RESUMEN
Herpesvirus infections are ubiquitous, with over 95% of the adult population infected by at least one strain. While most of these infections resolve without treatment in healthy individuals, they can cause significant morbidity and mortality in immunocompromised, stem cell, or organ transplant patients. Current nucleoside standards of care provide meaningful benefit but are limited due to poor tolerability, resistance, and generally narrow spectrum of activity. Herpesviruses share a conserved DNA polymerase, the inhibition of which is validated as an effective strategy to disrupt viral replication. By utilizing a non-nucleoside inhibitor of the viral DNA polymerase, we sought to develop agents covering multiple herpesviruses (e.g., CMV, VZV, HSV1/2, EBV, and HHV6). Herein is described the invention of an oxazolidinone class of broad-spectrum non-nucleoside herpes antiviral inhibitors. A lead compound (42) with potent biochemical and broad-spectrum cellular activity was found to be efficacious in murine models against both HSV-1 and CMV infection.
RESUMEN
Interests in covalent drugs have grown in modern drug discovery as they could tackle challenging targets traditionally considered "undruggable". The identification of covalent binders to target proteins typically involves directly measuring protein covalent modifications using high-resolution mass spectrometry. With a continually expanding library of compounds, conventional mass spectrometry platforms such as LC-MS and SPE-MS have become limiting factors for high-throughput screening. Here, we introduce a prototype high-resolution acoustic ejection mass spectrometry (AEMS) system for the rapid screening of a covalent modifier library comprising â¼10,000 compounds against a 50 kDa-sized target proteinâWerner syndrome helicase. The screening samples were arranged in a 1536-well format. The sample buffer containing high-concentration salts was directly analyzed without any cleanup steps, minimizing sample preparation efforts and ensuring protein stability. The entire AEMS analysis process could be completed within a mere 17 h. An automated data analysis tool facilitated batch processing of the sample data and quantitation of the formation of various covalent protein-ligand adducts. The screening results displayed a high degree of fidelity, with a Z' factor of 0.8 and a hit rate of 2.3%. The identified hits underwent orthogonal testing in a biochemical activity assay, revealing that 75% were functional antagonists of the target protein. Notably, a comparative analysis with LC-MS showcased the AEMS platform's low risk of false positives or false negatives. This innovative platform has enabled robust high-throughput covalent modifier screening, featuring a 10-fold increase in library size and a 10- to 100-fold increase in throughput when compared with similar reports in the existing literature.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Espectrometría de Masas , Espectrometría de Masas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Bibliotecas de Moléculas Pequeñas/química , Humanos , Acústica , Descubrimiento de Drogas/métodos , LigandosRESUMEN
The measurement of receptor occupancy (RO) using positron emission tomography (PET) has been instrumental in guiding discovery and development of CNS directed therapeutics. We and others have investigated muscarinic acetylcholine receptor 4 (M4) positive allosteric modulators (PAMs) for the treatment of symptoms associated with neuropsychiatric disorders. In this article, we describe the synthesis, in vitro, and in vivo characterization of a series of central pyridine-related M4 PAMs that can be conveniently radiolabeled with carbon-11 as PET tracers for the in vivo imaging of an allosteric binding site of the M4 receptor. We first demonstrated its feasibility by mapping the receptor distribution in mouse brain and confirming that a lead molecule 1 binds selectively to the receptor only in the presence of the orthosteric agonist carbachol. Through a competitive binding affinity assay and a number of physiochemical properties filters, several related compounds were identified as candidates for in vivo evaluation. These candidates were then radiolabeled with 11C and studied in vivo in rhesus monkeys. This research eventually led to the discovery of the clinical radiotracer candidate [11C]MK-6884.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Piridinas/farmacología , Receptor Muscarínico M4/agonistas , Animales , Células CHO , Radioisótopos de Carbono/química , Radioisótopos de Carbono/farmacología , Cricetulus , Humanos , Macaca mulatta , Agonistas Muscarínicos/química , Enfermedades Neurodegenerativas/diagnóstico por imagen , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Tomografía de Emisión de Positrones , Piridinas/química , Receptor Muscarínico M4/metabolismoRESUMEN
A series of novel tetracyclic core-containing HCV NS5A inhibitors has been discovered. Incorporation of tetrahydropyran-substituted amino acid moiety improved their potency and yielded HCV NS5A inhibitors with a minimum potency shift from the GT1a strain compared to other genotypes and mutants. Compounds 53 and 54 showed the best potency profile and had reasonable half-times in rat PK studies. However, further optimization of their oral bioavailability is still needed in order to advance them for further development. [BMCL ABSTRACT] ©2000 Elsevier Science Ltd. All rights reserved.
Asunto(s)
Antivirales/farmacología , Descubrimiento de Drogas , Genotipo , Hepacivirus/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/química , Antivirales/farmacocinética , Semivida , RatasRESUMEN
Inhibition of NS5A has emerged as an attractive strategy to intervene in hepatitis C virus (HCV) replication. Ruzasvir (formerly MK-8408) was developed as a novel NS5A inhibitor to improve upon the potency and barrier to resistance of early compounds. Ruzasvir inhibited HCV RNA replication with 50% effective concentrations (EC50s) of 1 to 4 pM in Huh7 or Huh7.5 cells bearing replicons for HCV genotype 1 (GT1) to GT7. The antiviral activity was modestly (10-fold) reduced in the presence of 40% normal human serum. The picomolar potency in replicon cells extended to sequences of clinical isolates available in public databases that were synthesized and tested as replicons. In GT1a, ruzasvir inhibited common NS5A resistance-associated substitutions (RASs), with the exception of M28G. De novo resistance selection studies identified pathways with certain amino acid substitutions at residues 28, 30, 31, and 93 across genotypes. Substitutions at position 93 were more common in GT1 to -4, while changes at position 31 emerged frequently in GT5 and -6. With the exception of GT4, the reintroduction of selected RASs conferred a ≥100-fold potency reduction in the antiviral activity of ruzasvir. Common RASs from other classes of direct-acting antiviral agents (DAAs) did not confer cross-resistance to ruzasvir. The interaction of ruzasvir with an NS3/4A protease inhibitor (grazoprevir) and an NS5B polymerase prodrug (uprifosbuvir) was additive to synergistic, with no evidence of antagonism or cytotoxicity. The antiviral profile of ruzasvir supported its further evaluation in human trials in combination with grazoprevir and uprifosbuvir.
Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Pirrolidinas/farmacología , Tiazoles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Amidas , Carbamatos , Línea Celular Tumoral , Ciclopropanos , Farmacorresistencia Viral/efectos de los fármacos , Quimioterapia Combinada/métodos , Genotipo , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/virología , Humanos , Inhibidores de Proteasas/farmacología , Quinoxalinas/farmacología , Replicón/efectos de los fármacos , Sulfonamidas , Uridina/análogos & derivados , Uridina/farmacologíaRESUMEN
We describe the discovery of MK-6169, a potent and pan-genotype hepatitis C virus NS5A inhibitor with optimized activity against common resistance-associated substitutions. SAR studies around the combination of changes to both the valine and aminal carbon region of elbasvir led to the discovery of a series of compounds with substantially improved potency against common resistance-associated substitutions in the major genotypes, as well as good pharmacokinetics in both rat and dog. Through further optimization of key leads from this effort, MK-6169 (21) was discovered as a preclinical candidate for further development.
Asunto(s)
Antivirales/farmacología , Descubrimiento de Drogas , Farmacorresistencia Viral/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/farmacocinética , Perros , Genotipo , Hepacivirus/genética , Hepacivirus/metabolismo , Masculino , Ratas , Distribución TisularRESUMEN
HCV NS5A inhibitors have shown impressive in vitro potency profiles in HCV replicon assays thus making them attractive components for inclusion in an all oral fixed dose combination regimen. Herein, we describe the discovery and characterization of silyl proline-containing HCV NS5A inhibitor MK-8325 with good pan-genotype activity and acceptable pharmacokinetic properties.
Asunto(s)
Antivirales/química , Compuestos Heterocíclicos de 4 o más Anillos/química , Prolina/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/farmacocinética , Antivirales/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Perros , Genotipo , Semivida , Haplorrinos , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Hepacivirus/fisiología , Compuestos Heterocíclicos de 4 o más Anillos/farmacocinética , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Ratas , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
We describe the research that led to the discovery of compound 40 (ruzasvir, MK-8408), a pan-genotypic HCV nonstructural protein 5A (NS5A) inhibitor with a "flat" GT1 mutant profile. This NS5A inhibitor contains a unique tetracyclic indole core while maintaining the imidazole-proline-valine Moc motifs of our previous NS5A inhibitors. Compound 40 is currently in early clinical trials and is under evaluation as part of an all-oral DAA regimen for the treatment of chronic HCV infection.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Polimorfismo Genético , Pirrolidinas/química , Pirrolidinas/farmacología , Tiazoles/química , Tiazoles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/farmacocinética , Línea Celular , Perros , Haplorrinos , Hepacivirus/genética , Compuestos Heterocíclicos de 4 o más Anillos/farmacocinética , Humanos , Pirrolidinas/farmacocinética , Ratas , Relación Estructura-Actividad , Tiazoles/farmacocinéticaRESUMEN
The discovery of potent and pan-genotypic HCV NS5A inhibitors faces many challenges including the significant diversity among genotypes, substantial potency shift conferred on some key resistance-associated variants, inconsistent SARs between different genotypes and mutants, and the lacking of models of inhibitor/protein complexes for rational inhibitor design. As part of ongoing efforts on HCV NS5A inhibition at Merck, we now describe the discovery of a novel series of chromane containing NS5A inhibitors. SAR studies around the "Z" group of the tetracyclic indole scaffold explored fused bicyclic rings as alternates to the phenyl group of elbasvir (1, MK-8742) and identified novel chromane and 2,3-dihydrobenzofuran derivatives as "Z" group replacements offered good potency across all genotypes. This effort, incorporating the C-1 fluoro substitution at the tetracyclic indole core, led to the discovery of a new series of NS5A inhibitors, such as compounds 14 and 25-28, with significantly improved potency against resistance-associated variants, such as GT2b, GT1a Y93H, and GT1a L31V. Compound 14 also showed reasonable PK exposures in preclinical species (rat and dog).
Asunto(s)
Antivirales/farmacología , Cromanos/farmacología , Descubrimiento de Drogas , Farmacorresistencia Viral/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/síntesis química , Antivirales/química , Cromanos/síntesis química , Cromanos/química , Perros , Relación Dosis-Respuesta a Droga , Masculino , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Ratas , Ratas Wistar , Relación Estructura-ActividadRESUMEN
Herein, we describe our research efforts to develop unique cores in molecules which function as HCV nonstructural protein 5A (NS5A) inhibitors. In particular, various fused tetracyclic cores were identified which showed genotype and mutant activities comparable to the indole-based tetracyclic core.
Asunto(s)
Indoles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/farmacología , Hepacivirus/efectos de los fármacosRESUMEN
We describe the impact of proline modifications, in our tetracyclic-indole based series of nonstructural protein 5A (NS5A) inhibitors, to their replicon profiles. This work identified NS5A inhibitors with an improved and flattened resistance profiles.
Asunto(s)
Antivirales/farmacología , Benzofuranos/farmacología , Imidazoles/farmacología , Indoles/química , Prolina/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/química , Benzofuranos/química , Imidazoles/química , Relación Estructura-ActividadRESUMEN
As part of an ongoing effort in NS5A inhibition at Merck we now describe our efforts for introducing substitution around the tetracyclic indole core of MK-8742. Fluoro substitution on the core combined with the fluoro substitutions on the proline ring improved the potency against GT1a Y93H significantly. However, no improvement on GT2b potency was achieved. Limiting the fluoro substitution to C-1 of the tetracyclic indole core had a positive impact on the potency against the resistance associated variants, such as GT1a Y93H and GT2b, and the PK profile as well. Compounds, such as 62, with reduced potency shifts between wild type GT1a to GT2b, GT1a Y93H, and GT1a L31V were identified.
Asunto(s)
Antivirales/farmacología , Benzofuranos/farmacología , Imidazoles/farmacología , Indoles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/química , Antivirales/farmacocinética , Benzofuranos/química , Benzofuranos/farmacocinética , Imidazoles/química , Imidazoles/farmacocinética , Indoles/química , Indoles/farmacocinética , Relación Estructura-ActividadRESUMEN
A matched and mixed capping SAR study was conducted on the tetracyclic indole class of HCV NS5A inhibitors to examine the influence of modifications of this region on the overall HCV virologic resistance profiles.
Asunto(s)
Antivirales/química , Hepacivirus/metabolismo , Indoles/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/metabolismo , Antivirales/farmacología , Área Bajo la Curva , Genotipo , Semivida , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Indoles/metabolismo , Indoles/farmacología , Curva ROC , Ratas , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismoRESUMEN
Herein we describe our research efforts around the aryl and heteroaryl substitutions at the aminal carbon of the tetracyclic indole-based HCV NS5A inhibitor MK-8742. A series of potent NS5A inhibitors are described, such as compounds 45-47, 54, 56, and 65, which showed improved potency against clinically relevant and resistance associated HCV variants. The improved potency profiles of these compounds demonstrated an SAR that can improve the potency against GT2b, GT1a Y93H, and GT1a L31V altogether, which was unprecedented in our previous efforts in NS5A inhibition.
Asunto(s)
Antivirales/farmacología , Benzofuranos/farmacología , Hepacivirus/efectos de los fármacos , Imidazoles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/síntesis química , Antivirales/química , Benzofuranos/síntesis química , Benzofuranos/química , Relación Dosis-Respuesta a Droga , Imidazoles/síntesis química , Imidazoles/química , Masculino , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
HCV NS5A inhibitors have demonstrated impressive in vitro potency profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed dose combination regimen for the treatment of HCV infection. Herein we describe our continued research efforts around the alkyl "Z group" modification of the tetracyclic indole-based NS5A inhibitor MK-8742, which led to the discovery of a series of potent NS5A inhibitors. Compounds 10 and 19 are of particular interests since they are as potent as our previous leads and have much improved rat pharmacokinetic profiles.
Asunto(s)
Antivirales/farmacología , Benzofuranos/farmacología , Hepacivirus/efectos de los fármacos , Imidazoles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/síntesis química , Antivirales/química , Benzofuranos/síntesis química , Benzofuranos/química , Relación Dosis-Respuesta a Droga , Hepatitis C/tratamiento farmacológico , Imidazoles/síntesis química , Imidazoles/química , Masculino , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
HCV NS5A inhibitors have demonstrated impressive in vitro virologic profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed-dose combination (FDC) regimen for the treatment of HCV infection. Merck's effort in this area identified MK-4882 and MK-8325 as early development leads. Herein, we describe the discovery of potent macrocyclic NS5A inhibitors bearing the MK-8325 or MK-4882 core structure.
Asunto(s)
Antivirales/farmacología , Descubrimiento de Drogas , Hepacivirus/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Compuestos Macrocíclicos/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/síntesis química , Antivirales/química , Relación Dosis-Respuesta a Droga , Hepatitis C/tratamiento farmacológico , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
HCV NS5A inhibitors have demonstrated impressive in vitro potency profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed dose combination regimen for the treatment of HCV infection. Herein, we describe research efforts that led to the discovery of a series of fused tricyclic core containing HCV NS5A inhibitors such as 24, 39, 40, 43, and 44 which have pan-genotype activity and are orally bioavailable in the rat.
Asunto(s)
Antivirales/farmacología , Descubrimiento de Drogas , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/síntesis química , Antivirales/química , Relación Dosis-Respuesta a Droga , Genotipo , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-Actividad , Proteínas no Estructurales Virales/genética , Replicación Viral/efectos de los fármacosRESUMEN
The pace and complexity of modern drug discovery places ever-increasing demands on scientists for data analysis and interpretation. Data flow programming and modern visualization tools address these demands directly. Three different requirements-one for allosteric modulator analysis, one for a specialized clotting analysis, and one for enzyme global progress curve analysis-are reviewed, and their execution in a combined data flow/visualization environment is outlined.
Asunto(s)
Recolección de Datos , Interpretación Estadística de Datos , Descubrimiento de Drogas/métodos , Procesamiento Automatizado de Datos/métodos , Farmacología/métodos , Estadística como Asunto/métodosRESUMEN
HCV NS5A inhibitors have shown impressive in vitro potency profiles in HCV replicon assays thus making them attractive components for inclusion in an all oral fixed dose combination treatment regimen. Herein we describe the research efforts that led to the discovery of silyl proline containing HCV NS5A inhibitors such as 7e and 8a with pan-genotype activity profile and acceptable pharmacokinetic properties.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Descubrimiento de Drogas , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Prolina/análogos & derivados , Silanos/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/síntesis química , Relación Dosis-Respuesta a Droga , Genotipo , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Silanos/farmacología , Relación Estructura-Actividad , Proteínas no Estructurales Virales/genética , Replicación Viral/efectos de los fármacosRESUMEN
We have been focused on identifying a structurally different next generation inhibitor to MK-5172 (our Ns3/4a protease inhibitor currently under regulatory review), which would achieve superior pangenotypic activity with acceptable safety and pharmacokinetic profile. These efforts have led to the discovery of a novel class of HCV NS3/4a protease inhibitors containing a unique spirocyclic-proline structural motif. The design strategy involved a molecular-modeling based approach, and the optimization efforts on the series to obtain pan-genotypic coverage with good exposures on oral dosing. One of the key elements in this effort was the spirocyclization of the P2 quinoline group, which rigidified and constrained the binding conformation to provide a novel core. A second focus of the team was also to improve the activity against genotype 3a and the key mutant variants of genotype 1b. The rational application of structural chemistry with molecular modeling guided the design and optimization of the structure-activity relationships have resulted in the identification of the clinical candidate MK-8831 with excellent pan-genotypic activity and safety profile.