RESUMO
PURPOSE: To investigate the incidence of cMET gene copy number changes and protein overexpression in Chinese gastric cancer (GC) and to preclinically test the hypothesis that the novel, potent and selective cMET small-molecule inhibitor volitinib, will deliver potent anti-tumor activity in cMET-dysregulated GC patient-derived tumor xenograft (PDX) models. EXPERIMENTAL DESIGN: A range of assays were used and included; in vitro cell line panel screening and pharmacodynamic (PD) analysis, cMET fluorescence in-situ hybridization (FISH) and immunohistochemical (IHC) tissue microarray (TMA) analysis of Chinese GC (n = 170), and anti-tumor efficacy testing and PD analysis of gastric PDX models using volitinib. RESULTS: The incidence of cMET gene amplification and protein overexpression within Chinese patient GC tumors was 6% and 13%, respectively. Volitinib displayed a highly selective profile across a gastric cell line panel, potently inhibiting cell growth only in those lines with dysregulated cMET (EC50 values 0.6 nM/L-12.5 nM/L). Volitinib treatment led to pharmacodynamic modulation of cMET signaling and potent tumor stasis in 3/3 cMET-dysregulated GC PDX models, but had negligible activity in a GC control model. CONCLUSIONS: This study provides an assessment of tumor cMET gene copy number changes and protein overexpression incidence in a cohort of Chinese GC patients. To our knowledge, this is the first study to demonstrate anti-tumor efficacy in a panel of cMET-dysregulated gastric cancer PDX models, using a novel selective cMET-inhibitor (volitinib). Thus, the translational science presented here provides strong rationale for the investigation of volitinib as a therapeutic option for patients with GC tumors harboring amplified cMET.
Assuntos
Amplificação de Genes , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Pirazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico , Triazinas/farmacologia , Animais , Povo Asiático , Linhagem Celular Tumoral , Estudos de Coortes , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-met/biossíntese , Proteínas Proto-Oncogênicas c-met/genética , Transdução de Sinais/genética , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
With the aim of improving HCV protease inhibitors reported in our previous manuscripts, we synthesized and evaluated a series of 1a-based tetrapeptidyl alpha-ketoamides with additional P4 modification. The promising analog discovered through this SAR, 5a, was further derivatized at P1' or P1 position. As a result of these efforts, we found that replacement of the P4 valine as seen in 1a with cyclohexylglycine (Chg) resulted in the discovery of 5a, 5c, and 5e endowed with improved cellular activity in comparison to 1a.
Assuntos
Amidas/síntese química , Antivirais/síntese química , Inibidores de Serina Proteinase/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Amidas/farmacologia , Antivirais/farmacologia , Replicon/efeitos dos fármacos , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-AtividadeRESUMO
We describe herein the design, syntheses, and biological evaluation of new series of P4 tetrazole and adipic acid, ester, amide capped tetrapeptidyl alpha-ketoamide based HCV protease inhibitors.
Assuntos
Amidas/farmacologia , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Amidas/síntese química , Amidas/química , Inibidores de Proteases/síntese química , Inibidores de Proteases/químicaRESUMO
The NS5B encoded by the hepatitis C virus genome is a RNA-dependent RNA polymerase essential to viral replication. The entire NS5B protein contains a catalytic domain followed by a regulatory motif and a membrane-anchor domain at its C-terminus. Reported here is the molecular cloning and expression of the full-length NS5B polymerase (NS5B-FL) in bacterial cells as a non-fusion protein. The non-tagged NS5B-FL was purified to homogeneity using sequential chromatographic columns and its identity was confirmed using anti-NS5B peptide antibodies and amino acid sequencing. Purified NS5B-FL demonstrated RNA-dependent RNA polymerase activity and was able to replicate a HCV RNA genome fragment through both copy-back and de novo mechanisms. Its biochemical properties were further characterized in comparison with a truncated form of NS5B polymerase with a deletion of 51 residues from its C-terminus.
Assuntos
Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificação , Sequência de Bases , Clonagem Molecular , Primers do DNA , Especificidade por Substrato , Proteínas não Estruturais Virais/metabolismoAssuntos
Desenho de Fármacos , Inibidores Enzimáticos , NF-kappa B/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Quinase I-kappa B , Estrutura Molecular , NF-kappa B/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de SinaisRESUMO
A convergent strategy for the synthesis of cyclic nucleotide-hybrid molecules on controlled pore glass is reported. A major advantage of the approach is the lack of restrictions on the sequence and structural variation, allowing the incorporation of modified ribonucleosides (such as 2'-OMe-ribonucleotides), as well as threoninol derivatives. This methodology allows a fully automated assembly by means of standard phosphoramidite chemistry and is based on a recently published procedure for the preparation of cyclic oligodinucleotides in the DNA series (M. Smietana, E. T. Kool, Angew. Chem. 2002, 114, 3856-3859; Angew. Chem. Int. Ed. Engl. 2002, 41, 3704-3707). A library of potential cyclic hybrid inhibitor compounds targeting hepatitis C virus NS5B enzyme (the replicating polymerase of HCV) was generated by means of the parallel-pool strategy. Screening of the library revealed that cyclic hybrid c(C(OME)EthenodA) was a significant inhibitor of NS5B, with an IC(50) of 40 microM. Preliminary structure-activity studies of this lead compound are described.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Oligonucleotídeos/síntese química , Oligonucleotídeos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/química , Butiratos/metabolismo , Ciclização , Avaliação Pré-Clínica de Medicamentos , Inibidores da Síntese de Ácido Nucleico , Nucleotídeos Cíclicos/química , Oligonucleotídeos/química , Transição de Fase , Ribonucleosídeos/síntese química , Ribonucleosídeos/química , Ribonucleosídeos/metabolismo , Relação Estrutura-AtividadeRESUMO
With the aim of discovering potent and selective HCV protease inhibitors, we synthesized and evaluated a series of 1a based tetrapeptidyl ketoamides with additional modification(s) at P1', P1, and P3 positions. As a result of this effort, we found that replacement of the P3 valine with tert-leucine resulted in the discovery of a series of inhibitors (e.g., 3a, 3c, and 4c) endowed with improved enzyme and/or cellular activity relative to 1a. When dosed to F-344 rats orally at 50mg/kg, 3a achieved 2.5x higher liver and plasma exposure in comparison to that detected with 1a.
Assuntos
Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Prolina/química , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/síntese química , Proteínas não Estruturais Virais/metabolismo , Animais , Compostos Bicíclicos com Pontes/química , Linhagem Celular Tumoral , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/virologia , Masculino , Prolina/farmacologia , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Inibidores de Serina Proteinase/farmacologiaRESUMO
We describe herein the design, syntheses and evaluation of a number of bicycloproline P2 bearing HCV protease inhibitors endowed with impressive enzyme potency, enzyme selectivity, cellular activity and favorable ADME profiles.
Assuntos
Hepacivirus/efeitos dos fármacos , Prolina/química , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Proteínas não Estruturais Virais/metabolismo , Animais , Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/farmacologia , Dipeptídeos/síntese química , Dipeptídeos/farmacologia , Hepacivirus/enzimologia , Humanos , Masculino , Prolina/farmacologia , Ratos , Ratos Sprague-Dawley , Inibidores de Serina Proteinase/síntese químicaRESUMO
We describe herein the synthesis and evaluation of two series of P-4 truncated tripeptidyl alpha-ketoamides as HCV serine protease inhibitors. The most promising compound disclosed in this communication 7b demonstrated enzyme binding affinity (K(i)) at 0.27 uM.
Assuntos
Amidas/farmacologia , Hepacivirus/efeitos dos fármacos , Oligopeptídeos/química , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/síntese química , Amidas/química , Linhagem Celular Tumoral , Hepacivirus/enzimologia , Humanos , Oligopeptídeos/farmacologia , Replicon/efeitos dos fármacos , Inibidores de Serina Proteinase/farmacologiaRESUMO
We describe herein the synthesis and biological evaluation of a series of tripeptidyl alpha-ketoamides as human rhinovirus (HRV) 3C protease inhibitors. The most potent inhibitor discussed in this manuscript, 4I, exhibited impressive enzyme inhibitory activity as well as antiviral activity against HRV-14.
Assuntos
Amidas/síntese química , Amidas/farmacologia , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Proteínas Virais/antagonistas & inibidores , Proteases Virais 3C , Amidas/química , Cisteína Endopeptidases , Avaliação Pré-Clínica de Medicamentos , Humanos , Inibidores de Proteases/química , Relação Estrutura-AtividadeRESUMO
The NS5B RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) is a key component of the viral replicase. Reported here is the three-dimensional structure of HCV NS5B polymerase, with the highlight on its C-terminal folding, determined by X-ray crystallography at 2.1-A resolution. Structural analysis revealed that a stretch of C-terminal residues of HCV NS5B inserted into the putative RNA binding cleft, where they formed a hydrophobic pocket and interacted with several important structural elements. This region was found to be conserved and unique to the RNA polymerases encoded by HCV and related viruses. Through biochemical analyses, we confirmed that this region interfered with the binding of HCV NS5B to RNA. Deletion of this fragment from HCV NS5B enhanced the RNA synthesis rate up to approximately 50-fold. These results provide not only direct experimental insights into the role of the C-terminal tail of HCV NS5B polymerase but also a working model for the RNA synthesis mechanism employed by HCV and related viruses.
Assuntos
Hepacivirus/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Sequência de Bases , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , RNA , RNA Polimerase Dependente de RNA/química , Proteínas não Estruturais Virais/metabolismoRESUMO
The activities of many protein kinases are regulated by phosphorylation. The phosphorylated protein kinases thus represent an important class of substrates for protein phosphatases. However, our ability to study the phosphatase-catalyzed substrate dephosphorylation has been limited in many cases by the difficulty in preparing sufficient amount of stoichiometrically phosphorylated kinases. We have applied the kinetic theory of substrate reaction during irreversible modification of enzyme activity to the study of phosphatase-catalyzed regulation of kinase activity. As an example, we measured the effect of the hematopoietic protein-tyrosine phosphatase (HePTP) on the reaction catalyzed by the fully activated, bisphosphorylated extracellular signal-regulated protein kinase 2 (ERK2/pTpY). Because only a catalytic amount of ERK2/pTpY is required, this method alleviates the need for large quantities of phospho-ERK2. Kinetic analysis of the ERK2/pTpY-catalyzed substrate reaction in the presence of HePTP leads to the determination of the rate constants for the HePTP-catalyzed dephosphorylation of free ERK2/pTpY and ERK2/pTpY*substrate(s) complexes. The data indicate that ERK2/pTpY is a highly efficient substrate for HePTP (k(cat)/K(m) = 3.05 x 10(6) M(-1) s(-1)). The data also show that binding of ATP to ERK2/pTpY has no effect on ERK2/pTpY dephosphorylation by HePTP. In contrast, binding of an Elk-1 peptide substrate to ERK2/pTpY completely blocks the HePTP action. This result indicates that phosphorylation of Tyr185 is important for ERK2 substrate recognition and that binding of the Elk-1 peptide substrate to ERK2/pTpY blocks the accessibility of pTyr185 to HePTP for dephosphorylation. Collectively, the results establish that the kinetic theory of irreversible enzyme modification can be applied to study the phosphatase catalyzed regulation of kinase activity.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Catálise , Regulação para Baixo , Ativação Enzimática , Células-Tronco Hematopoéticas/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular , Cinética , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Dados de Sequência Molecular , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/química , Especificidade por SubstratoRESUMO
Recent studies have demonstrated that bacteria possess an essential protein translocation system similar to mammalian signal recognition particle (SRP). Here we have identified the Ffh, a homologue of the mammalian SRP54 subunit from S. pneumoniae. Ffh is a 58-kDa protein with three distinct domains: an N-terminal hydrophilic domain (N-domain), a G-domain containing GTP/GDP binding motifs, and a C-terminal methionine-rich domain (M-domain). The full-length Ffh and a truncated protein containing N and G domains (Ffh-NG) were overexpressed in E. coli and purified to homogeneity. The full-length Ffh has an intrinsic GTPase activity with k(cat) of 0.144 min(-1), and the K(m) for GTP is 10.9 microM. It is able to bind to 4.5S RNA specifically as demonstrated by gel retardation assay. The truncated Ffh-NG has approximately the same intrinsic GTPase activity to the full-length Ffh, but is unable to bind to 4.5S RNA, indicating that the NG domain is sufficient for supporting intrinsic GTP hydrolysis, and that the M domain is required for RNA binding. The interaction of S. pneumoniae Ffh with its receptor, FtsY, resulted in a 20-fold stimulation in GTP hydrolysis. The stimulation was further demonstrated to be independent of the 4.5S RNA. In addition, a similar GTPase stimulation is also observed between Ffh-NG and FtsY, suggesting that the NG domain is sufficient and the M domain is not required for GTPase stimulation between Ffh and FtsY.
Assuntos
Proteínas de Bactérias/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Streptococcus pneumoniae/enzimologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , GTP Fosfo-Hidrolases/genética , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA Bacteriano , RNA Ribossômico/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Alinhamento de Sequência , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/genética , Streptococcus pneumoniae/química , Streptococcus pneumoniae/genéticaRESUMO
Eukaryotic initiation factor 4A (eIF4A) is an ATP-dependent RNA helicase and is homologous to the non-structural protein 3 (NS3) helicase domain encoded by hepatitis C virus (HCV). Reported here is the comparative characterization of human eIF4A and HCV NS3 helicase in an effort to better understand viral and cellular helicases of superfamily II and to assist in designing specific inhibitors against HCV infections. Both eIF4A and HCV NS3 helicase domain were expressed in bacterial cells as histidine-tagged proteins and purified to homogeneity. Purified eIF4A exhibited RNA-unwinding activity and acted on RNA or RNA/DNA but not DNA duplexes. In the absence of cellular cofactors, eIF4A operated unwinding in both the 3' to 5' and 5' to 3' directions, and was able to unwind blunt-ended RNA duplex, suggesting that bidirectionality is an intrinsic property of eIF4A. In contrast, HCV NS3 helicase showed unidirectional 3' to 5' unwinding of RNA and RNA/DNA, as well as of DNA duplexes. With respect to NTPase activity, eIF4A hydrolysed only ATP or dATP in the presence of RNAs, whereas HCV NS3 helicase could hydrolyse all ribo- and deoxyribo-NTPs in an RNA-independent manner. In parallel, only ATP or dATP could drive the unwinding activity of eIF4A whereas HCV NS3 could function with all eight standard NTPs and dNTPs. The observed differences in their substrate specificity may prove to be useful in designing specific inhibitors targeting HCV NS3 helicase but not human eIF4A.
Assuntos
Trifosfato de Adenosina/metabolismo , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas não Estruturais Virais/metabolismo , Adenosina Trifosfatases/metabolismo , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Primers do DNA/metabolismo , DNA Complementar/metabolismo , Fator de Iniciação 4A em Eucariotos , Hepacivirus/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Reação em Cadeia da Polimerase , Ligação Proteica , Estrutura Terciária de Proteína , RNA/metabolismo , Especificidade por Substrato , Temperatura , Fatores de Tempo , Proteínas não Estruturais Virais/químicaRESUMO
The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, K(d), of approximately 22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 A. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data.