RESUMO
The Hippo signaling pathway acts as a brake on regeneration in many tissues. This cascade of kinases culminates in the phosphorylation of the transcriptional cofactors Yap and Taz, whose concentration in the nucleus consequently remains low. Various types of cellular signals can reduce phosphorylation, however, resulting in the accumulation of Yap and Taz in the nucleus and subsequently in mitosis. We earlier identified a small molecule, TRULI, that blocks the final kinases in the pathway, Lats1 and Lats2, and thus elicits proliferation of several cell types that are ordinarily postmitotic and aids regeneration in mammals. In the present study, we present the results of chemical modification of the original compound and demonstrate that a derivative, TDI-011536, is an effective blocker of Lats kinases in vitro at nanomolar concentrations. The compound fosters extensive proliferation in retinal organoids derived from human induced pluripotent stem cells. Intraperitoneal administration of the substance to mice suppresses Yap phosphorylation for several hours and induces transcriptional activation of Yap target genes in the heart, liver, and skin. Moreover, the compound initiates the proliferation of cardiomyocytes in adult mice following cardiac cryolesions. After further chemical refinement, related compounds might prove useful in protective and regenerative therapies.
Assuntos
Inibidores de Proteínas Quinases , Proteínas Serina-Treonina Quinases , Regeneração , Animais , Proliferação de Células/efeitos dos fármacos , Coração/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas , Regeneração Hepática/efeitos dos fármacos , Regeneração Hepática/genética , Regeneração Hepática/fisiologia , Camundongos , Organoides/fisiologia , Fosforilação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Regeneração/efeitos dos fármacos , Regeneração/genética , Retina/fisiologia , Fenômenos Fisiológicos da Pele/efeitos dos fármacos , Fenômenos Fisiológicos da Pele/genética , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Proteínas de Sinalização YAP/metabolismoRESUMO
PTC725 is a small molecule NS4B-targeting inhibitor of hepatitis C virus (HCV) genotype (gt) 1 RNA replication that lacks activity against HCV gt2. We analyzed the Los Alamos HCV sequence database to predict susceptible/resistant HCV gt's according to the prevalence of known resistance-conferring amino acids in the NS4B protein. Our analysis predicted that HCV gt3 would be highly susceptible to the activity of PTC725. Indeed, PTC725 was shown to be active against a gt3 subgenomic replicon with a 50% effective concentration of â¼5 nM. De novo resistance selection identified mutations encoding amino acid substitutions mapping to the first predicted transmembrane region of NS4B, a finding consistent with results for PTC725 and other NS4B-targeting compounds against HCV gt1. This is the first report of the activity of an NS4B targeting compound against HCV gt3. In addition, we have identified previously unreported amino acid substitutions selected by PTC725 treatment which further demonstrate that these compounds target the NS4B first transmembrane region.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Indóis/farmacologia , Sulfonamidas/farmacologia , Proteínas não Estruturais Virais/genética , Substituição de Aminoácidos , Linhagem Celular Tumoral , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Genoma Viral , Genótipo , Humanos , Mutação , Replicon/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismoRESUMO
A novel series of 2-(4-sulfonamidophenyl)-indole 3-carboxamides was identified and optimized for activity against the HCV genotype 1b replicon resulting in compounds with potent and selective activity. Further evaluation of this series demonstrated potent activity across HCV genotypes 1a, 2a and 3a. Compound 4z had reduced activity against HCV genotype 1b replicons containing single mutations in the NS4B coding sequence (F98C and V105M) indicating that NS4B is the target. This novel series of 2-(4-sulfonamidophenyl)-indole 3-carboxamides serves as a promising starting point for a pan-genotype HCV discovery program.
Assuntos
Antivirais/química , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Indóis/química , Indóis/farmacologia , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Hepacivirus/química , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C/tratamento farmacológico , Humanos , Dados de Sequência Molecular , Mutação , Replicon/efeitos dos fármacos , Sulfonamidas/química , Sulfonamidas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
In the search for a second generation HCV protease inhibitor, molecular modeling studies of the X-ray crystal structure of Boceprevir1 bound to the NS3 protein suggest that expansion into the S4 pocket could provide additional hydrophobic Van der Waals interactions. Effective replacement of the P4 tert-butyl with a cyclohexylmethyl ligand led to inhibitor 2 with improved enzyme and replicon activities. Subsequent modeling and SAR studies led to the pyridine 38 and sulfone analogues 52 and 53 with vastly improved PK parameters in monkeys, forming a new foundation for further exploration.
Assuntos
Antivirais/química , Prolina/análogos & derivados , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Área Sob a Curva , Disponibilidade Biológica , Cristalografia por Raios X , Haplorrinos , Modelos Moleculares , Prolina/química , Prolina/farmacocinética , Prolina/farmacologia , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/farmacologia , Ratos , Relação Estrutura-AtividadeRESUMO
Hepatitis C Virus (HCV) infection is the major cause of chronic liver disease, leading to cirrhosis and hepatocellular carcinoma, which affects more than 200 million people worldwide. Currently the only therapeutic regimens are subcutaneous interferon-alpha or PEG-interferon alone or in combination with oral ribavirin. Although combination therapy is reasonably successful with the majority of genotypes, its efficacy against the predominant genotype (genotype 1) is moderate at best, with only approximately 50% of the patients showing sustained virological response. We recently disclosed the discovery of Boceprevir, SCH 503034 (1), which is a novel, potent, selective, orally bioavailable NS3 protease inhibitor that has been shown to be efficacious in humans and is currently undergoing clinical trials. As second generation compounds, we have further explored various novel structures with the aim of improving enzyme and cellular binding activities of 1. Herein, we disclose our efforts toward the identification of a novel P(3) sulfonamide-capped inhibitor that demonstrated improved binding and cellular activity compared to 1. X-ray structure of one of these inhibitors bound to the enzyme revealed a hydrogen bond of the P(3) sulfonamide group to Cys-159 which resulted in improved binding and cellular potency.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Sulfonamidas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Animais , Antivirais/química , Antivirais/farmacocinética , Cristalografia por Raios X , Descoberta de Drogas , Humanos , Modelos Moleculares , Ligação Proteica , Ratos , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacocinética , Replicação Viral/efeitos dos fármacosRESUMO
HCV infection is considered a silent epidemic because most people infected do not develop acute symptoms. Instead, the disease progresses to a chronic state leading to cirrhosis and hepatocarcinoma. Novel therapies are needed to combat this major health threat. The HCV NS3 serine protease has been the target of continuous investigation because of its pivotal role in viral replication. Herein, we present the P1-P3 macrocyclization approach followed for identification of HCV NS3 inhibitors as potential backup candidates to our first generation drug candidate, Sch 503034 (1). Different P1-P3 linkers were investigated to identify novel macrocyclic scaffolds. SAR exploration of P3-caps in the macrocyclic cores allowed the identification of l-serine derived macrocycle 32 (Ki* = 3 nM, EC90 = 30 nM) and allo-threonine derived macrocycle 36 (Ki* = 3 nM, EC90 = 30 nM) as potent HCV NS3 protease inhibitors.
Assuntos
Compostos Macrocíclicos/síntese química , Inibidores de Serina Proteinase/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/síntese química , Antivirais/farmacologia , Desenho de Fármacos , Compostos Macrocíclicos/farmacologia , Inibidores de Serina Proteinase/farmacologiaRESUMO
Hepatitis C virus (HCV) infection is the major cause of chronic liver disease, leading to cirrhosis and hepatocellular carcinoma, and affects more than 200 million people worldwide. Although combination therapy of interferon-alpha and ribavirin is reasonably successful in treating majority of genotypes, its efficacy against the predominant genotype (genotype 1) is moderate at best, with only about 40% of the patients showing sustained virological response. Herein, the SAR leading to the discovery of a series of ketoamide derived P(1)-P(3) macrocyclic inhibitors that are more potent than the first generation clinical candidate, boceprevir (1, Sch 503034), is discussed. The optimization of these macrocyclic inhibitors identified a P(3) imide capped analogue 52 that was 20 times more potent than 1 and demonstrated good oral pharmacokinetics in rats. X-ray structure of 52 bound to NS3 protease and biological data are also discussed.
Assuntos
Amidas/farmacologia , Descoberta de Drogas , Compostos Macrocíclicos/farmacologia , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Amidas/química , Cristalografia por Raios X , Ligação de Hidrogênio , Compostos Macrocíclicos/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Inibidores de Proteases/química , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
The structures of both native and S139A holo-HCV NS3/4A protease domain were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contributions to the binding energy arise from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease, which is currently in clinical trials.
Assuntos
Hepacivirus/enzimologia , Prolina/análogos & derivados , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Modelos Moleculares , Estrutura Molecular , Prolina/químicaRESUMO
Parallel artificial membrane permeability assay (PAMPA) and Caco-2 cells have been frequently used for the evaluation of in vitro permeability of new chemical entities. In this study we evaluated the correlation between permeability, assessed by both methods, and the cellular potency of 34 novel hepatitis C virus (HCV) protease inhibitors. Two types of assays were used to determine the potency of HCV protease inhibitors: a cell-free assay that evaluates the intrinsic affinity (K(i)) between the protease and the inhibitor and a cell-based replicon assay that determines the inhibitors' IC90. When the K(i)/IC90 ratios were compared with the PAMPA permeability and the Caco-2 permeability by linear regression analysis, a reasonable correlation was found between the K(i)/IC90 ratio and PAMPA permeability (r2=0.76) but not with Caco-2 permeability (r2=0.29). Correlations were also assessed between K(i)/IC90 ratios and the following physico-chemical properties: logP (r2=0.41), logD (r2=0.58), clogP (r2=0.13), and mlogP (r2=0.30). These results suggest that passive permeability may play a role in the uptake and cellular activity of these HCV protease inhibitors, and that PAMPA was more predictive of cellular activity than physico-chemical properties or Caco-2 permeability.
Assuntos
Permeabilidade da Membrana Celular , Membranas Artificiais , Inibidores de Proteases/metabolismo , Células CACO-2 , Difusão , Hepacivirus , Humanos , Proteínas Recombinantes/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
Starting from a pentapeptide Hepatitis C virus NS3 protease inhibitor, a number of alpha-ketoamide inhibitors based on novel dichlorocyclopropylproline P2 core were synthesized and investigated for their HCV NS3 serine protease activity. The key intermediate 3,4-dichlorocyclopropylproline was obtained through a dichloro carbene insertion to 3,4-dehydroproline. The size of the molecules was reduced significantly through a series of truncations of the initial pentapeptide. By varying P1 side chain in length and size, potency and selectivity were improved. A variety of aliphatic carbamate and urea capping groups were examined. In general, compounds with urea cappings were more potent and selective than their carbamate counterparts. The most potent compound was a tert-butyl urea analog. Variations at P3 position were also investigated. Among the three residues incorporated, tert-leucine was clearly superior, leading to compounds that had excellent enzyme potency and selectivity. The most potent compound achieved cell-based replicon assay EC50 of 40 nM. The most promising compound of all had excellent potency in both enzyme (Ki* = 9 nM) and replicon assays (EC50 = 100 nM). Its bioavailabilities were above 10% in all three animal species (rats, monkeys, and dogs). It has provided a lead for future investigations.
Assuntos
Hepacivirus/efeitos dos fármacos , Prolina/análogos & derivados , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacocinética , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Disponibilidade Biológica , Haplorrinos , Prolina/farmacologia , Ratos , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-Atividade , Ureia/análogos & derivadosRESUMO
DNA ligase catalyzes the closure of single-strand nicks in double-stranded DNA that arise during replication and recombination. Inhibition of bacterial ligase is expected to cause chromosome degradation and cell death, making it an attractive target for new antibacterials. The prototypical bacterial ligase couples the hydrolysis of NAD(+) to phosphodiester bond formation between an adjacent 3'OH and 5'-terminal phosphate of nicked duplex DNA. The first step is the reversible formation of a ligase-adenylate from the reaction between apoenzyme and NAD(+). Inhibitors that compete with NAD(+) are expected to be bacterial specific because eukaryotic DNA ligases use ATP and differ in the sequence composition of their adenylation domain. We report here a high-throughput assay that measures the adenylation reaction specifically by monitoring ligase-AMP formation via scintillation proximity technologies. Escherichia coli DNA ligase was biotinylated in vivo; after reaction with radiolabeled NAD(+), ligase-[(3)H]AMP could be captured onto the streptavidin-coated surface of the solid scintillant. The method was ideal for high-throughput screening because it required minimal manipulations and generated a robust signal with minimal scatter. Certain adenosine analogs were found to inhibit the adenylation assay and had similar potency of inhibition in a DNA ligation assay.
Assuntos
Técnicas de Química Analítica/métodos , DNA Ligases/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Monofosfato de Adenosina/biossíntese , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Biotina , DNA/genética , DNA/metabolismo , DNA Ligases/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Cinética , Ligantes , NAD/metabolismo , Reprodutibilidade dos Testes , Contagem de Cintilação , EstreptavidinaRESUMO
The structures of both the native holo-HCV NS3/4A protease domain and the protease domain with a serine 139 to alanine (S139A) mutation were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contribution to the binding energy arises from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets [the numbering of the subsites is as defined in Berger, A.; Schechter, I. Philos. Trans. R. Soc. London, Ser. B 1970, 257, 249-264]. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease that is currently in clinical trials.
Assuntos
Antivirais/síntese química , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/química , Hepacivirus/enzimologia , Prolina/análogos & derivados , Inibidores de Serina Proteinase/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Antivirais/química , Sítios de Ligação , Cristalografia por Raios X , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Moleculares , Prolina/síntese química , Prolina/química , Conformação Proteica , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Current hepatitis C virus (HCV) therapies may cure approximately 60% of infections. They are often contraindicated or poorly tolerated, underscoring the need for safer and more effective drugs. A novel, alpha-ketoamide-derived, substrate-based inhibitor of the HCV serine protease (SCH446211) was developed. Compared with earlier reported inhibitors of similar chemical class, it has a P1'-P2' extension which provides extended interaction with the protease active site. The aim of this study was to evaluate the in vitro antiviral activity of SCH446211. METHODS: Binding constant of SCH446211 to HCV NS3 protease was measured with the chromogenic substrate in vitro cleavage assay. Cell-based activity of SCH446211 was evaluated in replicon cells, which are Huh-7 hepatoma cells stably transfected with a subgenomic HCV RNA as reported previously. After 72 h of incubation with SCH446211, viral transcription and protein expression were measured by real-time RT-PCR (TaqMan), quantitative in situ hybridization, immunoblot and indirect immunofluorescence. RESULTS: The binding constant of SCH446211 to HCV NS3 protease was 3.8 +/- 0.4 nM. HCV replication and protein expression were inhibited by SCH446211 in replicon cells as consistently shown by four techniques. In particular, based on quantitative real-time RT-PCR measurements, the IC50 and IC90 of SCH446211 were estimated to be 40 +/- 20 and 100 +/- 20 nM (n = 17), respectively. Long-term culture of replicon cells with SCH446211 reduced replicon RNA to <0.1 copy per cell. SCH446211 did not show cellular toxicity at concentrations up to 50 microM. CONCLUSIONS: SCH446211 is a potent inhibitor of HCV protease in vitro. Its extended interaction with the HCV NS3 protease active site is associated with potent in vitro antiviral activity. This observation is potentially a useful guide for development of future potent inhibitors against HCV NS3 protease.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Oligopeptídeos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Hepacivirus/genética , RNA Viral/análise , RepliconRESUMO
Hepatitis C virus (HCV) infection is the major cause of chronic liver disease, leading to cirrhosis and hepatocellular carcinoma, which affects more than 170 million people worldwide. Currently the only therapeutic regimens are subcutaneous interferon-alpha or polyethylene glycol (PEG)-interferon-alpha alone or in combination with oral ribavirin. Although combination therapy is reasonably successful with the majority of genotypes, its efficacy against the predominant genotype (genotype 1) is moderate at best, with only about 40% of the patients showing sustained virological response. Herein, the SAR leading to the discovery of 70 (SCH 503034), a novel, potent, selective, orally bioavailable NS3 protease inhibitor that has been advanced to clinical trials in human beings for the treatment of hepatitis C viral infections is described. X-ray structure of inhibitor 70 complexed with the NS3 protease and biological data are also discussed.
Assuntos
Antivirais/síntese química , Hepacivirus/enzimologia , Prolina/análogos & derivados , Proteínas não Estruturais Virais/antagonistas & inibidores , Administração Oral , Animais , Antivirais/química , Antivirais/farmacocinética , Área Sob a Curva , Sítios de Ligação , Disponibilidade Biológica , Cristalografia por Raios X , Cães , Haplorrinos , Estrutura Molecular , Prolina/síntese química , Prolina/química , Prolina/farmacocinética , Ratos , Relação Estrutura-Atividade , Distribuição Tecidual , Proteínas não Estruturais Virais/químicaRESUMO
Synthesis and HCV NS3 serine protease inhibitory activity of 4-hydroxyproline derived macrocyclic inhibitors and SAR around this macrocyclic core is described in this communication. X-ray structure of inhibitor 38 bound to the protease is discussed.
Assuntos
Hepatite C/enzimologia , Compostos Macrocíclicos/farmacologia , Inibidores de Serina Proteinase/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Difração de Raios XRESUMO
Introduction of various modified prolines at P(2) and optimization of the P(1) side chain led to the discovery of SCH6 (24, Table 2), a potent ketoamide inhibitor of the HCV NS3 serine protease. In addition to excellent enzyme potency (K(i)*= 3.8 nM), 24 was also found to be a potent inhibitor of HCV subgenomic RNA replication with IC(50) and IC(90) of 40 and 100 nM, respectively. Recently, antiviral activity of 24 was demonstrated with inhibition of the full-length genotype 2a HCV genome. In addition, 24 was found to restore the responsiveness of the interferon regulatory factor 3 (IRF-3) in cells containing HCV RNA replicons.
Assuntos
Amidas/química , Amidas/farmacologia , Genoma Viral/genética , Hepacivirus/efeitos dos fármacos , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Serina Endopeptidases/metabolismo , Animais , Haplorrinos , Hepacivirus/enzimologia , Hepacivirus/genética , Modelos Moleculares , Estrutura Molecular , RNA Viral/genética , Ratos , Serina Endopeptidases/química , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismoRESUMO
The hepatitis C virus (HCV) NS3 protease is essential for viral replication. It has been a target of choice for intensive drug discovery research. On the basis of an active pentapeptide inhibitor, 1, we envisioned that macrocyclization from the P2 proline to P3 capping could enhance binding to the backbone Ala156 residue and the S4 pocket. Thus, a number of P2 proline-based macrocyclic alpha-ketoamide inhibitors were prepared and investigated in an HCV NS3 serine protease continuous assay (K(i*)). The biological activity varied substantially depending on factors such as the ring size, number of amino acid residues, number of methyl substituents, type of heteroatom in the linker, P3 residue, and configuration at the proline C-4 center. The pentapeptide inhibitors were very potent, with the C-terminal acids and amides being the most active ones (24, K(i*) = 8 nM). The tetrapeptides and tripeptides were less potent. Sixteen- and seventeen-membered macrocyclic compounds were equally potent, while fifteen-membered analogues were slightly less active. gem-Dimethyl substituents at the linker improved the potency of all inhibitors (the best compound was 45, K(i*) = 6 nM). The combination of tert-leucine at P3 and dimethyl substituents at the linker in compound 47 realized a selectivity of 307 against human neutrophil elastase. Compound 45 had an IC(50) of 130 nM in a cellular replicon assay, while IC(50) for 24 was 400 nM. Several compounds had excellent subcutaneous AUC and bioavailability in rats. Although tripeptide compound 40 was 97% orally bioavailable, larger pentapeptides generally had low oral bioavailability. The X-ray crystal structure of compounds 24 and 45 bound to the protease demonstrated the close interaction of the macrocycle with the Ala156 methyl group and S4 pocket. The strategy of macrocyclization has been proved to be successful in improving potency (>20-fold greater than that of 1) and in structural depeptization.
Assuntos
Antivirais/síntese química , Compostos Macrocíclicos/síntese química , Prolina/análogos & derivados , Prolina/síntese química , Inibidores de Serina Proteinase/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/farmacologia , Disponibilidade Biológica , Cristalografia por Raios X , Ciclização , Hepacivirus/efeitos dos fármacos , Humanos , Elastase de Leucócito/antagonistas & inibidores , Compostos Macrocíclicos/farmacologia , Modelos Moleculares , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Prolina/farmacologia , Serina Endopeptidases/química , Inibidores de Serina Proteinase/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/química , Replicação ViralRESUMO
The NS3 protease of hepatitis C virus (HCV) has emerged as one of the best characterized targets for next-generation HCV therapy. The tetrapeptide 1 and pentapeptide 2 are alpha-ketoamide-type HCV serine protease inhibitors with modest potency. We envisioned that the 1,2,3,4-tetrahydroisoquinoline-3-carboxylamide (Tic) moiety could be cyclized to the P3 capping group. The resulting macrocycle could enhance the binding through its extra contact with the Ala156 methyl group. Macrocyclization could also provide a less peptidic HCV inhibitor. Synthesis started from dipeptide 5, which was obtained via a coupling of two amino acid derivatives. The N-terminal was capped as hept-6-enoylamide to give 6. Hydroboration of the double bond afforded alcohol 7, the precursor to the macrocycle 8. The macrocyclization was achieved under Mitsunobu conditions (PPh(3), ADDP). The macrocyclic acid 9 was then combined with appropriate right-hand fragments 12, 14, or 16, which was prepared from common intermediate 11. Finally, oxidation of alpha-hydroxyamide provided target molecule alpha-ketoamides 17, 18, and 21. The C-terminal esters were then elaborated to carboxylic acids 19 and 20, and amides 20 and 23. The inhibitors 17-23 were tested in HCV NS3 protease continuous assay. Tripeptide 17 was more potent than the larger acyclic tetrapeptide 1. The tetrapeptides 18-20 were as active as 17. Most significantly, the pentapeptides (21-23) were much better inhibitors (K(i) = 0.015-0.26 microM). The carboxylic acid (22) and amide (23) were 57-80 times more potent than the acyclic analogue 2. The X-ray crystal structure of compound 23 bound to the protease revealed that the macrocycle adopted a donutlike conformation and had close contact with the Ala156 methyl group. The ketone carbonyl formed a reversible covalent bond with Ser139. The n-propyl of P1 novaline and the aromatic ring of P2' phenylglycine formed a C-shaped clamp around the Lys136 side chain.
Assuntos
Ácidos Carboxílicos/síntese química , Compostos Macrocíclicos/síntese química , Tetra-Hidroisoquinolinas/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Compostos Aza/síntese química , Sítios de Ligação , Ácidos Carboxílicos/química , Cristalografia por Raios X , Compostos Macrocíclicos/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Tetra-Hidroisoquinolinas/química , Proteínas não Estruturais Virais/químicaRESUMO
Prolonged hepatitis C infection is the leading cause for cirrhosis of the liver and hepatocellular carcinoma. The etiological agent HCV virus codes a single polyprotein of approximately 3000 amino acids that is processed with the help of a serine protease NS3A to produce structural and non-structural proteins required for viral replication. Inhibition of NS3 protease can potentially be used to develop drugs for treatment of HCV infections. Herein, we report the development of a series of novel NS3 serine protease inhibitors derived from 2-aza-bicyclo[2.2.1]-heptane carboxylic acid with potential therapeutic use for treatment of HCV infections.
Assuntos
Compostos Bicíclicos com Pontes , Hepacivirus/efeitos dos fármacos , Replicon/efeitos dos fármacos , Inibidores de Serina Proteinase , Proteínas não Estruturais Virais/antagonistas & inibidores , Sítios de Ligação , Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/química , Compostos Bicíclicos com Pontes/farmacologia , Hepacivirus/química , Hepacivirus/enzimologia , Estrutura Molecular , Ligação Proteica , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/química , Difração de Raios XRESUMO
Depeptidization efforts of the P(3)-P(2) region of P(3) capped alpha-ketoamide inhibitor of HCV NS3 serine protease 1 are reported. We clearly established that N-methylation of the P(2) nitrogen and modification of the P(2)' carboxylic acid terminus were essential for activity in the replicon assay.