ABSTRACT
AIMS: The aim of this study was to evaluate the abuse potential of dasotraline, a novel dopamine and norepinephrine reuptake inhibitor with slow absorption (tmax, 10-12h) and elimination (t1/2=47-77 h) that is in development for the treatment of attention deficit hyperactivity disorder (ADHD). METHODS: Recreational stimulant users (N=48) who had specific experience with cocaine, and who were able to distinguish methylphenidate (60 mg) versus placebo in a qualification session, were randomized, in a 6-period, double-blind, crossover design, to receive single doses of dasotraline 8 mg, 16 mg, and 36 mg, methylphenidate (MPH) 40 mg and 80 mg, and placebo. The primary endpoint was the Drug Liking Visual Analog Scale (VAS) score at the time of peak effect (Emax). RESULTS: There were no significant differences between the 3 doses of dasotraline and placebo on the drug liking VAS at Emax, and on most secondary endpoints. Both doses of MPH had significantly higher VAS-drug liking scores at Emax relative to both placebo (P<0.001 for all comparisons) and dasotraline 8 mg (P<0.001), 16 mg (P<0.001) and 36 mg (P<0.01). The increase in heart rate for MPH and dasotraline 36 mg showed a time-course that closely matched subject-rated measures such as Any Effects VAS. CONCLUSIONS: In this study, dasotraline was found to have low potential for abuse, which may be, in part, related to its established pharmacokinetics (PK) profile, which is characterized by slow absorption and gradual elimination.
Subject(s)
1-Naphthylamine/analogs & derivatives , Central Nervous System Stimulants/adverse effects , Drug Users/psychology , Methylphenidate/adverse effects , Substance-Related Disorders , 1-Naphthylamine/adverse effects , 1-Naphthylamine/pharmacokinetics , 1-Naphthylamine/pharmacology , Adult , Central Nervous System Stimulants/pharmacokinetics , Central Nervous System Stimulants/pharmacology , Cross-Over Studies , Dose-Response Relationship, Drug , Double-Blind Method , Female , Heart Rate/drug effects , Humans , Male , Methylphenidate/pharmacokinetics , Methylphenidate/pharmacology , Middle AgedABSTRACT
Essential tremor (ET) is a common movement disorder. Animal studies show that histaminergic modulation may affect the pathological processes involved in the generation of ET. Histamine-3 receptor inverse agonists (H3RIA) have demonstrated attenuating effects on ET in the harmaline rat model. In this double-blind, three-way cross-over, single-dose, double-dummy study the effects of 25 mg of a novel H3RIA (MK-0249) and a stable alcohol level (0.6 g L(-1)) were compared with placebo, in 18 patients with ET. Tremor was evaluated using laboratory tremorography, portable tremorography and a clinical rating scale. The Leeds Sleep Evaluation Questionnaire (LSEQ) and a choice reaction time (CRT) test were performed to evaluate potential effects on sleep and attention, respectively. A steady state of alcohol significantly diminished tremor as assessed by laboratory tremorography, portable tremorography and clinical ratings compared with placebo. A high single MK-0249 dose was not effective in reducing tremor, but caused significant effects on the LSEQ and the CRT test. These results suggest that treatment with a single dose of MK-0249 does not improve tremor in alcohol-responsive patients with ET, whereas stable levels of alcohol as a positive control reproduced the commonly reported tremor-diminishing effects of alcohol.
Subject(s)
Essential Tremor/drug therapy , Ethanol/metabolism , Histamine Agonists/therapeutic use , Quinazolinones/therapeutic use , Attention/drug effects , Cross-Over Studies , Double-Blind Method , Essential Tremor/metabolism , Female , Histamine Agonists/pharmacokinetics , Humans , Male , Middle Aged , Quinazolinones/pharmacokinetics , Reaction Time/drug effects , Receptors, Histamine H3/metabolismABSTRACT
The neuropeptide TIP39 was recently purified from bovine hypothalamus based on the ability of the peptide to activate the parathyroid hormone 2 receptor (PTH2R) ( Nat. Neurosci. 2 (1999) 941). PTH2R is abundantly expressed in the nervous system, and its expression pattern suggests that it may play a role in modulation of pituitary function and in nociception. Towards understanding the physiological role of TIP39 and PTH2R, we cloned human, mouse and rat TIP39 gene. Our results revealed that: (1) the mature peptide is processed from a precursor; (2) TIP39 peptide is highly conserved among species; and (3) TIP39 from all species activates adenylyl cyclase and elevates intracellular calcium levels through PTH2R. We also defined and compared the structure-activity relationship of TIP39 on both activation of adenylyl cyclase and calcium mobilization pathways through PTH2R, finding common and differential determinants of TIP39 that are required for these pathways. Furthermore, we observed that TIP39 elevates intracellular calcium levels in primary dorsal root ganglion neurons whereas the peptide inactive on PTH2R do not, suggesting that TIP39 may activate these neurons important for nociception in vivo through PTH2R-dependent mechanisms.
Subject(s)
Neuropeptides/genetics , Receptors, Parathyroid Hormone/physiology , Amino Acid Sequence , Animals , Base Sequence , Calcium Signaling , Cells, Cultured , Cloning, Molecular , Cyclic AMP/biosynthesis , DNA, Complementary , Embryo, Mammalian/cytology , Ganglia, Spinal/cytology , Ganglia, Spinal/physiology , Humans , In Situ Hybridization , Mice , Molecular Sequence Data , Neuropeptides/chemistry , Neuropeptides/pharmacology , Rats , Receptor, Parathyroid Hormone, Type 2 , Receptors, Parathyroid Hormone/drug effects , Signal Transduction , Species Specificity , Structure-Activity RelationshipABSTRACT
Neuropathic pain is induced by injury or disease of the nervous system. Studies aimed at understanding the molecular pathophysiology of neuropathic pain have so far focused on a few known molecules and signaling pathways in neurons. However, the pathophysiology of neuropathic pain appears to be very complex and remains poorly understood. A global understanding of the molecular mechanisms involved in neuropathic pain is needed for a better understanding of the pathophysiology and treatment of neuropathic pain. Towards this end, we examined global gene expression changes as well as the pathobiology at the cellular level in a spinal nerve ligation neuropathic pain model using DNA microarray, quantitative real-time PCR and immunohistochemistry. We found that the behavioral hypersensitivity that is manifested in the persistent pain state is accompanied by previously undescribed changes in gene expression. In the DRG, we found regulation of: (1) immediate early genes; (2) genes such as ion channels and signaling molecules that contribute to the excitability of neurons; and (3) genes that are indicative of secondary events such as neuroinflammation. In addition, we studied gene regulation in both injured and uninjured DRG by quantitative PCR, and observed differential gene regulation in these two populations of DRGs. Furthermore, we demonstrated unexpected co-regulation of many genes, especially the activation of neuroinflammation markers in both the PNS and CNS. The results of our study provide a new picture of the molecular mechanisms that underlie the complexity of neuropathic pain and suggest that chronic pain shares common pathobiology with progressive neurodegenerative disease.
Subject(s)
Gene Expression/physiology , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/pathology , Pain/metabolism , Pain/pathology , Animals , Chronic Disease , Ganglia, Spinal/injuries , Ganglia, Spinal/metabolism , Ganglia, Spinal/pathology , Ligation , Male , Rats , Rats, Sprague-Dawley , Spinal Cord/metabolism , Spinal Cord/pathology , Spinal Nerves/injuries , Spinal Nerves/metabolism , Spinal Nerves/pathologyABSTRACT
Calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), amylin and calcitonin (CT) are structurally and functionally related neuropeptides. It has recently been shown that the molecular pharmacology of CGRP and ADM is determined by coexpression of one of three receptor activity-modifying proteins (RAMPs) with calcitonin receptor-like receptor (CRLR). Furthermore, RAMP proteins have also been shown to govern the pharmacology of the calcitonin receptor, which in association with RAMP1 or RAMP3, binds amylin with high affinity. In this study, we have cloned the rat RAMP family and characterized the pharmacology of rat CGRP and ADM receptors. Rat RAMP1, RAMP2 and RAMP3 shared 72%, 69% and 85% homology with their respective human homologues. As expected CRLR-RAMP1 coexpression conferred sensitivity to CGRP, whilst association of RAMP2 or RAMP3 with CRLR conferred high affinity ADM binding. Using specific oligonucleotides we have determined the expression of RAMP1, RAMP2 and RAMP3 mRNAs in the rat central nervous system by in situ hybridization. The localization of RAMP mRNAs was heterogeneous. RAMP1 mRNA was predominantly expressed in cortex, caudate putamen and olfactory tubercles; RAMP2 mRNA was most abundant in hypothalamus; and RAMP3 was restrictively expressed in thalamic nuclei. Interestingly, in specific brain areas only a single RAMP mRNA was often detected, suggesting mutual exclusivity in expression. These data allow predictions to be made of where each RAMP protein may heterodimerize with its partner G-protein-coupled receptor(s) at the cellular level and consequently advance current understanding of cellular sites of action of CGRP, ADM, amylin and CT. Furthermore, these localization data suggest that the RAMP family may associate and modify the behaviour of other, as yet unidentified neurotransmitter receptors.
Subject(s)
Central Nervous System/metabolism , Membrane Proteins/genetics , Neuropeptides/metabolism , RNA, Messenger/metabolism , Receptors, Calcitonin/metabolism , Adrenomedullin , Amino Acid Sequence/physiology , Amyloid/metabolism , Animals , Calcitonin/metabolism , Calcitonin Gene-Related Peptide/metabolism , Calcitonin Receptor-Like Protein , Cells, Cultured , Cloning, Molecular , DNA, Complementary/isolation & purification , Diencephalon/metabolism , Intracellular Signaling Peptides and Proteins , Islet Amyloid Polypeptide , Male , Membrane Proteins/metabolism , Mesencephalon/metabolism , Molecular Sequence Data , Peptides/metabolism , Rats , Rats, Sprague-Dawley , Receptor Activity-Modifying Protein 1 , Receptor Activity-Modifying Protein 2 , Receptor Activity-Modifying Protein 3 , Receptor Activity-Modifying Proteins , Rhombencephalon/metabolism , Sequence Homology, Amino Acid , Spinal Cord/metabolism , Telencephalon/metabolismABSTRACT
The synthesis, structure-activity relationships, and biological properties of a novel series of imidazole-containing inhibitors of farnesyltransferase are described. Starting from a 3-aminopyrrolidinone core, a systematic series of modifications provided 5h, a non-thiol, non-peptide farnesyltransferase inhibitor with excellent bioavailability in dogs. Compound 5h was found to have an unusually favorable ratio of cell potency to intrinsic potency, compared with other known FTIs. It exhibited excellent potency against a range of tumor cell lines in vitro and showed full efficacy in the K-rasB transgenic mouse model.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Imidazoles/chemical synthesis , Lactams/chemical synthesis , Nitriles/chemical synthesis , Pyrrolidinones/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Binding Sites , Binding, Competitive , Biological Availability , Cell Line, Transformed , Dogs , Drug Design , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Farnesyltranstransferase , Genes, ras , Imidazoles/chemistry , Imidazoles/pharmacology , Lactams/chemistry , Lactams/pharmacology , Mice , Mice, Transgenic , Models, Molecular , Neoplasms, Experimental/pathology , Nitriles/chemistry , Nitriles/pharmacology , Pyrrolidinones/chemistry , Pyrrolidinones/pharmacology , Radioligand Assay , Stereoisomerism , Structure-Activity RelationshipABSTRACT
A series of amino acid-based linkers was used to investigate the effects of various substituents upon the potency, pharmacokinetic properties, and conformation of macrocyclic farnesyl-protein transferase inhibitors (FTIs). As a result of the studies described herein, highly potent FTIs with improved pharmacokinetic profiles have been identified.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Alkyl and Aryl Transferases/drug effects , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/pharmacokinetics , Amino Acids/chemistry , Animals , Cells, Cultured , Dogs , Enzyme Inhibitors/chemical synthesis , Half-Life , Inhibitory Concentration 50 , Metabolic Clearance Rate/physiology , Molecular Conformation , Protein Binding/drug effects , RatsABSTRACT
It remains unknown whether the extent of vasoactive response to exogenous calcitonin gene-related peptide (CGRP) varies among different regional vascular beds. It is also unclear whether endogenous CGRP plays a functional role in regulating basal vascular activity. To address these two issues, experiments were conducted in 27 anesthetized rats instrumented with a carotid flow probe and catheters in a jugular vein, left ventricle (LV), and femoral artery, and in 6 conscious dogs, chronically instrumented with LV pressure gauge, aortic and atrial catheters, and ascending aortic, coronary, carotid, and renal flow probes. In both species, administration of human alpha-CGRP (0.1-0.5 microg/kg, i.v.) induced a dose-dependent peripheral vasodilation that was completely abolished by pretreatment with alpha-CGRP[8-37] (30 microg/kg/min, i.v.), a competitive antagonist of CGRP receptors. Regional blood flow measured by the radioactive microsphere technique in rats showed that the alpha-CGRP (0.3 microg/kg, i.v.)-induced increase in blood flow was greater (p < 0.05) in the heart (+53 +/- 16%) than in the brain (+14 +/- 6%). In the presence of beta-adrenergic receptor blockade with propranolol, however, the increases in blood flow in these two vascular beds were identical. In conscious dogs, alpha-CGRP (0.3 microg/kg, i.v.) produced similar increases in coronary (+24 +/- 6%), carotid (+26 +/- 3%), and renal (+26 +/- 6%) blood flow, which were different from the patterns induced by other vasodilators; at an equivalent level of reduction in mean arterial pressure and total peripheral resistance, alpha-CGRP increased coronary and carotid blood flow significantly less (p < 0.05) than adenosine or nitroprusside. Unlike alpha-CGRP, adenosine and nitroprusside, as expected, induced pronounced differential blood flow changes in these vascular beds. Neither systemic hemodynamics nor regional blood flow distribution was altered by the administration of a pharmacological blocking dose of alpha-CGRP[8-37] in the two species. Thus, we conclude that endogenous alpha-CGRP does not play an important role in cardiovascular regulation under normal, resting conditions, although exogenous alpha-CGRP induces a marked, comparable vasorelaxation in different regional vascular beds.
Subject(s)
Calcitonin Gene-Related Peptide/pharmacology , Hemodynamics/drug effects , Adenosine/pharmacology , Animals , Blood Pressure/drug effects , Carotid Arteries/drug effects , Dogs , Female , Heart Rate/drug effects , Humans , Male , Nitroprusside/pharmacology , Rats , Rats, Sprague-Dawley , Regional Blood Flow/drug effects , Species Specificity , Vasodilator Agents/pharmacologySubject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Alkyl and Aryl Transferases/chemistry , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Farnesyltranstransferase , Magnetic Resonance Spectroscopy , Molecular Conformation , Structure-Activity RelationshipABSTRACT
Imidazolemethyl diaryl ethers are potent inhibitors of farnesyl-protein transferase. The SNAr displacement reaction used to prepare these diaryl ethers was amenable to rapid parallel synthesis of FPTase inhibitors. The use of a broad range of commercially available phenols quickly identified compounds which proved active in cells.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Phenyl Ethers/pharmacology , Alkyl and Aryl Transferases/metabolism , Animals , Binding, Competitive , Cell Line , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Imidazoles/chemistry , Inhibitory Concentration 50 , Peptide Library , Phenyl Ethers/chemical synthesis , Rats , Structure-Activity RelationshipABSTRACT
A series of aryloxy substituted piperazinones with dual farnesyltransferase/geranylgeranyltransferase-I inhibitory activity was prepared. These compounds were found to have potent inhibitory activity in vitro and are promising agents for the inhibition of Ki-Ras signaling.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Piperazines/pharmacology , Drug Design , Enzyme Inhibitors/chemistry , Farnesyltranstransferase , Genes, ras/drug effects , Piperazines/chemistry , Polymers/chemistry , Signal Transduction/drug effects , Structure-Activity RelationshipABSTRACT
The cerebral circulation is innervated by calcitonin gene-related peptide (CGRP) containing fibers originating in the trigeminal ganglion. During a migraine attack, there is a release of CGRP in conjunction with the head pain, and triptan administration abolishes both the CGRP release and the pain at the same time. In the search for a novel treatment of migraine, a non-peptide CGRP antagonist has long been sought. Here, we present data on a human cell line and human and guinea-pig isolated cranial arteries for such an antagonist, Compound 1 (4-(2-Oxo-2,3-dihydro-benzoimidazol-1-yl)-piperidine-1-carboxylic acid [1-(3,5-dibromo-4-hydroxy-benzyl)-2-oxo-2-(4-phenyl-piperazin-1-yl)-ethyl]-amide). On SK-N-MC cell membranes, radiolabelled CGRP binding was displaced by both CGRP-(8-37) and Compound 1, yielding pK(i) values of 8.9 and 7.8, respectively. Functional studies with SK-N-MC cells showed that CGRP-induced cAMP production was antagonised by both CGRP-(8-37) and Compound 1 with pA(2) values of 7.8 and 7.7, respectively. Isolated human and guinea pig cerebral arteries were studied with a sensitive myograph technique. CGRP induced a concentration-dependent relaxation in human cerebral arteries which was antagonized by both CGRP-(8-37) and Compound 1 in a competitive manner. In guinea pig basilar arteries, CGRP-(8-37) antagonised the CGRP-induced relaxation while Compound 1 had a weak blocking effect. The clinical studies of non-peptide CGRP antagonists are awaited with great interest.
Subject(s)
Calcitonin Gene-Related Peptide Receptor Antagonists , Cerebral Arteries/drug effects , Piperazines/pharmacology , Piperidines/pharmacology , Animals , Binding, Competitive , Calcitonin Gene-Related Peptide/pharmacology , Cell Membrane/metabolism , Cerebral Arteries/metabolism , Cerebral Arteries/physiology , Cyclic AMP/metabolism , Dose-Response Relationship, Drug , Guinea Pigs , Humans , In Vitro Techniques , Peptide Fragments/pharmacology , Tumor Cells, Cultured , Vasodilation/drug effectsABSTRACT
The evaluation of SAR associated with the insertion of carbonyl groups at various positions of N-arylpiperazinone farnesyltransferase inhibitors is described herein. 1-Aryl-2,3-diketopiperazine derivatives exhibited the best balance of potency and pharmacokinetic profile relative to the parent 1-aryl-2-piperazinones.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Piperazines/pharmacology , Animals , Dogs , Enzyme Inhibitors/pharmacokinetics , Farnesyltranstransferase , Structure-Activity RelationshipABSTRACT
Cellular transformation by Ras oncoproteins requires the posttranslation modification of farnesylation in a reaction catalyzed by farnesyl protein transferase (FPTase). Thus, inhibitors of FPTase have been developed as potential anticancer agents. However, recent studies with selective inhibitors of FPTase have shown that Ki4B-Ras retains its ability to transform cells by undergoing alternative prenylation by the related geranylgeranyl protein transferase I (GGPTase-I) in human tumor cells. We have developed a high-performance liquid chromatography/mass spectrometry assay for the detection and quantitation of the different processing states of Ki4B-Ras isolated from PSN-1 cells (a human pancreatic cell line with an activating Gly12 to Arg mutation) treated with the prenyltransferase inhibitor, L-778,123. Recently tested in the clinic, L-778,123 is a potent inhibitor of FPTase (in vitro IC50 = 2 nM) with some activity against GGPTase-I (in vitro IC50 = 98 nM). We find primarily farnesylated-Ki4B-Ras in vehicle-treated PSN-1 cells, a mixture of farnesylated- and geranylgeranylated-Ki4B-Ras in cells treated with nanomolar concentrations of L-778,123, and a mixture of unprocessed, farnesylated, and geranylgeranylated-Ki4B-Ras in cells treated with micromolar concentrations of compound. Of importance, this technique does not require metabolic labeling and may be used as a pharmacodynamic assay for Ki4B-Ras processing in mouse models.
Subject(s)
Dimethylallyltranstransferase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Gas Chromatography-Mass Spectrometry/methods , Imidazoles/pharmacology , Proto-Oncogene Proteins p21(ras)/analysis , Tumor Cells, Cultured/drug effects , Alkyl and Aryl Transferases/antagonists & inhibitors , Alkyl and Aryl Transferases/metabolism , Farnesyltranstransferase , Humans , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/enzymology , Protein Prenylation , Recombinant Proteins/metabolism , Tumor Cells, Cultured/enzymologyABSTRACT
[reaction: see text] Synthesis of the 8-amino-5,6,7,8-tetrahydroimidazo[1,5-a]pyridine ring system was accomplished by intramolecular cyclization of an iminium ion, derived from condensation of an amine and a substituted gamma-(1-imidazolyl)butyraldehyde. The reaction was used to produce conformationally restricted farnesyltransferase inhibitor analogues which exhibit improved in vivo metabolic stability.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Imidazoles/chemical synthesis , Pyridines/chemical synthesis , Administration, Oral , Animals , Dogs , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Farnesyltranstransferase , Imidazoles/chemistry , Imidazoles/pharmacokinetics , Imidazoles/pharmacology , Indicators and Reagents , Models, Molecular , Molecular Conformation , Pyridines/chemistry , Pyridines/pharmacokinetics , Pyridines/pharmacology , Structure-Activity RelationshipABSTRACT
For Ras oncoproteins to transform mammalian cells, they must be posttranslationally modified with a farnesyl group in a reaction catalyzed by the enzyme farnesyl:protein transferase (FPTase). Inhibitors of FPTase have therefore been developed as potential anticancer agents. These compounds reverse many of the malignant phenotypes of Ras-transformed cells in culture and inhibit the growth of tumor xenografts in nude mice. Furthermore, the FPTase inhibitor (FTI) L-744,832 causes tumor regression in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice and tumor stasis in MMTV-N-ras mice. Although these data support the further development of FTIs, it should be noted that Ki-ras is the ras gene most frequently mutated in human cancers. Moreover, Ki-RasB binds more tightly to FPTase than either Ha- or N-Ras, and thus higher concentrations of FTIs that are competitive with the protein substrate may be required to inhibit Ki-Ras processing. Given the unique biochemical and biological features of Ki-RasB, it is important to evaluate the efficacy of FTIs or any other modulator of oncogenic Ras function in model systems expressing this Ras oncoprotein. We have developed strains of transgenic mice carrying the human Ki-rasB cDNA with an activating mutation (G12V) under the control of the MMTV enhancer/promoter. The predominant pathological feature that develops in these mice is the stochastic appearance of mammary adenocarcinomas. High levels of the Ki-rasB transgene RNA are detected in these tumors. Treatment of MMTV-Ki-rasB mice with L-744,832 caused inhibition of tumor growth in the absence of systemic toxicity. Although FPTase activity was inhibited in tumors from the treated mice, unprocessed Ki-RasB was not detected. These results demonstrate the utility of the MMTV-Ki-rasB transgenic mice for testing potential anticancer agents. Additionally, the data suggest that although the FTI L-744,832 can inhibit tumor growth in this model, Ki-Ras may not be the sole mediator of the biological effects of the FTI.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Antineoplastic Agents/therapeutic use , Enzyme Inhibitors/therapeutic use , Genes, ras , Growth Inhibitors/therapeutic use , Mammary Neoplasms, Animal/drug therapy , Mammary Tumor Virus, Mouse , Methionine/analogs & derivatives , Animals , Disease Models, Animal , Farnesyltranstransferase , Female , Humans , Methionine/therapeutic use , Mice , Mice, Transgenic , Phenotype , TransgenesABSTRACT
The design and syntheses of non-thiol inhibitors of farnesyl-protein transferase are described. Optimization of cysteine-substituted diarylethers led to highly potent imidazole-containing diarylethers and diarylsulfones. Polar diaryl linkers dramatically improved potency and gave highly cell active compounds.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Imidazoles/chemistry , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Ethers/chemistry , Humans , Imidazoles/pharmacology , Magnetic Resonance Spectroscopy , Molecular Structure , Sulfones/chemistrySubject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Imidazoles/chemical synthesis , Piperazines/chemical synthesis , Animals , Enzyme Inhibitors/pharmacology , Farnesyltranstransferase , Humans , Imidazoles/pharmacology , Mice , Mice, Nude , Models, Molecular , Piperazines/pharmacology , Rats , Tumor Cells, CulturedABSTRACT
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 cell-transforming 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-, D- and noncoded amino acids, has been screened for inhibitors of FPTase activity. The tetrapeptide, NH2-D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH2 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, NH2-D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH2 was determined by NMR spectroscopy. Distance constraints were derived from two-dimensional transferred nuclear Overhauser effect (TRNOE) experiments. Ligand competition experiments identified the NOEs that originate from the active-site 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 reverse-turn conformation most closely approximating a type II' beta-turn. The resolved conformation of the inhibitor represents a distinctly different structural motif from that determined for Ras-competitive 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.
Subject(s)
Alkyl and Aryl Transferases/metabolism , Enzyme Inhibitors/chemistry , Oligopeptides/chemistry , Alkyl and Aryl Transferases/antagonists & inhibitors , Enzyme Inhibitors/metabolism , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Structure , Oligopeptides/metabolismABSTRACT
Inhibitors of farnesyl protein transferase (FPTase) based upon a pseudotripeptide template are described that comprise an imidazole group substituted with a hydrophobic substituent. (1, 5)-Disubstitution of the imidazole group is shown to be the optimal array that leads to potent and selective inhibitors of FPTase. A variety of aryl and isoprenyl substituents are shown to afford effective inhibitors, and the mechanism by which these compounds inhibit FPTase has been investigated. The biochemical behavior of these compounds suggests that they bind to FPTase at the site usually occupied by the protein substrate. In experiments in cell culture, the methyl ester prodrugs of these inhibitors are cell permeant and potently inhibit the posttranslational modification of H-Ras protein. Additionally, these molecules revert the phenotype of ras transformed cells as evidenced by their ability to slow the growth of ras transformed cell lines in soft agar. One of the inhibitors, as its methyl prodrug, was evaluated in two in vivo models of tumor growth. The compound selectively inhibited the growth of tumors derived from H-ras transformed cells, in nude mice, and caused the regression of preexisting tumors in an H-ras transgenic animal model.