. The compounds were synthesized starting from 6-amino-1,3-dimethyluracil via different reaction sequences involving (cyano)acetylation, Vilsmeier formylation, or reaction with diethyl ethoxymethylenemalonate (EMME). The most potent and selective compound of the present series was 6-carbethoxy-1,2,3,4-tetrahydro-1,3-dimethyl-5-(2-naphthylmethyl)aminopyrido[2,3-d]pyrimidine-2,4-dione (11c) with a Ki value of 5 nM at rat and 25 nM at human A1 receptors. The compound was more than 60-fold selective versus A3 and more than 300-fold selective versus A2A receptors. It showed an over 300-fold improvement with respect to the lead compound. In GTPgammaS binding studies at membranes of Chinese hamster ovary cells recombinantly expressing the human adenosine A1 receptor, 11c behaved as an antagonist with inverse agonistic activity. A regioisomer of 11c, 6-carbethoxy-1,2,3,4-tetrahydro-1,3-dimethyl-7-(2- naphthylmethyl)aminopyrido[2,3-d]pyrimidine-2,4-dione (7a) in which the 2-naphthylmethylamino substituent at position 5 of 11c was moved to the 7-position, was a relatively potent (Ki=226 nM) and selective (>20-fold) A3 ligand. In the series of compounds lacking an electron-withdrawing ethoxycarbonyl or cyano substituent in the 6-position, compounds with high affinity for adenosine A2A receptors were identified, such as 1,2,3,4-tetrahydro-1,3-dimethyl-5-(1-naphthyl)aminopyrido[2,3-d]pyrimidine-2,4-dione 16b (Ki human A2A=81.3 nM, Ki human A1=153 nM, and Ki human A3>10,000 nM).     

Binding of dexetimide and levetimide to [3H](+)pentazocine- and [3H]1,3-di(2-tolyl)guanidine-defined sigma recognition sites.

The potent antimuscarinic benzetimide and its resolved stereoisomers dexetimide and levetimide were tested for their affinities at sigma sites labelled by [3H](+)pentazocine or [3H]1,3-di(2-tolyl)guanidine. Levetimide was a potent and stereoselective inhibitor of [3H](+)pentazocine binding, with a Ki of 2.2 nM, while dexetimide was nine-fold less potent (Ki = 19 nM). Dexetimide and levetimide potently inhibited [3H]DTG binding although without stereoselectivity (Ki values of 65 and 103 nM, respectively). Levetimide may be a useful tool with which to investigate sigma recognition sites and sigma subtypes.     

Investigating the stereochemistry of binding to HIV-1 protease with inhibitors containing isomers of 4-amino-3-hydroxy-5-phenylpentanoic acid

A series of inhibitors containing all possible isomers of 4-amino-3-hydroxy-5-phenylpentanoic acid was synthesized and tested for inhibition of HIV-1 protease. Incorporation of the (3S,4S) isomer of the t-butyloxycarbonyl protected amino acid into the sequence Glu-Phe resulted in a potent inhibitor of HIV-1 protease (Ki = 63 nM). This inhibitor is at least 47-times more potent than the inhibitors containing other isomers of 4-amino-3-hydroxy-5-phenylpentanoic acid, indicating that the (3S,4S) isomer is the preferred isomer for binding to HIV-1 protease.     

Selection of a potent inhibitor of trihydroxynaphthalene reductase by sorting disease control data

Compounds that control rice blast, but not other crop diseases, were selected for testing as inhibitors of trihydroxynaphthalene reductase of the fungal melanin biosynthetic pathway. A potent inhibitor of the enzyme (2) (Ki = 25 nM) was identified. An X-ray structure of the enzyme-NADPH-2 complex was determined at 2.1 A resolution.     

C(4)-alkyl substituted furanyl cyclobutenediones as potent, orally bioavailable CXCR2 and CXCR1 receptor antagonists

A novel series of cyclobutenedione centered C(4)-alkyl substituted furanyl analogs was developed as potent CXCR2 and CXCR1 antagonists. Compound 16 exhibits potent inhibitory activities against IL-8 binding to the receptors (CXCR2 Ki=1 nM, IC(50)=1.3 nM; CXCR1 Ki=3 nM, IC(50)=7.3 nM), and demonstrates potent inhibition against both Gro-alpha and IL-8 induced hPMN migration (chemotaxis: CXCR2 IC(50)=0.5 nM, CXCR1 IC(50)=37 nM). In addition, 16 has shown good oral pharmacokinetic profiles in rat, mouse, monkey, and dog.     

Inactivation of chymotrypsin and human skin chymase: kinetics of time-dependent inhibition in the presence of substrate

The reaction of chymase, a chymotryptic proteinase from human skin, and bovine pancreatic chymotrypsin with a number of time-dependent inhibitors has been studied. An integrated equation, relating product formation with time, has been derived for the reaction of enzymes with time-dependent inhibitors in the presence of substrate. This is based on a two-step model in which a rapidly reversible, non-covalent complex (EI) is formed prior to a tighter, less readily reversible complex (EI)*). The equation depends on the simplifying assumption [I] much greater than [E], but is applicable to reversible and irreversible slow-binding and tight-binding inhibitors whether or not they show saturation kinetics. The method has been applied to the reaction of chymase and chymotrypsin with the tetrapeptide aldehyde, chymostatin, basic pancreatic trypsin inhibitor and Ala-Ala-Phe-chloromethylketone (AAPCK). The irreversible inhibitor, AAPCK, showed the expected saturation kinetics for both enzymes and the apparent first-order rate constants (k2) and dissociation constants (Ki) for the non-covalent complexes were determined. Chymostatin was a much more potent inhibitor which failed to show a saturation effect. The second-order rate constant of inactivation (k2/Ki), the first-order reactivation rate constant (k-2), and the dissociation constant of the covalent complex (Ki*) were determined. Basic pancreatic trypsin inhibitor, a potent inhibitor of chymotrypsin, had similar kinetics to chymostatin but failed to inhibit chymase. The applicability of the two-step model and the integrated equation to slow- and tight-binding inhibitors is discussed in relation to a number of examples from the literature.     

alpha-Bromo ketone substrate analogues are powerful reversible inhibitors of carboxypeptidase A

The aldehyde (RS)-2-benzyl-4-oxobutanoic acid, which is 25% hydrated at pH 7.5, has recently been shown to be a strong reversible competitive inhibitor of carboxypeptidase A [Ki = 0.48 nM; Galardy, R. E., & Kortylewicz, Z. P. (1984) Biochemistry 23, 2083-2087]. The ketone analogue of this aldehyde (RS)-2-benzyl-4-oxopentanoic acid (IV) is not detectably hydrated under the same conditions and is 1500-fold less potent (Ki = 730 microM). The ketone homologue (RS)-2-benzyl-5-oxohexanoic acid (XIII) is also a weak inhibitor (Ki = 1.3 mM). The alpha-monobrominated derivatives of these two ketones are, however, strong competitive inhibitors with Ki's of 0.57 microM and 1.3 microM, respectively. Oximes derived from the aldehyde, the ketones IV and XIII, and a homologue of the aldehyde are weak inhibitors with Ki's ranging from 480 to 7900 microM. The inhibition of carboxypeptidase A by the alpha-monobrominated ketones is reversible and independent of the time (up to 6 h) of incubation of enzyme and inhibitor together. Bromoacetone at a concentration of 30 mM does not inhibit carboxypeptidase A. Incubation of an equimolar mixture of 2-benzyl-4-bromo-5-oxohexanoic acid (XV) and enzyme for 1 h led to the recovery of 82% of XV, demonstrating that it is the major species reversibly bound during assay of inhibition. Taken together, these results indicate that tight binding of carbonyl inhibitors to carboxypeptidase A requires specific binding of inhibitor functional groups such as benzyl and an electrophilic carbonyl carbon such as that of an alpha-bromo ketone or aliphatic aldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective modification of CaaX peptides with ortho-substituted anilinogeranyl lipids by protein farnesyl transferase: competitive substrates and potent inhibitors from a library of farnesyl diphosphate analogues

Protein farnesyl transferase (FTase) catalyzes transfer of a 15-carbon farnesyl group from farnesyl diphosphate (FPP) to a conserved cysteine in the C-terminal Ca1a2X motif of a range of proteins ("C" refers to the cysteine, "a" to any aliphatic amino acid, and "X" to any amino acid), and the lipid chain interacts with, and forms part of, the Ca1a2X peptide binding site. Here, we employed a library of anilinogeranyl diphosphate (AGPP) derivatives to examine whether altering the interacting surface between the two substrates could be exploited to generate Ca1a2X peptide selective FPP analogues. Analysis of transfer kinetics to dansyl-GCVLS peptide revealed that AGPP analogues with substituents smaller than or equal in size to a thiomethyl group supported FTase function, while analogues with larger substituents did not. Analogues with small meta-substitutions on the aniline ring such as iodo and cyano increased reactivity with dansyl-GCVLS and provided analogues that were effective FPP competitors. Other analogues with ortho-substitutions on the aniline were potent dansyl-GCVLS modification FTase inhibitors (Ki in the 2.4-18 nM range). Both meta- and para-trifluoromethoxy-AGPP are transferred to dansyl-GCVLS while the ortho-substituted isomer was a potent farnesyl transferase inhibitor (FTI) with an inhibition constant Ki = 3.0 nM. In contrast, ortho-trifluoromethoxy-AGPP was efficiently transferred to dansyl-GCVIM. Competition for dansyl-GCVLS and dansyl-GCVIM peptides by FPP and ortho-trifluoromethoxy-AGPP gave both analogue and farnesyl modified dansyl-GCVIM but only farnesylated dansyl-GCVLS. We provide evidence that competitive modification of dansyl-GCVIM by ortho-trifluoromethoxy-AGPP stems from a prechemical step discrimination between the competing peptides by the FTase-analogue complex. These results show that subtle changes engineered into the isoprenoid structure can alter the reactivity and FPP competitiveness of the analogues, which may be important for the development of prenylated protein function inhibitors.