ABSTRACT
Respiratory syncytial virus (RSV) is an RNA virus infecting the upper and lower respiratory tract and is recognized as a major respiratory health threat, particularly to older adults, immunocompromised individuals, and young children. Around 64 million children and adults are infected every year worldwide. Despite two vaccines and a new generation monoclonal antibody recently approved, no effective antiviral treatment is available. In this manuscript, we present the medicinal chemistry efforts resulting in the identification of compound 28 (JNJ-8003), a novel RSV non-nucleoside inhibitor displaying subnanomolar activity in vitro as well as prominent efficacy in mice and a neonatal lamb models.
Subject(s)
Antiviral Agents , Pyridines , Animals , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/chemical synthesis , Humans , Mice , Pyridines/pharmacology , Pyridines/chemistry , Pyridines/chemical synthesis , Respiratory Syncytial Virus Infections/drug therapy , Respiratory Syncytial Virus Infections/virology , Structure-Activity Relationship , Sheep , Drug Discovery , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/chemical synthesis , Respiratory Syncytial Virus, Human/drug effects , Respiratory Syncytial Viruses/drug effectsABSTRACT
Respiratory syncytial virus (RSV) causes respiratory disease and complications in infants, the elderly and the immunocompromised. While three vaccines and two prophylactic monoclonal antibodies are now available, only one antiviral, ribavirin, is currently approved for treatment. This review aims to summarize the current state of treatments directly targeting RSV. Two major viral processes are attractive for RSV-specific antiviral drug discovery and development as they play essential roles in the viral cycle: the entry/fusion process carried out by the fusion protein and the replication/transcription process carried out by the polymerase complex constituted of the L, P, N and M2-1 proteins. For each viral target resistance mutations to small molecules of different chemotypes seem to delineate definite binding pockets in the fusion proteins and in the large proteins. Elucidating the mechanism of action of these inhibitors thus helps to understand how the fusion and polymerase complexes execute their functions. While many inhibitors have been studied, few are currently in clinical development for RSV treatment: one is in phase III, three in phase II and two in phase I. Progression was halted for many others because of strategic decisions, low enrollment, safety, but also lack of efficacy. Lessons can be learnt from the halted programs to increase the success rate of the treatments currently in development.
Subject(s)
Antiviral Agents , Respiratory Syncytial Virus Infections , Respiratory Syncytial Virus, Human , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Respiratory Syncytial Virus Infections/drug therapy , Respiratory Syncytial Virus Infections/virology , Humans , Respiratory Syncytial Virus, Human/drug effects , Respiratory Syncytial Virus, Human/genetics , Drug Discovery , Virus Replication/drug effects , Animals , Drug Resistance, Viral , Drug DevelopmentABSTRACT
Respiratory syncytial virus (RSV) is a major cause of hospitalization in infants, the elderly, and immune-compromised patients. While a half-life extended monoclonal antibody and 2 vaccines have recently been approved for infants and the elderly, respectively, options to prevent disease in immune-compromised patients are still needed. Here, we describe spiro-azetidine oxindoles as small molecule RSV entry inhibitors displaying favorable potency, developability attributes, and long-acting PK when injected as an aqueous suspension, suggesting their potential to prevent complications following RSV infection over a period of 3 to 6 months with 1 or 2 long-acting intramuscular (IM) or subcutaneous (SC) injections in these immune-compromised patients.
Subject(s)
Antiviral Agents , Azetidines , Oxindoles , Respiratory Syncytial Virus Infections , Spiro Compounds , Humans , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Virus Infections/drug therapy , Animals , Oxindoles/chemistry , Oxindoles/pharmacology , Spiro Compounds/chemistry , Spiro Compounds/pharmacology , Spiro Compounds/pharmacokinetics , Spiro Compounds/administration & dosage , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/administration & dosage , Azetidines/chemistry , Azetidines/pharmacology , Azetidines/administration & dosage , Azetidines/pharmacokinetics , Pre-Exposure Prophylaxis/methods , Injections, Intramuscular , Indoles/chemistry , Indoles/administration & dosage , Indoles/pharmacology , Injections, Subcutaneous , Respiratory Syncytial Virus, Human/drug effects , Virus Internalization/drug effectsABSTRACT
Acute myelogenous leukemia (AML), a heterogeneous disease of the blood and bone marrow, is characterized by the inability of myeloblasts to differentiate into mature cell types. Dihydroorotate dehydrogenase (DHODH) is an enzyme well-known in the pyrimidine biosynthesis pathway and preclinical findings demonstrated that DHODH is a metabolic vulnerability in AML as inhibitors can induce differentiation across multiple AML subtypes. As a result of virtual screening and structure-based drug design approaches, a novel series of isoquinolinone DHODH inhibitors was identified. Further lead optimization afforded JNJ-74856665 as an orally bioavailable, potent, and selective DHODH inhibitor with favorable physicochemical properties selected for clinical development in patients with AML and myelodysplastic syndromes (MDS).
Subject(s)
Dihydroorotate Dehydrogenase , Enzyme Inhibitors , Leukemia, Myeloid, Acute , Oxidoreductases Acting on CH-CH Group Donors , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Humans , Leukemia, Myeloid, Acute/drug therapy , Animals , Structure-Activity Relationship , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/therapeutic use , Enzyme Inhibitors/pharmacokinetics , Drug Discovery , Rats , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/pharmacokinetics , Quinolones/chemistry , Quinolones/pharmacology , Quinolones/therapeutic use , Quinolones/pharmacokinetics , Quinolones/chemical synthesis , Cell Line, Tumor , Molecular Docking SimulationABSTRACT
Dihydroorotate dehydrogenase (DHODH) is a mitochondrial enzyme that affects many aspects essential to cell proliferation and survival. Recently, DHODH has been identified as a potential target for acute myeloid leukemia therapy. Herein, we describe the identification of potent DHODH inhibitors through a scaffold hopping approach emanating from a fragment screen followed by structure-based drug design to further improve the overall profile and reveal an unexpected novel binding mode. Additionally, these compounds had low P-gp efflux ratios, allowing for applications where exposure to the brain would be required.
ABSTRACT
The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 Å resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs.
Subject(s)
Respiratory Syncytial Virus, Human , RNA-Dependent RNA Polymerase/chemistry , Protein Binding , RNA/metabolism , Nucleotides/metabolismABSTRACT
In continuation of our efforts of finding novel nucleoside inhibitors for the treatment of viral diseases, we initiated a discovery research program aimed at identifying novel nucleos(t)ide inhibitors for emerging diseases like Dengue and Chikungunya. Based on the previously reported 2'-spiro-oxetane uridine derivatives active against Hepatitis C Virus (HCV), we envisaged its sulfur analogue as an interesting congener both from a synthetic as well as biological point of view. Surprisingly, we found the 2'-spirothietane uridine derivatives not only to be active against HCV and Dengue virus (DENV), viruses belonging to the flavivirus family, but also to demonstrate activity against alphaviruses like Chikungunya virus (CHIKV) and Sindbis virus (SINV).
ABSTRACT
Acute myelogenous leukemia (AML), a disease of the blood and bone marrow, is characterized by the inability of myeloblasts to differentiate into mature cell types. Dihydroorotate dehydrogenase (DHODH) is an enzyme well-known in the pyrimidine biosynthesis pathway; however, small molecule DHODH inhibitors were recently shown to induce differentiation in multiple AML subtypes. Using virtual screening and structure-based drug design approaches, a new series of N-heterocyclic 3-pyridyl carboxamide DHODH inhibitors were discovered. Two lead compounds, 19 and 29, have potent biochemical and cellular DHODH activity, favorable physicochemical properties, and efficacy in a preclinical model of AML.
Subject(s)
Dihydroorotate Dehydrogenase , Leukemia, Myeloid, Acute , Dihydroorotate Dehydrogenase/antagonists & inhibitors , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Humans , Leukemia, Myeloid, Acute/drug therapyABSTRACT
The existence of a druggable binding pocket is a prerequisite for computational drug-target interaction studies including virtual screening. Retrospective studies have shown that extended sampling methods like Markov State Modeling and mixed-solvent simulations can identify cryptic pockets relevant for drug discovery. Here, we apply a combination of mixed-solvent molecular dynamics (MD) and time-structure independent component analysis (TICA) to four retrospective case studies: NPC2, the CECR2 bromodomain, TEM-1, and MCL-1. We compare previous experimental and computational findings to our results. It is shown that the successful identification of cryptic pockets depends on the system and the cosolvent probes. We used alternative TICA internal features such as the unbiased backbone coordinates or backbone dihedrals versus biased interatomic distances. We found that in the case of NPC2, TEM-1, and MCL-1, the use of unbiased features is able to identify cryptic pockets, although in the case of the CECR2 bromodomain, more specific features are required to properly capture a pocket opening. In the perspective of virtual screening applications, it is shown how docking studies with the parent ligands depend critically on the conformational state of the targets.
Subject(s)
Drug Discovery , Molecular Dynamics Simulation , Binding Sites , Ligands , Molecular Docking Simulation , Retrospective Studies , Solvents/chemistryABSTRACT
Dihydroorotate dehydrogenase (DHODH) enzymatic activity impacts many aspects critical to cell proliferation and survival. Recently, DHODH has been identified as a target for acute myeloid differentiation therapy. In preclinical models of AML, the DHODH inhibitor Brequinar (BRQ) demonstrated potent anti-leukemic activity. Herein we describe a carboxylic acid isostere study of Brequinar which revealed a more potent non-carboxylic acid derivative with improved cellular potency and good pharmacokinetic properties.
Subject(s)
Antineoplastic Agents/pharmacology , Biphenyl Compounds/pharmacology , Carboxylic Acids/pharmacology , Enzyme Inhibitors/pharmacology , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Biphenyl Compounds/chemistry , Carboxylic Acids/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dihydroorotate Dehydrogenase , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , Humans , Mice , Microsomes, Liver/chemistry , Microsomes, Liver/metabolism , Molecular Structure , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Structure-Activity RelationshipABSTRACT
OBJECTIVES: To characterize antiviral activity of the capsid assembly modulator (CAM-N) JNJ-56136379 against HBV genotypes and variants carrying amino acid substitutions in the core protein. METHODS: Anti-HBV activity of JNJ-56136379 was investigated against a diverse panel of 53 HBV clinical isolates (genotypes A-H). The impact of core amino acid substitutions using site-directed mutants (SDMs) was assessed in a transient replication assay. RESULTS: JNJ-56136379 median 50% effective concentration (EC50) values across all genotypes were 10-33 nM versus 17 nM (genotype D reference). JNJ-56136379 remained active against isolates carrying nucleos(t)ide analogue resistance mutations (median EC50 2-25 nM) or basal core promoter (BCP) ± precore (PC) mutations (median EC50 13-20 nM) or PC mutations (median EC50 11 nM), representing activity against isolates from HBeAg-positive and -negative hepatitis B patients. Core amino acid substitutions in the CAM-binding pocket, when tested as SDMs at positions 23, 25, 30, 33, 37, 106, 110, 118, 124, 127 and 128, reduced JNJ-56136379 anti-HBV activity; EC50 fold increases ranged from 3.0 (S106T) to 85 (T33N). All substitutions were rare in a public database of >7600 HBV core sequences (frequencies 0.01%-0.3%). Nucleos(t)ide analogues retained full activity against these core SDMs. CONCLUSIONS: JNJ-56136379, a potent HBV CAM-N, currently in Phase 2 clinical development, was generally fully active against an extensive panel of genotype A-H clinical isolates, regardless of the presence of nucleos(t)ide analogue resistance or BCP/PC mutations. JNJ-56136379 activity was reduced by some core amino acid substitutions in the CAM-binding pocket.
Subject(s)
Hepatitis B virus , Hepatitis B, Chronic , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Capsid , Capsid Proteins , DNA, Viral , Genotype , Hepatitis B e Antigens/therapeutic use , Hepatitis B virus/genetics , Hepatitis B, Chronic/drug therapy , Humans , MutationABSTRACT
Following the elucidation of the human genome, chemogenomics emerged in the beginning of the twenty-first century as an interdisciplinary research field with the aim to accelerate target and drug discovery by making best usage of the genomic data and the data linkable to it. What started as a systematization approach within protein target families now encompasses all types of chemical compounds and gene products. A key objective of chemogenomics is the establishment, extension, analysis, and prediction of a comprehensive SAR matrix which by application will enable further systematization in drug discovery. Herein we outline future perspectives of chemogenomics including the extension to new molecular modalities, or the potential extension beyond the pharma to the agro and nutrition sectors, and the importance for environmental protection. The focus is on computational sciences with potential applications for compound library design, virtual screening, hit assessment, analysis of phenotypic screens, lead finding and optimization, and systems biology-based prediction of toxicology and translational research.
Subject(s)
Computational Biology/methods , Databases, Chemical , Drug Design , Drug Discovery/methods , Proteins/metabolism , Computer Simulation , HumansABSTRACT
The Open PHACTS Discovery Platform integrates several public databases, which can be of interest when annotating the results of a phenotypic screening campaign. Workflow tools provide easy-to-customize possibilities to access the platform. Here, we describe how to create such workflows for two different workflow tools (KNIME and Pipeline Pilot), including a protocol to annotate compounds (e.g., phenotypic screening hits) with compound classification, known protein targets, and classifications of the targets.
Subject(s)
Computational Biology/methods , Drug Discovery , Software , Drug Discovery/methods , Molecular Sequence Annotation , Phenotype , Web Browser , WorkflowABSTRACT
Protocols for the design of kinase-focused compound libraries are presented. Kinase-focused compound libraries can be differentiated based on the design goal. Depending on whether the library should be a discovery library specific for one particular kinase, a general discovery library for multiple distinct kinase projects, or even phenotypic screening, there exists today a variety of in silico methods to design candidate compound libraries. We address the following scenarios: 1) Datamining of SAR databases and kinase focused vendor catalogues; 2) Predictions and virtual screening; 3) Structure-based design of combinatorial kinase inhibitors; 4) Design of covalent kinase inhibitors; 5) Design of macrocyclic kinase inhibitors; and 6) Design of allosteric kinase inhibitors and activators.
Subject(s)
Drug Discovery/methods , Protein Kinase Inhibitors/chemistry , Small Molecule Libraries/chemistry , Structure-Activity Relationship , Data Mining/methods , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/pharmacologyABSTRACT
The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies.
Subject(s)
B-Lymphocytes/metabolism , Phosphoproteins/genetics , Proteome/genetics , Receptor, ErbB-3/genetics , Receptor, ErbB-4/genetics , Signal Transduction , Amino Acid Sequence , Animals , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , Cell Line , Gene Expression Regulation , Gene Regulatory Networks , Genetic Engineering , Humans , Mice , Molecular Sequence Data , Neuregulin-1/metabolism , Neuregulin-1/pharmacology , Phosphoproteins/metabolism , Phosphorylation , Protein Binding , Protein Interaction Mapping , Proteome/metabolism , Receptor, ErbB-3/metabolism , Receptor, ErbB-4/metabolismABSTRACT
Likely due to conformational rearrangements, small molecule inhibitors may stabilize the active conformation of protein kinases and paradoxically promote tumorigenesis. We combined limited proteolysis with stable isotope labeling MS to monitor protein conformational changes upon binding of small molecules. Applying this method to the human serine/threonine kinase B-Raf, frequently mutated in cancer, we found that binding of ATP or its nonhydrolyzable analogue AMP-PNP, but not ADP, stabilized the structure of both B-Raf(WT) and B-Raf(V600E). The ATP-competitive type I B-Raf inhibitor vemurafenib and the type II inhibitor sorafenib stabilized the kinase domain (KD) but had distinct effects on the Ras-binding domain. Stabilization of the B-Raf(WT) KD was confirmed by hydrogen/deuterium exchange MS and molecular dynamics simulations. Our results are further supported by cellular assays in which we assessed cell viability and phosphorylation profiles in cells expressing B-Raf(WT) or B-Raf(V600E) in response to vemurafenib or sorafenib. Our data indicate that an overall stabilization of the B-Raf structure by specific inhibitors activates MAPK signaling and increases cell survival, helping to explain clinical treatment failure. We also applied our method to monitor conformational changes upon nucleotide binding of the pseudokinase KSR1, which holds high potential for inhibition in human diseases.
Subject(s)
Isotope Labeling/methods , Protein Kinase Inhibitors/chemistry , Protein Kinases/chemistry , Proteomics/methods , Proto-Oncogene Proteins B-raf/chemistry , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Adenylyl Imidodiphosphate/chemistry , Adenylyl Imidodiphosphate/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Deuterium Exchange Measurement , Humans , Indoles/chemistry , Indoles/pharmacology , MAP Kinase Signaling System/drug effects , Mass Spectrometry/methods , Molecular Dynamics Simulation , Mutation , Niacinamide/analogs & derivatives , Niacinamide/chemistry , Niacinamide/pharmacology , Peptides/analysis , Phenylurea Compounds/chemistry , Phenylurea Compounds/pharmacology , Phosphorylation/drug effects , Protein Binding , Protein Kinase Inhibitors/pharmacology , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Proteolysis , Proteomics/instrumentation , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Sorafenib , Sulfonamides/chemistry , Sulfonamides/pharmacology , Trypsin/chemistry , VemurafenibABSTRACT
Blocking the interaction between the p53 tumor suppressor and its regulatory protein MDM2 is a promising therapeutic concept under current investigation in oncology drug research. We report here the discovery of the first representatives of a new class of small molecule inhibitors of this protein-protein interaction: the dihydroisoquinolinones. Starting from an initial hit identified by virtual screening, a derivatization program has resulted in compound 11, a low nanomolar inhibitor of the p53-MDM2 interaction showing significant cellular activity. Initially based on a binding mode hypothesis, this effort was then guided by a X-ray co-crystal structure of MDM2 in complex with one of the synthesized analogs. The X-ray structure revealed an unprecedented binding mode for p53-MDM2 inhibitors.
Subject(s)
Isoquinolines/chemistry , Isoquinolines/pharmacology , Protein Interaction Maps/drug effects , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolism , Crystallography, X-Ray , Humans , Molecular Docking Simulation , Protein Binding/drug effects , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Structure-Activity Relationship , Tumor Suppressor Protein p53/antagonists & inhibitorsABSTRACT
Proteins are dynamic molecules; they undergo crucial conformational changes induced by post-translational modifications and by binding of cofactors or other molecules. The characterization of these conformational changes and their relation to protein function is a central goal of structural biology. Unfortunately, most conventional methods to obtain structural information do not provide information on protein dynamics. Therefore, mass spectrometry-based approaches, such as limited proteolysis, hydrogen-deuterium exchange, and stable-isotope labeling, are frequently used to characterize protein conformation and dynamics, yet the interpretation of these data can be cumbersome and time consuming. Here, we present PepShell, a tool that allows interactive data analysis of mass spectrometry-based conformational proteomics studies by visualization of the identified peptides both at the sequence and structure levels. Moreover, PepShell allows the comparison of experiments under different conditions, including different proteolysis times or binding of the protein to different substrates or inhibitors.
Subject(s)
Data Display , Mass Spectrometry/methods , Protein Conformation , Proteins/chemistry , Proteomics/methods , SoftwareABSTRACT
The explored kinome was extended with broad profiling using the DiscoveRx and Millipore assay panels. The analysis of the profiling of 3368 selected inhibitors on 456 kinases in the DiscoveRx format delivered several insights. First, the coverage depended on the threshold of the selectivity parameter. Second, the relation between hit confirmation rates and inhibitor selectivity showed unexpectedly that higher selectivity can increase the likelihood of false positives. Third, comparing the coverage of a focused to that of a random library showed that the design based on a maximum number of scaffolds was superior to a limited number of scaffolds. Therefore, selective compounds can be used in target validation, enable the jumpstarting of new kinase drug discovery projects, and chart new biological space via phenotypic screening.