RESUMO
Farnesyl:protein transferase (FPTase) inhibitors (FTIs) were originally developed as potential anticancer agents targeting the ras oncogene and are currently in clinical trials. Whereas FTIs inhibit the farnesylation of Ha-Ras, they do not completely inhibit the prenylation of Ki-Ras, the allele most frequently mutated in human cancers. Whereas farnesylation of Ki-Ras is blocked by FTIs, Ki-Ras remains prenylated in FTI-treated cells because of its modification by the related prenyltransferase, geranylgeranyl:protein transferase type I (GGPTase-I). Hence, cells transformed with Ki-ras tend to be more resistant to FTIs than Ha-ras-transformed cells. To determine whether Ki-ras-transformed cells can be targeted by combining an FTI with a GGPTase-I inhibitor (GGTI), we evaluated potent, selective FTIs, GGTIs, and dual prenylation inhibitors (DPIs) that have both FTI and GGTI activity. We find that in human PSN-1 pancreatic tumor cells, which harbor oncogenic Ki-ras, and in other tumor lines having either wild-type or oncogenic Ki-ras, treatment with an FTI/GGTI combination or with a DPI blocks Ki-Ras prenylation and induces markedly higher levels of apoptosis relative to FTI or GGTI alone. We demonstrate that these compounds can inhibit their enzyme targets in mice by monitoring pancreatic and tumor tissues from treated animals for inhibition of prenylation of Ki-Ras, HDJ2, a substrate specific for FPTase, and Rap1A, a substrate specific for GGPTase-I. Continuous infusion (72 h) of varying doses of GGTI in conjunction with a high, fixed dose of FTI causes a dose-dependent inhibition of Ki-Ras prenylation. However, a 72-h infusion of a GGTI, at a dose sufficient to inhibit Ki-Ras prenylation in the presence of an FTI, causes death within 2 weeks of the infusion when administered either as monotherapy or in combination with an FTI. DPIs are also lethal after a 72-h infusion at doses that inhibit Ki-Ras prenylation. Because 24 h infusion of a high dose of DPI is tolerated and inhibits Ki-Ras prenylation, we compared the antitumor efficacy from a 24-h FTI infusion to that of a DPI in a nude mouse/PSN-1 tumor cell xenograft model and in Ki-ras transgenic mice with mammary tumors. The FTI and DPI were dosed at a level that provided comparable inhibition of FPTase. The FTI and the DPI displayed comparable efficacy, causing a decrease in growth rate of the PSN-1 xenograft tumors and tumor regression in the transgenic model, but neither treatment regimen induced a statistically significant increase in tumor cell apoptosis. Although FTI/GGTI combinations elicit a greater apoptotic response than either agent alone in vitro, the toxicity associated with GGTI treatment in vivo limits the duration of treatment and, thus, may limit the therapeutic benefit that might be gained by inhibiting oncogenic Ki-Ras through dual prenyltransferase inhibitor therapy.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores Enzimáticos/farmacologia , Alquil e Aril Transferases/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/toxicidade , Farnesiltranstransferase , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologia , Prenilação de Proteína/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/metabolismoRESUMO
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.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Genes ras , Inibidores do Crescimento/uso terapêutico , Neoplasias Mamárias Animais/tratamento farmacológico , Vírus do Tumor Mamário do Camundongo , Metionina/análogos & derivados , Animais , Modelos Animais de Doenças , Farnesiltranstransferase , Feminino , Humanos , Metionina/uso terapêutico , Camundongos , Camundongos Transgênicos , Fenótipo , TransgenesAssuntos
Alquil e Aril Transferases , Clonagem Molecular/métodos , Transferases/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Cromatografia por Troca Iônica/métodos , Primers do DNA , Epitopos , Escherichia coli , Humanos , Indicadores e Reagentes , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos , Reação em Cadeia da Polimerase/métodos , Biossíntese de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Transferases/imunologia , Transferases/isolamento & purificaçãoAssuntos
Alquil e Aril Transferases , Antineoplásicos/síntese química , Inibidores Enzimáticos/síntese química , Piperazinas/síntese química , Piperazinas/farmacologia , Transferases/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase , Camundongos , Camundongos Nus , Conformação Molecular , Estrutura Molecular , Transplante de Neoplasias , Fosfatos de Poli-Isoprenil/metabolismo , Prenilação de Proteína , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Sarcoma Experimental/tratamento farmacológico , Sarcoma Experimental/patologia , Sesquiterpenos , Proteínas ras/metabolismoRESUMO
The N-termini of members of the T-cell factor (Tcf) and lymphocyte-enhancement factor (Lef) protein families bind to beta-catenin, forming bipartite transcription factors which regulate expression of genes involved in organismal development and the growth of normal and malignant colon epithelium. Elevated levels of Tcf4:beta-catenin are found in colon tumor cells with mutations in the adenomatous polyposis coli (APC) gene. The elevated levels of Tcf4:beta-catenin result in increased transcription of genes, including c-myc, important for the growth of these tumor cells. Here we analyze the interaction between beta-catenin and Tcf4 and show that the N-terminal 53 amino acids of Tcf4 bind with high affinity to beta-catenin. We show that this high-affinity interaction involves multiple contact points including Tcf4 Asp-16, which is essential for beta-catenin binding. In addition to Tcf/Lef family members, beta-catenin binds to APC and cadherins. We found that the binding of beta-catenin to Tcf4, APC, or E-cadherin was mutually exclusive. These results are discussed with regard to how beta-catenin interacts with its binding partners and to the potential for identifying specific, small molecule inhibitors of these interactions.
Assuntos
Aminoácidos/metabolismo , Proteínas do Citoesqueleto/metabolismo , Transativadores , Fatores de Transcrição/metabolismo , Proteína da Polipose Adenomatosa do Colo , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos/análise , Aminoácidos/genética , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Ligação Competitiva , Caderinas/química , Caderinas/genética , Caderinas/metabolismo , Sequência Conservada , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Ensaio de Imunoadsorção Enzimática , Humanos , Concentração Inibidora 50 , Leucina/genética , Leucina/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Solubilidade , Fatores de Transcrição TCF , Proteína 2 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/química , Fatores de Transcrição/genética , beta CateninaRESUMO
Protein farnesyltransferase (FTase) catalyzes the attachment of a farnesyl lipid group to the cysteine residue located in the C-terminal tetrapeptide of many essential signal transduction proteins, including members of the Ras superfamily. Farnesylation is essential both for normal functioning of these proteins, and for the transforming activity of oncogenic mutants. Consequently FTase is an important target for anti-cancer therapeutics. Several FTase inhibitors are currently undergoing clinical trials for cancer treatment. Here, we present the crystal structure of human FTase, as well as ternary complexes with the TKCVFM hexapeptide substrate, CVFM non-substrate tetrapeptide, and L-739,750 peptidomimetic with either farnesyl diphosphate (FPP), or a nonreactive analogue. These structures reveal the structural mechanism of FTase inhibition. Some CaaX tetrapeptide inhibitors are not farnesylated, and are more effective inhibitors than farnesylated CaaX tetrapeptides. CVFM and L-739,750 are not farnesylated, because these inhibitors bind in a conformation that is distinct from the TKCVFM hexapeptide substrate. This non-substrate binding mode is stabilized by an ion pair between the peptide N terminus and the alpha-phosphate of the FPP substrate. Conformational mapping calculations reveal the basis for the sequence specificity in the third position of the CaaX motif that determines whether a tetrapeptide is a substrate or non-substrate. The presence of beta-branched amino acids in this position prevents formation of the non-substrate conformation; all other aliphatic amino acids in this position are predicted to form the non-substrate conformation, provided their N terminus is available to bind to the FPP alpha-phosphate. These results may facilitate further development of FTase inhibitors.
Assuntos
Alquil e Aril Transferases/química , Mimetismo Molecular , Oligopeptídeos/farmacologia , Alquil e Aril Transferases/antagonistas & inibidores , Sequência de Aminoácidos , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Moleculares , Oligopeptídeos/química , Conformação Proteica , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/químicaRESUMO
The roles of 11 conserved amino acids of the beta-subunit of human farnesyl:protein transferase (FTase) were examined by performing kinetic and biochemical analyses of site-directed mutants. This biochemical information along with the x-ray crystal structure of rat FTase indicates that residues His-248, Arg-291, Lys-294, and Trp-303 are involved with binding and utilization of the substrate farnesyl diphosphate. Our data confirm structural evidence that amino acids Cys-299, Asp-297, and His-362 are ligands for the essential Zn2+ ion and suggest that Asp-359 may also play a role in Zn2+ binding. Additionally, we demonstrate that Arg-202 is important for binding the essential C-terminal carboxylate of the protein substrate.
Assuntos
Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/biossíntese , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Arginina , Sequência de Bases , Sequência Conservada , Cristalografia por Raios X , Farnesiltranstransferase , Histidina , Humanos , Cinética , Lisina , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TriptofanoRESUMO
We have isolated cDNAs encoding the alpha and beta subunits of human farnesyl-protein transferase (FPTase). The proteins encoded by these two cDNAs are 93-95% identical to the corresponding subunits of bovine and rat FPTase and show regions of homology with proteins encoded by Saccharomyces cerevisiae prenyl-protein transferase genes. Human FPTase expressed in Escherichia coli from a translationally coupled operon had kinetic properties similar to those of FPTase isolated from bovine brain. Examination of farnesyl diphosphate binding indicated that while neither individual subunit was capable of isoprenoid binding, a radiolabeled farnesyl diphosphate analog could be specifically photo-cross-linked to the beta subunit of FPTase holoenzyme. To further analyze subunit structure-function and to detect functional similarities with yeast prenyl-protein transferases (FPTase and two geranylgeranyl-protein transferases), amino acid changes homologous to those found in mutant yeast prenyl-protein transferase subunits were made in the subunits of human FPTase. Substitutions in either the alpha or beta subunits that decrease the activity of yeast prenyl-protein transferases were also observed to impair human FPTase. Kinetic analyses showed that these mutant human FPTases have Km and kcat values that are altered with respect to wild-type human FPTase.