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1.
RSC Med Chem ; 15(6): 2146-2159, 2024 Jun 19.
Article in English | MEDLINE | ID: mdl-38911172

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of coronavirus disease (COVID-19) since its emergence in December 2019. As of January 2024, there has been over 774 million reported cases and 7 million deaths worldwide. While vaccination efforts have been successful in reducing the severity of the disease and decreasing the transmission rate, the development of effective therapeutics against SARS-CoV-2 remains a critical need. The main protease (Mpro) of SARS-CoV-2 is an essential enzyme required for viral replication and has been identified as a promising target for drug development. In this study, we report the identification of novel Mpro inhibitors, using a combination of deep reinforcement learning for de novo drug design with 3D pharmacophore/shape-based alignment and privileged fragment match count scoring components followed by hit expansions and molecular docking approaches. Our experimentally validated results show that 3 novel series exhibit potent inhibitory activity against SARS-CoV-2 Mpro, with IC50 values ranging from 1.3 µM to 2.3 µM and a high degree of selectivity. These findings represent promising starting points for the development of new antiviral therapies against COVID-19.

2.
J Med Chem ; 67(11): 9465-9484, 2024 Jun 13.
Article in English | MEDLINE | ID: mdl-38753983

ABSTRACT

Clostridioides difficile (C. difficile) is one of the leading causes of healthcare-associated infections worldwide. The increasing incidence of strains resistant to currently available therapies highlights the need for alternative treatment options with a novel mode of action. Oxazolidinones that are connected to a quinolone moiety with a pyrrolidine linker, such as compound 1, are reported to exhibit potent broadspectrum antibacterial activity. In an effort to optimize this class of compounds for the treatment of C. difficile infection (CDI), we have identified cadazolid (9), a first-in-class quinoxolidinone antibiotic, which is a potent inhibitor of C. difficile protein synthesis. In order to achieve narrow-spectrum coverage of clinically most relevant strains without affecting the gut microbiota, an emphasis was placed on abolishing activity against commensals of the intestinal microbiome while retaining good coverage of pathogenic C. difficile, including hypervirulent and epidemic strains.


Subject(s)
Anti-Bacterial Agents , Clostridioides difficile , Clostridium Infections , Microbial Sensitivity Tests , Structure-Activity Relationship , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/therapeutic use , Anti-Bacterial Agents/chemical synthesis , Clostridioides difficile/drug effects , Clostridium Infections/drug therapy , Animals , Humans , Drug Discovery , Gastrointestinal Microbiome/drug effects , Mice , Oxazolidinones
3.
Acta Crystallogr D Struct Biol ; 80(Pt 2): 123-136, 2024 Feb 01.
Article in English | MEDLINE | ID: mdl-38289714

ABSTRACT

To identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate-binding pocket. Of 631 soaked fragments, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (three hits) or at crystal-packing interfaces (two hits). Notably, two fragments with a pose that was sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. The structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Catalytic Domain , Coronavirus 3C Proteases , Crystallography, X-Ray
4.
PLoS One ; 17(3): e0264450, 2022.
Article in English | MEDLINE | ID: mdl-35259191

ABSTRACT

Eidolon helvum bats are reservoir hosts for highly pathogenic lyssaviruses often showing limited disease upon natural infection. An enhanced antiviral interferon (IFN) response combined with reduced inflammation might be linked to the apparent virus tolerance in bats. Lyssavirus phosphoproteins inhibit the IFN response with virus strain-specific efficiency. To date, little is known regarding the lyssavirus P-dependent anti-IFN countermeasures in bats, mainly due to a lack of in vitro tools. By using E. helvum bat cell cultures in a newly established bat-specific IFN-promoter activation assay, we analyzed the IFN-ß inhibitory activity of multiple lyssavirus P in E. helvum compared to human cells. Initial virus infection studies with a recently isolated E. helvum-borne Lagos bat virus street strain from Ghana showed enhanced LBV propagation in an E. helvum lung cell line compared to human A549 lung cells at later time points suggesting effective viral countermeasures against cellular defense mechanisms. A direct comparison of the IFN-ß inhibitory activity of the LBV-GH P protein with other lyssavirus P proteins showed that LBV-GH P and RVP both strongly inhibited the bat IFN-ß promotor activation (range 75-90%) in EidLu/20.2 and an E. helvum kidney cell line. Conversely, LBV-GH P blocked the activation of the human IFN-ß promoter less efficiently compared to a prototypic Rabies virus P protein (range LBV P 52-68% vs RVP 71-95%) in two different human cell lines (HEK-293T, A549). The same pattern was seen for two prototypic LBV P variants suggesting an overall reduced LBV P IFN-ß inhibitory activity in human cells as compared to E. helvum bat cells. Increased IFN-ß inhibition by lyssavirus P in reservoir host cells might be a result of host-specific adaptation processes towards an enhanced IFN response in bat cells.


Subject(s)
Chiroptera , Lyssavirus , Rhabdoviridae Infections , Animals , Antibodies, Viral , Humans , Interferon-beta , Nigeria , Phosphoproteins
5.
ChemMedChem ; 16(5): 891-897, 2021 03 03.
Article in English | MEDLINE | ID: mdl-33236408

ABSTRACT

Ribosomal protein synthesis is an important target in antibacterial drug discovery. Numerous natural products have served as starting points for the development of antibiotics. We report here the total synthesis of xenocoumacin 1, a natural product that binds to 16S ribosomal RNA at a highly conserved region, as well as analogues thereof. Preliminary structure-activity relationship studies were aimed at understanding and modulating the selectivity between eukaryotic and prokaryotic ribosomes. Modifications were mainly tolerated in the aromatic region. Whole-cell activity against Gram-negative bacteria is limited by efflux and penetration, as demonstrated in genetically modified strains of E. coli. Analogues with high selectivity for eukaryotic ribosomes were identified, but it was not possible to obtain inhibitors selective for bacterial protein synthesis. Achieving high selectivity (albeit not the desired one) was thus possible despite the high homology between eukaryotic and prokaryotic ribosomes in the binding region.


Subject(s)
Anti-Bacterial Agents/pharmacology , Benzopyrans/pharmacology , Escherichia coli/drug effects , Ribosomal Proteins/antagonists & inhibitors , Anti-Bacterial Agents/chemistry , Benzopyrans/chemistry , Crystallography, X-Ray , Dose-Response Relationship, Drug , Escherichia coli/metabolism , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Ribosomal Proteins/biosynthesis , Structure-Activity Relationship
6.
J Med Chem ; 63(1): 66-87, 2020 01 09.
Article in English | MEDLINE | ID: mdl-31804826

ABSTRACT

UDP-3-O-((R)-3-hydroxymyristoyl)-N-glucosamine deacetylase (LpxC) is as an attractive target for the discovery and development of novel antibacterial drugs to address the critical medical need created by multidrug resistant Gram-negative bacteria. By using a scaffold hopping approach on a known family of methylsulfone hydroxamate LpxC inhibitors, several hit series eliciting potent antibacterial activities against Enterobacteriaceae and Pseudomonas aeruginosa were identified. Subsequent hit-to-lead optimization, using cocrystal structures of inhibitors bound to Pseudomonas aeruginosa LpxC as guides, resulted in the discovery of multiple chemical series based on (i) isoindolin-1-ones, (ii) 4,5-dihydro-6H-thieno[2,3-c]pyrrol-6-ones, and (iii) 1,2-dihydro-3H-pyrrolo[1,2-c]imidazole-3-ones. Synthetic methods, antibacterial activities and relative binding affinities, as well as physicochemical properties that allowed compound prioritization are presented. Finally, in vivo properties of lead molecules which belong to the most promising pyrrolo-imidazolone series, such as 18d, are discussed.


Subject(s)
Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/therapeutic use , Enzyme Inhibitors/therapeutic use , Escherichia coli Infections/drug therapy , Gram-Negative Bacteria/drug effects , Hydroxamic Acids/therapeutic use , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacokinetics , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacokinetics , Escherichia coli/drug effects , Female , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/pharmacokinetics , Klebsiella pneumoniae/drug effects , Mice, Inbred ICR , Microbial Sensitivity Tests , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/enzymology , Pyrroles/chemical synthesis , Pyrroles/pharmacokinetics , Pyrroles/therapeutic use
7.
J Med Chem ; 63(1): 88-102, 2020 01 09.
Article in English | MEDLINE | ID: mdl-31804829

ABSTRACT

LpxC inhibitors were optimized starting from lead compounds with limited efficacy and solubility and with the goal to provide new options for the treatment of serious infections caused by Gram-negative pathogens in hospital settings. To enable the development of an aqueous formulation for intravenous administration of the drug at high dose, improvements in both solubility and antibacterial activity in vivo were prioritized early on. This lead optimization program resulted in the discovery of compounds such as 13 and 30, which exhibited high solubility and potent efficacy against Gram-negative pathogens in animal infection models.


Subject(s)
Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/therapeutic use , Enzyme Inhibitors/therapeutic use , Escherichia coli Infections/drug therapy , Hydroxamic Acids/therapeutic use , Administration, Intravenous , Animals , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacokinetics , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacokinetics , Gram-Negative Bacteria/drug effects , Hepatocytes/metabolism , Hydroxamic Acids/administration & dosage , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/pharmacokinetics , Mice , Microbial Sensitivity Tests , Molecular Structure , Rats , Solubility
8.
Sci Rep ; 9(1): 5634, 2019 04 04.
Article in English | MEDLINE | ID: mdl-30948752

ABSTRACT

Oxazolidinones are synthetic antibiotics used for treatment of infections caused by Gram-positive bacteria. They target the bacterial protein synthesis machinery by binding to the peptidyl transferase centre (PTC) of the ribosome and interfering with the peptidyl transferase reaction. Cadazolid is the first member of quinoxolidinone antibiotics, which are characterized by combining the pharmacophores of oxazolidinones and fluoroquinolones, and it is evaluated for treatment of Clostridium difficile gastrointestinal infections that frequently occur in hospitalized patients. In vitro protein synthesis inhibition by cadazolid was shown in Escherichia coli and Staphylococcus aureus, including an isolate resistant against linezolid, the prototypical oxazolidinone antibiotic. To better understand the mechanism of inhibition, we determined a 3.0 Å cryo-electron microscopy structure of cadazolid bound to the E. coli ribosome in complex with mRNA and initiator tRNA. Here we show that cadazolid binds with its oxazolidinone moiety in a binding pocket in close vicinity of the PTC as observed previously for linezolid, and that it extends its unique fluoroquinolone moiety towards the A-site of the PTC. In this position, the drug inhibits protein synthesis by interfering with the binding of tRNA to the A-site, suggesting that its chemical features also can enable the inhibition of linezolid-resistant strains.


Subject(s)
Oxazolidinones/metabolism , Oxazolidinones/pharmacology , Protein Synthesis Inhibitors/pharmacology , Acetamides/pharmacology , Anti-Bacterial Agents/pharmacology , Clostridium Infections/drug therapy , Cryoelectron Microscopy/methods , Escherichia coli/metabolism , Fluoroquinolones/pharmacology , Humans , Microbial Sensitivity Tests , Peptidyl Transferases/antagonists & inhibitors , RNA, Transfer, Met/metabolism , Ribosomes/metabolism , Staphylococcus aureus/metabolism
9.
Angew Chem Int Ed Engl ; 57(34): 11020-11024, 2018 08 20.
Article in English | MEDLINE | ID: mdl-29943519

ABSTRACT

The macrocyclic antibiotic mangrolide A has been described to exhibit potent activity against a number of clinically important Gram-negative pathogens. Reported is the first enantioselective total synthesis of mangrolide A and derivatives. Salient features of this synthesis include a highly convergent macrocycle preparation, stereoselective synthesis of the disaccharide moiety, and two ß-selective glycosylations. The synthesis of mangrolide A and its analogues enabled the re-examination of its activity against bacterial pathogens, and only minimal activity was observed.


Subject(s)
Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Macrocyclic Compounds/chemical synthesis , Macrocyclic Compounds/pharmacology , Anti-Bacterial Agents/chemistry , Carbohydrate Sequence , Drug Resistance, Bacterial , Glycosylation , Macrocyclic Compounds/chemistry , Microbial Sensitivity Tests , Stereoisomerism
10.
Article in English | MEDLINE | ID: mdl-28584149

ABSTRACT

Cadazolid (CDZ) is a new antibiotic currently in clinical development for the treatment of Clostridium difficile infections. CDZ interferes with the bacterial protein synthesis machinery. The aim of the present study was to identify resistance mechanisms for CDZ and compare the results to those obtained for linezolid (LZD) in C. difficile by whole-genome sequencing (WGS) of strains generated by in vitro passages and to those obtained for LZD-resistant clinical isolates. Clones of C. difficile 630 selected with CDZ during 46 passages had a maximally 4-fold increase in CDZ MIC, while the LZD MIC for clones selected with LZD increased up to 16-fold. CDZ cross-resistance with LZD was maximally 4-fold, and no cross-resistance with other antibiotics tested was observed. Our data suggest that there are different resistance mechanisms for CDZ and LZD in C. difficile Mutations after passages with CDZ were found in rplD (ribosomal protein L4) as well as in tra and rmt, whereas similar experiments with LZD showed mutations in rplC (ribosomal protein L3), reg, and tpr, indicating different resistance mechanisms. Although high degrees of variation between the sequenced genomes of the clinical isolates were observed, the same mutation in rplC was found in two clinical isolates with high LZD MICs. No mutations were found in the 23S rRNA genes, and attempts to isolate the cfr gene from resistant clinical isolates were unsuccessful. Analysis of 50% inhibitory concentrations (IC50s) determined in in vitro transcription/translation assays performed with C. difficile cell extracts from passaged clones correlated well with the MIC values for all antibiotics tested, indicating that the ribosomal mutations are causing the resistant phenotype.


Subject(s)
Anti-Bacterial Agents/pharmacology , Clostridioides difficile/drug effects , Clostridioides difficile/genetics , Drug Resistance, Bacterial/genetics , Linezolid/pharmacology , Oxazolidinones/pharmacology , Protein Synthesis Inhibitors/pharmacology , Ribosomal Proteins/genetics , Amino Acid Sequence , Clostridioides difficile/isolation & purification , Drug Resistance, Bacterial/physiology , Genome, Bacterial/genetics , Humans , Microbial Sensitivity Tests , RNA, Ribosomal, 23S/genetics , Ribosomal Protein L3 , Sequence Analysis, DNA
11.
J Med Chem ; 60(9): 3755-3775, 2017 05 11.
Article in English | MEDLINE | ID: mdl-28406299

ABSTRACT

Our strategy to combat resistant bacteria consisted of targeting the GyrB/ParE ATP-binding sites located on bacterial DNA gyrase and topoisomerase IV and not utilized by marketed antibiotics. Screening around the minimal ethyl urea binding motif led to the identification of isoquinoline ethyl urea 13 as a promising starting point for fragment evolution. The optimization was guided by structure-based design and focused on antibacterial activity in vitro and in vivo, culminating in the discovery of unprecedented substituents able to interact with conserved residues within the ATP-binding site. A detailed characterization of the lead compound highlighted the potential for treatment of the problematic fluoroquinolone-resistant MRSA, VRE, and S. pneumoniae, and the possibility to offer patients an intravenous-to-oral switch therapy was supported by the identification of a suitable prodrug concept. Eventually, hERG K-channel block was identified as the main limitation of this chemical series, and efforts toward its minimization are reported.


Subject(s)
Anti-Bacterial Agents/pharmacology , Isoquinolines/pharmacology , Animals , Anti-Bacterial Agents/chemistry , Area Under Curve , Drug Discovery , Gram-Negative Bacteria/drug effects , Half-Life , Hydrogen Bonding , Isoquinolines/chemistry , Isoquinolines/pharmacokinetics , Isoquinolines/therapeutic use , Microbial Sensitivity Tests , Potassium Channels/drug effects , Rats , Respiratory Tract Infections/drug therapy , Respiratory Tract Infections/microbiology , Solubility , Urea/chemistry
12.
J Med Chem ; 60(9): 3776-3794, 2017 05 11.
Article in English | MEDLINE | ID: mdl-28406300

ABSTRACT

There is an urgent unmet medical need for novel antibiotics that are effective against a broad range of bacterial species, especially multidrug resistant ones. Tetrahydropyran-based inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) display potent activity against Gram-positive pathogens and no target-mediated cross-resistance with fluoroquinolones. We report our research efforts aimed at expanding the antibacterial spectrum of this class of molecules toward difficult-to-treat Gram-negative pathogens. Physicochemical properties (polarity and basicity) were considered to guide the design process. Dibasic tetrahydropyran-based compounds such as 6 and 21 are potent inhibitors of both DNA gyrase and topoisomerase IV, displaying antibacterial activities against Gram-positive and Gram-negative pathogens (Staphylococcus aureus, Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii). Compounds 6 and 21 are efficacious in clinically relevant murine infection models.


Subject(s)
Anti-Bacterial Agents/pharmacology , Gram-Negative Bacteria/drug effects , Pyrans/pharmacology , Topoisomerase Inhibitors/chemical synthesis , Topoisomerase Inhibitors/pharmacology , Animals , Anti-Bacterial Agents/adverse effects , Anti-Bacterial Agents/chemical synthesis , Guinea Pigs , Humans , Microbial Sensitivity Tests , Myocytes, Cardiac/drug effects , Pyrans/adverse effects , Pyrans/chemical synthesis , Topoisomerase Inhibitors/adverse effects
13.
Antimicrob Agents Chemother ; 60(1): 628-31, 2016 01.
Article in English | MEDLINE | ID: mdl-26503650

ABSTRACT

The promotion of colonization with vancomycin-resistant enterococci (VRE) is one potential side effect during treatment of Clostridium difficile-associated diarrhea (CDAD), resulting from disturbances in gut microbiota. Cadazolid (CDZ) is an investigational antibiotic with potent in vitro activity against C. difficile and against VRE and is currently in clinical development for the treatment of CDAD. We report that CDZ treatment did not lead to intestinal VRE overgrowth in mice.


Subject(s)
Anti-Bacterial Agents/adverse effects , Clostridioides difficile/drug effects , Diarrhea/drug therapy , Enterocolitis, Pseudomembranous/drug therapy , Oxazolidinones/pharmacology , Vancomycin/adverse effects , Aminoglycosides/pharmacology , Animals , Anti-Bacterial Agents/administration & dosage , Clostridioides difficile/growth & development , Clostridioides difficile/pathogenicity , Colony Count, Microbial , Diarrhea/etiology , Diarrhea/microbiology , Diarrhea/pathology , Enterocolitis, Pseudomembranous/etiology , Enterocolitis, Pseudomembranous/microbiology , Enterocolitis, Pseudomembranous/pathology , Fidaxomicin , Intestines/drug effects , Intestines/microbiology , Intestines/pathology , Metronidazole/pharmacology , Mice , Microbial Sensitivity Tests , Streptococcal Infections/drug therapy , Streptococcal Infections/microbiology , Streptococcal Infections/pathology , Treatment Outcome , Vancomycin/administration & dosage , Vancomycin Resistance , Vancomycin-Resistant Enterococci/drug effects , Vancomycin-Resistant Enterococci/growth & development , Vancomycin-Resistant Enterococci/pathogenicity
15.
Lancet Infect Dis ; 15(5): 559-64, 2015 May.
Article in English | MEDLINE | ID: mdl-25863564

ABSTRACT

BACKGROUND: Scientific evidence suggests that dromedary camels are the intermediary host for the Middle East respiratory syndrome coronavirus (MERS-CoV). However, the actual number of infections in people who have had contact with camels is unknown and most index patients cannot recall any such contact. We aimed to do a nationwide serosurvey in Saudi Arabia to establish the prevalence of MERS-CoV antibodies, both in the general population and in populations of individuals who have maximum exposure to camels. METHODS: In the cross-sectional serosurvey, we tested human serum samples obtained from healthy individuals older than 15 years who attended primary health-care centres or participated in a national burden-of-disease study in all 13 provinces of Saudi Arabia. Additionally, we tested serum samples from shepherds and abattoir workers with occupational exposure to camels. Samples were screened by recombinant ELISA and MERS-CoV seropositivity was confirmed by recombinant immunofluorescence and plaque reduction neutralisation tests. We used two-tailed Mann Whitney U exact tests, χ(2), and Fisher's exact tests to analyse the data. FINDINGS: Between Dec 1, 2012, and Dec 1, 2013, we obtained individual serum samples from 10,009 individuals. Anti-MERS-CoV antibodies were confirmed in 15 (0·15%; 95% CI 0·09-0·24) of 10,009 people in six of the 13 provinces. The mean age of seropositive individuals was significantly younger than that of patients with reported, laboratory-confirmed, primary Middle Eastern respiratory syndrome (43·5 years [SD 17·3] vs 53·8 years [17·5]; p=0·008). Men had a higher antibody prevalence than did women (11 [0·25%] of 4341 vs two [0·05%] of 4378; p=0·028) and antibody prevalence was significantly higher in central versus coastal provinces (14 [0·26%] of 5479 vs one [0·02%] of 4529; p=0·003). Compared with the general population, seroprevalence of MERS-CoV antibodies was significantly increased by 15 times in shepherds (two [2·3%] of 87, p=0·0004) and by 23 times in slaughterhouse workers (five [3·6%] of 140; p<0·0001). INTERPRETATION: Seroprevalence of MERS-CoV antibodies was significantly higher in camel-exposed individuals than in the general population. By simple multiplication, a projected 44,951 (95% CI 26,971-71,922) individuals older than 15 years might be seropositive for MERS-CoV in Saudi Arabia. These individuals might be the source of infection for patients with confirmed MERS who had no previous exposure to camels. FUNDING: European Union, German Centre for Infection Research, Federal Ministry of Education and Research, German Research Council, and Ministry of Health of Saudi Arabia.


Subject(s)
Antibodies, Viral/blood , Camelus/virology , Coronavirus Infections/epidemiology , Coronavirus Infections/transmission , Middle East Respiratory Syndrome Coronavirus/immunology , Abattoirs , Adolescent , Adult , Aged , Animal Husbandry , Animals , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Cross-Sectional Studies , Female , Humans , Male , Middle Aged , Occupations , Prevalence , Saudi Arabia/epidemiology , Seroepidemiologic Studies
16.
J Virol ; 89(11): 5876-82, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25787289

ABSTRACT

UNLABELLED: The hepatitis C virus (HCV; genus Hepacivirus) is a highly relevant human pathogen. Unique hepaciviruses (HV) were discovered recently in animal hosts. The direct ancestor of HCV has not been found, but the genetically most closely related animal HVs exist in horses. To investigate whether other peridomestic animals also carry HVs, we analyzed sera from Ghanaian cattle for HVs by reverse transcription-PCR (RT-PCR). Nine of 106 specimens from different sampling sites contained HV RNA (8.5%) at median viral loads of 1.6 × 10(5) copies/ml. Infection seemed unrelated to cattle age and gender. Near-full-genome sequencing of five representative viruses confirmed taxonomic classifications. Cattle HVs formed two distinct phylogenetic lineages that differed by up to 17.7% on the nucleotide level in the polyprotein-encoding region, suggesting cocirculation of different virus subtypes. A conserved microRNA122-binding site in the 5' internal ribosomal entry site suggested liver tropism of cattle HVs. Phylogenetic analyses suggested the circulation of HVs in cattle for several centuries. Cattle HVs were genetically highly divergent from all other HVs, including HCV. HVs from genetically related equine and bovine hosts were not monophyletic, corroborating host shifts during the evolution of the genus Hepacivirus. Similar to equine HVs, the genetic diversity of cattle HVs was low compared to that of HCV genotypes. This suggests an influence of the human-modified ecology of peridomestic animals on virus diversity. Further studies should investigate the occurrence of cattle HVs in other geographic areas and breeds, virus pathogenicity in cattle, and the potential exposure of human risk groups, such as farmers, butchers, and abattoir workers. IMPORTANCE: HCV (genus Hepacivirus) is a major human pathogen, causing liver failure and cancer. Unique hepaciviruses (HVs) were discovered over the last few years in animals, but the direct ancestor of HCV has not been found. The animal HV most closely related to HCV so far originated from horses, suggesting that other livestock animals also harbor HVs. Therefore, we investigated African cattle and discovered previously unknown HVs at high prevalence and viral loads. Because of the agricultural importance of cattle, it may be relevant to investigate HV pathogenicity. The frequent exposure of humans to cattle also may warrant investigations of the zoonotic potential of these viruses. Evolutionary analyses suggested that cattle HVs have existed for centuries. Despite the genetic relatedness of their animal hosts, HVs from cattle and horses were not phylogenetically related, corroborating frequent host shifts during the evolution of the genus Hepacivirus.


Subject(s)
Cattle Diseases/virology , Hepacivirus/classification , Hepacivirus/isolation & purification , Hepatitis C/veterinary , Animals , Cattle , Cluster Analysis , Genetic Variation , Genome, Viral , Genotype , Ghana , Hepacivirus/genetics , Hepatitis C/virology , Molecular Sequence Data , Phylogeny , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Sequence Homology , Serum/virology , Viral Load
17.
J Infect Dis ; 211(6): 889-97, 2015 Mar 15.
Article in English | MEDLINE | ID: mdl-25057042

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) infection is associated with a high case-fatality rate, and the potential pandemic spread of the virus is a public health concern. The spike protein of MERS-CoV (MERS-S) facilitates viral entry into host cells, which depends on activation of MERS-S by cellular proteases. Proteolytic activation of MERS-S during viral uptake into target cells has been demonstrated. However, it is unclear whether MERS-S is also cleaved during S protein synthesis in infected cells and whether cleavage is required for MERS-CoV infectivity. Here, we show that MERS-S is processed by proprotein convertases in MERS-S-transfected and MERS-CoV-infected cells and that several RXXR motifs located at the border between the surface and transmembrane subunit of MERS-S are required for efficient proteolysis. However, blockade of proprotein convertases did not impact MERS-S-dependent transduction of target cells expressing high amounts of the viral receptor, DPP4, and did not modulate MERS-CoV infectivity. These results show that MERS-S is a substrate for proprotein convertases and demonstrate that processing by these enzymes is dispensable for S protein activation. Efforts to inhibit MERS-CoV infection by targeting host cell proteases should therefore focus on enzymes that process MERS-S during viral uptake into target cells.


Subject(s)
Coronavirus/physiology , Proprotein Convertases/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , HEK293 Cells , Humans , Molecular Sequence Data , Protease Inhibitors/pharmacology , Protein Processing, Post-Translational , Proteolysis , Spike Glycoprotein, Coronavirus/chemistry , Virus Internalization
18.
J Med Chem ; 58(2): 927-42, 2015 Jan 22.
Article in English | MEDLINE | ID: mdl-25494934

ABSTRACT

Novel antibacterial drugs that are effective against infections caused by multidrug resistant pathogens are urgently needed. In a previous report, we have shown that tetrahydropyran-based inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) display potent antibacterial activity and exhibit no target-mediated cross-resistance with fluoroquinolones. During the course of our optimization program, lead compound 5 was deprioritized due to adverse findings in cardiovascular safety studies. In the effort of mitigating these findings and optimizing further the pharmacological profile of this class of compounds, we have identified a subseries of tetrahydropyran-based molecules that are potent DNA gyrase and topoisomerase IV inhibitors and display excellent antibacterial activity against Gram positive pathogens, including clinically relevant resistant isolates. One representative of this class, compound 32d, elicited only weak inhibition of hERG K(+) channels and hNaV1.5 Na(+) channels, and no effects were observed on cardiovascular parameters in anesthetized guinea pigs. In vivo efficacy in animal infection models has been demonstrated against Staphylococcus aureus and Streptococcus pneumoniae strains.


Subject(s)
Anti-Bacterial Agents/chemical synthesis , Gram-Positive Bacteria/drug effects , Pyrans/chemical synthesis , Topoisomerase II Inhibitors/chemical synthesis , Animals , Anti-Bacterial Agents/pharmacology , Guinea Pigs , Hemodynamics/drug effects , Humans , Male , Mice , Microbial Sensitivity Tests , Pyrans/pharmacology , Rats , Rats, Wistar , Structure-Activity Relationship , Topoisomerase II Inhibitors/pharmacology
19.
Emerg Infect Dis ; 20(4): 552-9, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24655412

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) has caused an ongoing outbreak of severe acute respiratory tract infection in humans in the Arabian Peninsula since 2012. Dromedary camels have been implicated as possible viral reservoirs. We used serologic assays to analyze 651 dromedary camel serum samples from the United Arab Emirates; 151 of 651 samples were obtained in 2003, well before onset of the current epidemic, and 500 serum samples were obtained in 2013. Recombinant spike protein-specific immunofluorescence and virus neutralization tests enabled clear discrimination between MERS-CoV and bovine CoV infections. Most (632/651, 97.1%) camels had antibodies against MERS-CoV. This result included all 151 serum samples obtained in 2003. Most (389/651, 59.8%) serum samples had MERS-CoV-neutralizing antibody titers >1,280. Dromedary camels from the United Arab Emirates were infected at high rates with MERS-CoV or a closely related, probably conspecific, virus long before the first human MERS cases.


Subject(s)
Antibodies, Neutralizing/immunology , Camelus/immunology , Camelus/virology , Coronavirus Infections/immunology , Coronavirus/immunology , Respiratory Tract Infections/immunology , Animals , Antibodies, Viral/immunology , Coronavirus Infections/epidemiology , Neutralization Tests/methods , Respiratory Tract Infections/epidemiology , Respiratory Tract Infections/virology , Syndrome , United Arab Emirates/epidemiology
20.
Antimicrob Agents Chemother ; 58(2): 892-900, 2014.
Article in English | MEDLINE | ID: mdl-24277020

ABSTRACT

Clostridium difficile is a leading cause of health care-associated diarrhea with significant morbidity and mortality, and new options for the treatment of C. difficile-associated diarrhea (CDAD) are needed. Cadazolid is a new oxazolidinone-type antibiotic that is currently in clinical development for treatment of CDAD. Here, we report the in vitro and in vivo antibacterial evaluation of cadazolid against C. difficile. Cadazolid showed potent in vitro activity against C. difficile with a MIC range of 0.125 to 0.5 µg/ml, including strains resistant to linezolid and fluoroquinolones. In time-kill kinetics experiments, cadazolid showed a bactericidal effect against C. difficile isolates, with >99.9% killing in 24 h, and was more bactericidal than vancomycin. In contrast to metronidazole and vancomycin, cadazolid strongly inhibited de novo toxin A and B formation in stationary-phase cultures of toxigenic C. difficile. Cadazolid also inhibited C. difficile spore formation substantially at growth-inhibitory concentrations. In the hamster and mouse models for CDAD, cadazolid was active, conferring full protection from diarrhea and death with a potency similar to that of vancomycin. These findings support further investigations of cadazolid for the treatment of CDAD.


Subject(s)
Anti-Bacterial Agents/pharmacology , Clostridioides difficile/drug effects , Clostridium Infections/drug therapy , Enterocolitis, Pseudomembranous/drug therapy , Oxazolidinones/pharmacology , Spores, Bacterial/drug effects , Acetamides/pharmacology , Animals , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/biosynthesis , Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/biosynthesis , Clostridioides difficile/growth & development , Clostridioides difficile/metabolism , Clostridium Infections/microbiology , Clostridium Infections/mortality , Cricetinae , Enterocolitis, Pseudomembranous/microbiology , Enterocolitis, Pseudomembranous/mortality , Enterotoxins/antagonists & inhibitors , Enterotoxins/biosynthesis , Female , Fluoroquinolones/pharmacology , Humans , Linezolid , Male , Metronidazole/pharmacology , Mice , Mice, Inbred C57BL , Microbial Sensitivity Tests , Spores, Bacterial/growth & development , Survival Analysis , Vancomycin/pharmacology
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