RESUMO
This paper describes an intermolecular cross-selective [2 + 2] photocycloaddition reaction of exocyclic arylidene oxetanes, azetidines, and cyclobutanes with simple electron-deficient alkenes. The reaction takes place under mild conditions using a commercially available Ir(III) photosensitizer upon blue light irradiation. This transformation provides access to a range of polysubstituted 2-oxaspiro[3.3]heptane, 2-azaspiro[3.3]heptane, and spiro[3.3]heptane motifs, which are of prime interest in medicinal chemistry as gem-dimethyl and carbonyl bioisosteres. A variety of further transformations of the initial cycloadducts are demonstrated to highlight the versatility of the products and enable selective access to either of a syn- or an anti-diastereoisomer through kinetic or thermodynamic epimerization, respectively. Mechanistic experiments and DFT calculations suggest that this reaction proceeds through a sensitized energy transfer pathway.
RESUMO
Intermolecular C-C bond-forming reactions are underdeveloped transformations in the field of biocatalysis. Here we report a photoenzymatic intermolecular hydroalkylation of olefins catalyzed by flavin-dependent 'ene'-reductases. Radical initiation occurs via photoexcitation of a rare high-order enzyme-templated charge-transfer complex that forms between an alkene, α-chloroamide, and flavin hydroquinone. This unique mechanism ensures that radical formation only occurs when both substrates are present within the protein active site. This active site can control the radical terminating hydrogen atom transfer, enabling the synthesis of enantioenriched γ-stereogenic amides. This work highlights the potential for photoenzymatic catalysis to enable new biocatalytic transformations via previously unknown electron transfer mechanisms.
Assuntos
Alcenos/química , Amidas/síntese química , Flavoproteínas/química , Oxirredutases/química , Alquilação/efeitos da radiação , Biocatálise/efeitos da radiação , Domínio Catalítico , Dinitrocresóis/química , Dinitrocresóis/efeitos da radiação , Flavoproteínas/efeitos da radiação , Luz , Modelos Químicos , Oxirredutases/efeitos da radiaçãoRESUMO
Targeted protein degradation as a therapeutic modality has seen dramatic progress and massive investment in recent years because of the convergence of two key scientific breakthroughs: optimization of first-generation peptidic proteolysis-targeted chimeras (PROTACs) into more drug-like molecules able to support in vivo proof of concept and the discovery that clinical molecules function as degraders by binding and repurposing the proteins cereblon and DCAF15. This provided clinical validation for the general approach through the cereblon modulator class of drugs and provided highly drug-like and ligand-efficient E3 ligase binders upon which to tether target-binding moieties. Increasingly rational and systematic approaches including biophysical and structural studies on ternary complexes are being leveraged as the field advances. In this Perspective we summarize the discoveries that have laid the foundation for future degradation therapeutics, focusing on those classes of small molecules that redirect E3 ubiquitin ligases to non-native substrates.
Assuntos
Proteólise/efeitos dos fármacos , Sítios de Ligação , Humanos , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Bibliotecas de Moléculas Pequenas/química , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Targeted protein degradation via small-molecule modulation of cereblon offers vast potential for the development of new therapeutics. Cereblon-binding therapeutics carry the safety risks of thalidomide, which caused an epidemic of severe birth defects characterized by forelimb shortening or phocomelia. Here we show that thalidomide is not teratogenic in transgenic mice expressing human cereblon, indicating that binding to cereblon is not sufficient to cause birth defects. Instead, we identify SALL4 as a thalidomide-dependent cereblon neosubstrate. Human mutations in SALL4 cause Duane-radial ray, IVIC, and acro-renal-ocular syndromes with overlapping clinical presentations to thalidomide embryopathy, including phocomelia. SALL4 is degraded in rabbits but not in resistant organisms such as mice because of SALL4 sequence variations. This work expands the scope of cereblon neosubstrate activity within the formerly 'undruggable' C2H2 zinc finger family and offers a path toward safer therapeutics through an improved understanding of the molecular basis of thalidomide-induced teratogenicity.
Assuntos
Regulação da Expressão Gênica , Peptídeo Hidrolases/química , Teratogênicos/química , Talidomida/química , Fatores de Transcrição/química , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Homozigoto , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas , Ligantes , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Peptídeo Hidrolases/genética , Proteólise , Coelhos , Testículo/metabolismo , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/metabolismo , Dedos de ZincoRESUMO
Myeloperoxidase (MPO) activity and subsequent generation of hypochlorous acid has been associated with the killing of host-invading microorganisms (e.g. bacteria, viruses, and fungi). However, during oxidative stress, high MPO activity can damage host tissue and is linked to several chronic inflammatory conditions. Herein, we describe the development of a novel biaryl, indole-pyrazole series of irreversible mechanism-based inhibitors of MPO. Derived from an indole-containing high-throughput screen hit, optimization efforts resulted in potent and selective 6-substituted indoles with good oral bioavailability and in vivo activity.
Assuntos
Inibidores Enzimáticos/metabolismo , Indóis/metabolismo , Peroxidase/metabolismo , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Meia-Vida , Indóis/química , Indóis/farmacocinética , Indóis/uso terapêutico , Camundongos , Peritonite/tratamento farmacológico , Peritonite/patologia , Peroxidase/antagonistas & inibidores , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacocinética , Relação Estrutura-AtividadeRESUMO
Myeloperoxidase (MPO) is a leukocyte-derived redox enzyme that has been linked to oxidative stress and damage in many inflammatory states, including cardiovascular disease. We have discovered aminopyridines that are potent mechanism-based inhibitors of MPO, with significant selectivity over the closely related thyroid peroxidase. 1-((6-Aminopyridin-3-yl)methyl)-3-(4-bromophenyl)urea (Aminopyridine 2) inhibited MPO in human plasma and blocked MPO-dependent vasomotor dysfunction ex vivo in rat aortic rings. Aminopyridine 2 also showed high oral bioavailability and inhibited MPO activity in vivo in a mouse model of peritonitis. Aminopyridine 2 could effectively be administered as a food admixture, making it an important tool for assessing the relative importance of MPO in preclinical models of chronic inflammatory disease.
Assuntos
Aminopiridinas/farmacologia , Inibidores Enzimáticos/farmacologia , Peroxidase/antagonistas & inibidores , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Disponibilidade Biológica , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-DawleyRESUMO
Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5' splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule-mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases.
Assuntos
Processamento Alternativo , Atrofia Muscular Espinal/tratamento farmacológico , RNA de Cadeia Dupla/agonistas , Ribonucleoproteína Nuclear Pequena U1/agonistas , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo , Animais , Sítios de Ligação , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/mortalidade , Atrofia Muscular Espinal/patologia , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteólise , Precursores de RNA/agonistas , Precursores de RNA/química , Precursores de RNA/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , Ribonucleoproteína Nuclear Pequena U1/química , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/metabolismo , Análise de Sobrevida , Proteína 2 de Sobrevivência do Neurônio Motor/química , Proteína 2 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Daclatasvir is a first-in-class, potent, and selective inhibitor of the hepatitis C virus nonstructural protein 5A replication complex. In support of nonclinical studies during discovery and exploratory development, liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance were used in connection with synthetic and radiosynthetic approaches to investigate the biotransformation of daclatasvir in vitro and in cynomolgus monkeys, dogs, mice, and rats. The results of these studies indicated that disposition of daclatasvir was accomplished mainly by the release of unchanged daclatasvir into bile and feces and, secondarily, by oxidative metabolism. Cytochrome P450s were the main enzymes involved in the metabolism of daclatasvir. Oxidative pathways included δ-oxidation of the pyrrolidine moiety, resulting in ring opening to an aminoaldehyde intermediate followed by an intramolecular reaction between the aldehyde and the proximal imidazole nitrogen atom. Despite robust formation of the resulting metabolite in multiple systems, rates of covalent binding to protein associated with metabolism of daclatasvir were modest (55.2-67.8 pmol/mg/h) in nicotinamide adenine dinucleotide phosphate (reduced form)-supplemented liver microsomes (human, monkey, rat), suggesting that intramolecular rearrangement was favored over intermolecular binding in the formation of this metabolite. This biotransformation profile supported the continued development of daclatasvir, which is now marketed for the treatment of chronic hepatitis C virus infection.
Assuntos
Biotransformação/fisiologia , Imidazóis/metabolismo , Pirrolidinas/metabolismo , Animais , Bile/metabolismo , Carbamatos , Cromatografia Líquida de Alta Pressão/métodos , Sistema Enzimático do Citocromo P-450/metabolismo , Cães , Haplorrinos , Hepatócitos/metabolismo , Humanos , Macaca fascicularis , Espectroscopia de Ressonância Magnética/métodos , Masculino , Espectrometria de Massas/métodos , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/metabolismo , Oxirredução , Ratos , Ratos Sprague-Dawley , Valina/análogos & derivadosRESUMO
6,6-Fused ring systems including tetrahydroisoquinolines and tetrahydropyrido[3,4-d]pyrimidines have been explored as possible replacements for the piperazine benzamide portion of the HIV-1 attachment inhibitor BMS-663068. In initial studies, the tetrahydroisoquinoline compounds demonstrate sub-nanomolar activity in a HIV-1 pseudotype viral infection assay used as the initial screen for inhibitory activity. Analysis of SARs and approaches to optimization for an improved drug-like profile are examined herein.
Assuntos
Compostos Aza/química , Benzamidas/química , Descoberta de Drogas , Inibidores da Fusão de HIV/química , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , Indóis/química , Piperazinas/química , Tetra-Hidroisoquinolinas/farmacologia , Ligação Viral/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , Proteína gp120 do Envelope de HIV/genética , Inibidores da Fusão de HIV/síntese química , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Células HeLa , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Tetra-Hidroisoquinolinas/síntese química , Tetra-Hidroisoquinolinas/química , Replicação Viral/efeitos dos fármacosRESUMO
The worldwide prevalence of chronic hepatitis C virus (HCV) infection is estimated to be approaching 200 million people. Current therapy relies upon a combination of pegylated interferon-alpha and ribavirin, a poorly tolerated regimen typically associated with less than 50% sustained virological response rate in those infected with genotype 1 virus. The development of direct-acting antiviral agents to treat HCV has focused predominantly on inhibitors of the viral enzymes NS3 protease and the RNA-dependent RNA polymerase NS5B. Here we describe the profile of BMS-790052, a small molecule inhibitor of the HCV NS5A protein that exhibits picomolar half-maximum effective concentrations (EC(50)) towards replicons expressing a broad range of HCV genotypes and the JFH-1 genotype 2a infectious virus in cell culture. In a phase I clinical trial in patients chronically infected with HCV, administration of a single 100-mg dose of BMS-790052 was associated with a 3.3 log(10) reduction in mean viral load measured 24 h post-dose that was sustained for an additional 120 h in two patients infected with genotype 1b virus. Genotypic analysis of samples taken at baseline, 24 and 144 h post-dose revealed that the major HCV variants observed had substitutions at amino-acid positions identified using the in vitro replicon system. These results provide the first clinical validation of an inhibitor of HCV NS5A, a protein with no known enzymatic function, as an approach to the suppression of virus replication that offers potential as part of a therapeutic regimen based on combinations of HCV inhibitors.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Imidazóis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Adolescente , Adulto , Animais , Antivirais/sangue , Antivirais/química , Antivirais/uso terapêutico , Carbamatos , Linhagem Celular , Chlorocebus aethiops , Farmacorresistência Viral , Feminino , Genótipo , Células HeLa , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Imidazóis/sangue , Imidazóis/química , Concentração Inibidora 50 , Masculino , Pessoa de Meia-Idade , Pirrolidinas , Fatores de Tempo , Valina/análogos & derivados , Células Vero , Carga Viral/efeitos dos fármacos , Adulto JovemRESUMO
Can classical and modern chemical C-H oxidation reactions complement biotransformation in the synthesis of drug metabolites? We have surveyed the literature in an effort to try to answer this important question of major practical significance in the pharmaceutical industry. Drug metabolites are required throughout all phases of the drug discovery and development process; however, their synthesis is still an unsolved problem. This Review, not intended to be comprehensive or historical, highlights relevant applications of chemical C-H oxidation reactions, electrochemistry and microfluidic technologies to drug templates in order to access drug metabolites, and also highlights promising reactions to this end. Where possible or appropriate, the contrast with biotransformation is drawn. In doing so, we have tried to identify gaps where they exist in the hope to spur further activity in this very important research area.
Assuntos
Preparações Farmacêuticas/química , Elementos de Transição/química , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Carbono/química , Catálise , Técnicas Eletroquímicas , Hidrogênio/química , Técnicas Analíticas Microfluídicas , Oxirredução , Preparações Farmacêuticas/metabolismoRESUMO
A previous disclosure from this lab highlighted the discovery of pyridyl amides as potent 11ß-HSD1 inhibitors. In order to build additional novelty and polarity into this chemotype, replacement of the hydrogen-bonding carbonyl (CO) pharmacophore with the bioisosteric sulfonyl (SO2) group was examined. Despite initial comparisons suggesting the corresponding sulfonamides exhibited weaker activity versus their carbonyl counterparts, further optimization was performed in an effort to identify various potent and unique leads for the program. Judicious incorporation of polar moieties resulted in the identification of compounds with enhanced potency and lipophilicity profiles, resulting in leads with superior aqueous solubility and liver microsomal stability.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Inibidores Enzimáticos/química , Doenças Metabólicas/tratamento farmacológico , Sulfonamidas/química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/uso terapêutico , Humanos , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Sulfonamidas/metabolismo , Sulfonamidas/uso terapêuticoRESUMO
Synthesis and SAR of 2-alkyloxazoles as class III phosphatidylinositol-4-kinase beta (PI4KIIIß) inhibitors is described. These compounds demonstrate that inhibition of PI4KIIIß leads to potent inhibition of HCV replication as observed in genotype (GT) 1a and 1b replicon and GT2a JFH1 virus assays in vitro.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Oxazóis/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Replicação Viral/efeitos dos fármacos , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Hepacivirus/enzimologia , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Oxazóis/síntese química , Oxazóis/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
Synthesis and structure-activity relationship of a series of substituted piperidinyl glycine 2-cyano-4,5-methano pyrroline DPP-IV inhibitors are described. Improvement of the inhibitory activity and chemical stability of this series of compounds was respectively achieved by the introduction of bulky groups at the 4-position and 1-position of the piperidinyl glycine, leading to a series of potent and stable DPP-IV inhibitors.
Assuntos
Dipeptidil Peptidase 4/química , Inibidores da Dipeptidil Peptidase IV/química , Piperidinas/química , Pirrolidinas/química , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/síntese química , Inibidores da Dipeptidil Peptidase IV/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ligação Proteica , Pirrolidinas/síntese química , Pirrolidinas/metabolismo , Relação Estrutura-Atividade , TemperaturaRESUMO
Myeloperoxidase (MPO) is a heme-containing peroxidase from phagocytic cells, which plays an important role in the innate immune response. The primary anti-microbial function of MPO is achieved by catalyzing the oxidation of halides by hydrogen peroxide (H2O2). Upon activation of phagocytes, MPO activity is detectable in both phagosomes and extracellularly, where it can remain or transcytose into interstitial compartments. Activated MPO leads to oxidative stress and tissue damage in many inflammatory states, including cardiovascular disease. Starting from a low molecular weight (LMW) high throughput screening (HTS) hit, here we report the discovery of a novel pyrrolidinone indole (IN-4) as a highly potent MPO inhibitor. This compound displays similar in vitro potency across peroxidation, plasma and NETosis assays. In a dilution/dialysis study, <5% of the original MPO activity was detected post-incubation of MPO with IN-4, suggesting irreversible enzyme inhibition. A fast MPO inactivation rate (kinact/Ki) and low partition ratio (k3/k4) make IN-4 kinetic properties attractive for an MPO inhibitor. This compound also displays significant selectivity over the closely related thyroid peroxidase (TPO), and is selective for extracellular MPO over intracellular (neutrophil) MPO. Moreover, IN-4 shows good exposure, low clearance and high oral bioavailability in mice, rats and dogs. The high in vitro MPO activity and high oral exposure observed with IN-4 result in a dose-dependent inhibition of MPO activity in three mouse models of inflammation. In conclusion, IN-4 is a novel, potent, mechanism-based and selective MPO inhibitor, which may be used as superior therapeutic agent to treat multiple inflammatory conditions, including cardiovascular disease.
Assuntos
Doenças Cardiovasculares , Peroxidase , Ratos , Camundongos , Animais , Cães , Peróxido de Hidrogênio , Antioxidantes , Indóis , PirrolidinonasRESUMO
CaMKK2 signals through AMPK-dependent and AMPK-independent pathways to trigger cellular outputs including proliferation, differentiation, and migration, resulting in changes to metabolism, bone mass accrual, neuronal function, hematopoiesis, and immunity. CAMKK2 is upregulated in tumors including hepatocellular carcinoma, prostate, breast, and gastric cancer, and genetic deletion in myeloid cells results in increased antitumor immunity in several syngeneic models. Validation of the biological roles of CaMKK2 has relied on genetic deletion or small molecule inhibitors with activity against several biological targets. We sought to generate selective inhibitors and degraders to understand the biological impact of inhibiting catalytic activity and scaffolding and the potential therapeutic benefits of targeting CaMKK2. We report herein selective, ligand-efficient inhibitors and ligand-directed degraders of CaMKK2 that were used to probe immune and tumor intrinsic biology. These molecules provide two distinct strategies for ablating CaMKK2 signaling in vitro and in vivo.
Assuntos
Proteínas Quinases Ativadas por AMP , Neoplasias Hepáticas , Masculino , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Cálcio , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina , LigantesRESUMO
The allosteric inhibitor of the mechanistic target of rapamycin (mTOR) everolimus reduces seizures in tuberous sclerosis complex (TSC) patients through partial inhibition of mTOR functions. Due to its limited brain permeability, we sought to develop a catalytic mTOR inhibitor optimized for central nervous system (CNS) indications. We recently reported an mTOR inhibitor (1) that is able to block mTOR functions in the mouse brain and extend the survival of mice with neuronal-specific ablation of the Tsc1 gene. However, 1 showed the risk of genotoxicity in vitro. Through structure-activity relationship (SAR) optimization, we identified compounds 9 and 11 without genotoxicity risk. In neuronal cell-based models of mTOR hyperactivity, both corrected aberrant mTOR activity and significantly improved the survival rate of mice in the Tsc1 gene knockout model. Unfortunately, 9 and 11 showed limited oral exposures in higher species and dose-limiting toxicities in cynomolgus macaque, respectively. However, they remain optimal tools to explore mTOR hyperactivity in CNS disease models.
Assuntos
Inibidores de MTOR , Sirolimo , Camundongos , Animais , Síndrome , Sistema Nervoso Central/metabolismo , Encéfalo/metabolismo , Serina-Treonina Quinases TOR , Trifosfato de AdenosinaRESUMO
Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multi-functional protein that is expressed in basally phosphorylated (p56) and in hyperphosphorylated (p58) forms. NS5A phosphorylation has been implicated in regulating multiple aspects of HCV replication. We recently reported the identification of a class of compounds that potently inhibit HCV RNA replication by targeting NS5A. Although the precise mechanism of inhibition of these compounds is not well understood, one activity that has been described is their ability to block expression of the hyperphosphorylated form of NS5A. Here, we report that an NS5A inhibitor impaired hyperphosphorylation without affecting basal phosphorylation at the C-terminal region of NS5A. This inhibitor activity did not require NS5A domains II and III and was distinct from that of a cellular kinase inhibitor that also blocked NS5A hyperphosphorylation, results that are consistent with an inhibitor-binding site within the N-terminal region of NS5A. In addition, we observed that an NS5A inhibitor promoted the accumulation of an HCV polyprotein intermediate, suggesting that inhibitor binding to NS5A may occur prior to the completion of polyprotein processing. Finally, we observed that NS5A p56 and p58 separated into different membrane fractions during discontinuous sucrose gradient centrifugation, consistent with these NS5A phosphoforms performing distinct replication functions. The p58 localization pattern was disrupted by an NS5A inhibitor. Collectively, our results suggest that NS5A inhibitors probably impact several aspects of HCV expression and regulation. These findings may help to explain the exceptional potency of this class of HCV replication complex inhibitors.