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Most libraries for fragment-based drug discovery are restricted to 1,000-10,000 compounds, but over 500,000 fragments are commercially available and potentially accessible by virtual screening. Whether this larger set would increase chemotype coverage, and

A key challenge in structure-based discovery is accounting for modulation of protein-ligand interactions by ordered and bulk solvent. To investigate this, we compared ligand binding to a buried cavity in Cytochrome c Peroxidase (CcP), where affinity is dom

Here, we report the near full-length genome sequence of a Vesiculovirus newjersey isolate obtained from a naturally infected cow ( Bos taurus ) in the state of Chiapas, Mexico. This sequence will support future efforts to improve our understanding of the evolutionary dynamics of this pathogen in endemic regions of Mexico., Competing Interests: The authors declare no conflict of interest.

Superinfection of cattle persistently infected with foot-and-mouth disease virus (FMDV), with a heterologous FMDV strain has been shown to generate novel recombinant viruses. In this study, we investigated the pathogenesis events within specific tissues associated with FMDV coinfections in cattle subjected to either simultaneous or serial exposure to two distinct strains of FMDV. Both strains of FMDV (one each of serotypes O and A) were similarly localized to the nasopharyngeal mucosa during the early stages of infection. However, while no recombinant FMDV genomes were recovered from simultaneously coinfected cattle, interserotypic recombinants were isolated from nasopharyngeal tissue samples obtained at 48 h after heterologous superinfection of a persistently infected FMDV carrier. Additionally, analysis of FMDV genomes obtained from replicate nasopharyngeal tissue samples demonstrated that adjacent segments of the mucosa were sometimes infected by distinct viruses, demonstrating a multifocal and heterogeneous distribution of FMDV infection during primary and persistent phases of infection. This work indicates that superinfection of FMDV carriers may be an important source of emergent recombinant strains of FMDV in areas where multiple strains are co-circulating. IMPORTANCE Foot-and-mouth disease (FMD) is a socioeconomically impactful livestock disease with a complex epidemiology and ecology. Although recombinant viruses have been identified in field samples, the mechanisms of emergence of those viruses have never been elucidated. This current study demonstrates how serial infection of cattle with two distinct serotypes of FMD virus (FMDV) leads to rapid generation of recombinant viruses in the upper respiratory tracts of infected animals. This finding is particularly relevant in relation to the management of persistently infected FMDV carrier cattle that can maintain subclinical FMDV infection for months to years after an initial infection. Such carrier animals may function as mixing vessels that facilitate the emergence of novel recombinant FMDV strains in areas where multiple virus strains are in circulation., Competing Interests: The authors declare no conflict of interest.

Codon deoptimization (CD) has been recently used as a possible strategy to derive foot-and-mouth disease (FMD) live-attenuated vaccine (LAV) candidates containing DIVA markers. However, reversion to virulence, or loss of DIVA, from possible recombination with wild-type (WT) strains has yet to be analyzed. An in vitro assay was developed to quantitate the levels of recombination between WT and a prospective A24-P2P3 partially deoptimized LAV candidate. By using two genetically engineered non-infectious RNA templates, we demonstrate that recombination can occur within non-deoptimized viral genomic regions (i.e., 3'end of P3 region). The sequencing of single plaque recombinants revealed a variety of genome compositions, including full-length WT sequences at the consensus level and deoptimized sequences at the sub-consensus/consensus level within the 3'end of the P3 region. Notably, after further passage, two recombinants that contained deoptimized sequences evolved to WT. Overall, recombinants featuring large stretches of CD or DIVA markers were less fit than WT viruses. Our results indicate that the developed assay is a powerful tool to evaluate the recombination of FMDV genomes in vitro and should contribute to the improved design of FMDV codon deoptimized LAV candidates.

We report the nearly full genome sequences of 14 isolates of serotype A foot-and-mouth disease virus and 5 isolates of serotype O, which were obtained from subclinically infected Asian buffalo in Pakistan in 2011 to 2012. Sequences from subclinically infected animals are rare and complement the more commonly available sequences from clinical cases.

We report the near full genome sequences of 18 isolates of foot-and-mouth disease virus serotype O and 6 isolates of serotype A obtained from outbreaks in Pakistan between 2011 and 2012. The scarcity of full-length FMDV sequences from this region enhances the importance of these genomes for understanding regional molecular epidemiology.

We report the near-full-length genome sequences of 22 isolates of foot-and-mouth disease virus (FMDV) serotype Asia-1, lineage Sindh-08, obtained from foot-and-mouth disease outbreaks in Pakistan between 2011 and 2012. The scarcity of full-length FMDV sequences from this region enhances the importance of these new genomes for understanding the regional molecular epidemiology.

We report the near-full-genome sequences of 49 isolates of serotype Asia-1 foot-and-mouth disease virus obtained from subclinically infected Asian buffalo in Islamabad Capital Region, Pakistan, in 2011 to 2012. Sequences from subclinically infected animals are exceedingly rare and complement the more commonly available sequences acquired from clinical cases.

Viral recombination contributes to the emergence of novel strains with the potential for altered host range, transmissibility, virulence, and immune evasion. For foot-and-mouth disease virus (FMDV), cell culture experiments and phylogenetic analyses of field samples have demonstrated the occurrence of recombination. However, the frequency of recombination and associated virus-host interactions within an infected host have not been determined. We have previously reported the detection of interserotypic recombinant FMDVs in oropharyngeal fluid (OPF) samples of 42% (5/12) of heterologously superinfected FMDV carrier cattle. The present investigation consists of a detailed analysis of the virus populations in these samples including identification and characterization of additional interserotypic minority recombinants. In every animal in which recombination was detected, recombinant viruses were identified in the OPF at the earliest sampling point after superinfection. Some recombinants remained dominant until the end of the experiment, whereas others were outcompeted by parental strains. Genomic analysis of detected recombinants suggests host immune pressure as a major driver of recombinant emergence as all recombinants had capsid-coding regions derived from the superinfecting virus to which the animals did not have detectable antibodies at the time of infection. In vitro analysis of a plaque-purified recombinant virus demonstrated a growth rate comparable to its parental precursors, and measurement of its specific infectivity suggested that the recombinant virus incurred no penalty in packaging its new chimeric genome. These findings have important implications for the potential role of persistently infected carriers in FMDV ecology and the emergence of novel strains.

The identification and isolation of highly infectious SARS-CoV-2-infected individuals is an important public health strategy. Rapid antigen detection tests (RADT) are promising tools for large-scale screenings due to timely results and feasibility for on-site testing. Nonetheless, the diagnostic performance of RADT in detecting infectious individuals is not yet fully determined. In this study, RT-qPCR and virus culture of RT-qPCR-positive samples were used to evaluate and compare the performance of the Standard Q COVID-19 Ag test in detecting SARS-CoV-2-infected and possibly infectious individuals. To this end, two combined oro- and nasopharyngeal swabs were collected at a routine SARS-CoV-2 diagnostic center. A total of 2,028 samples were tested, and 118 virus cultures were inoculated. SARS-CoV-2 infection was detected in 210 samples by RT-qPCR, representing a positive rate of 10.36%. The Standard Q COVID-19 Ag test yielded a positive result in 92 (4.54%) samples resulting in an overall sensitivity and specificity of 42.86 and 99.89%, respectively. For adjusted C T values of <20 ( n  = 14), <25 ( n  = 57), and <30 ( n  = 88), the RADT reached sensitivities of 100, 98.25, and 88.64%, respectively. All 29 culture-positive samples were detected by the RADT. Although the overall sensitivity was low, the Standard Q COVID-19 Ag test reliably detected patients with high RNA loads. In addition, negative RADT results fully corresponded with the lack of viral cultivability in Vero E6 cells. These results indicate that RADT can be a valuable tool for the detection of individuals with high RNA loads that are likely to transmit SARS-CoV-2.

BceF is a bacterial tyrosine kinase (BY-kinase) from Burkholderia cepacia , a Gram-negative bacterium accountable for respiratory infections in immunocompromised and cystic fibrosis patients. BceF is involved in the production of exopolysaccharides secreted to the biofilm matrix and promotes resistant and aggressive infections. BY-kinases share no homology with mammalian kinases, and thereby offer a means to develop novel and specific antivirulence drugs. Here, we report the crystal structure of the BceF kinase domain at 1.85 Å resolution. The isolated BceF kinase domain is assembled as a dimer in solution and crystallized as a dimer in the asymmetric unit with endogenous adenosine-diphosphate bound at the active sites. The low enzymatic efficiency measured in solution may be explained by the partial obstruction of the active sites at the crystallographic dimer interface. This study provides insights into self-assembly and the specific activity of isolated catalytic domains. Several unique variations around the active site compared to other BY-kinases may allow for structure-based design of specific inhibitors to target Burkholderia cepacia virulence.

Dopamine D1 receptor (D1R) is an important drug target implicated in many psychiatric and neurological disorders. Selective agonism of D1R are sought to be the therapeutic strategy for these disorders. Most selective D1R agonists share a dopamine-like catechol moiety in their molecular structure, and their therapeutic potential is therefore limited by poor pharmacological properties in vivo. Recently, a class of non-catechol D1R selective agonists with a distinct scaffold and pharmacological properties were reported. Here, we report the crystal structure of D1R in complex with stimulatory G protein (Gs) and a non-catechol agonist Compound 1 at 3.8 Å resolution. The structure reveals the ligand bound to D1R in an extended conformation, spanning from the orthosteric site to extracellular loop 2 (ECL2). Structural analysis reveals that the unique features of D1R ligand binding pocket explains the remarkable selectivity of this scaffold for D1R over other aminergic receptors, and sheds light on the mechanism for D1R activation by the non-catechol agonist.

AMHRII, the anti-Müllerian hormone receptor, is selectively expressed in normal sexual organs but is also re-expressed in gynecologic cancers. Hence, we developed murlentamab, a humanized glyco-engineered anti-AMHRII monoclonal antibody currently in clinical trial. Low-fucosylated antibodies are known to increase the antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) potency of effector cells, but some preliminary results suggest a more global murlentamab-dependent activation of the immune system. In this context, we demonstrate here that the murlentamab opsonization of AMHRII-expressing ovarian tumor cells, in the presence of unstimulated- or tumor-associated macrophage (TAM)-like macrophages, significantly promotes macrophage-mediated ADCC and shifts the whole microenvironment towards a pro-inflammatory and anti-tumoral status, thus triggering anti-tumor activity. We also report that murlentamab orients both unstimulated- and TAM-like macrophages to an M1-like phenotype characterized by a strong expression of co-stimulation markers, pro-inflammatory cytokines and chemokines, favoring T cell recruitment and activation. Moreover, we show that murlentamab treatment shifts CD4 + Th1/Th2 balance towards a Th1 response and activates CD8 + T cells. Altogether, these results suggest that murlentamab, through naïve macrophage orientation and TAM reprogrammation, stimulates the anti-tumor adaptive immune response. Those mechanisms might contribute to the sustained clinical benefit observed in advanced cancer patients treated with murlentamab. Finally, the enhanced murlentamab activity in combination with pembrolizumab opens new therapeutic perspectives.

Foot-and-mouth disease virus (FMDV) infects hosts as a population of closely related viruses referred to as a quasispecies. The behavior of this quasispecies has not been described in detail in natural host species. In this study, virus samples collected from vaccinated and non-vaccinated cattle up to 35 days post-experimental infection with FMDV A24-Cruzeiro were analyzed by deep-sequencing. Vaccination induced significant differences compared to viruses from non-vaccinated cattle in substitution rates, entropy, and evidence for adaptation. Genomic variation detected during early infection reflected the diversity inherited from the source virus (inoculum), whereas by 12 days post infection, dominant viruses were defined by newly acquired mutations. Mutations conferring recognized fitness gain occurred and were associated with selective sweeps. Persistent infections always included multiple FMDV subpopulations, suggesting distinct foci of infection within the nasopharyngeal mucosa. Subclinical infection in vaccinated cattle included very early bottlenecks associated with reduced diversity within virus populations. Viruses from both animal cohorts contained putative antigenic escape mutations. However, these mutations occurred during later stages of infection, at which time transmission is less likely to occur. This study improves upon previously published work by analyzing deep sequences of samples, allowing for detailed characterization of FMDV populations over time within multiple hosts.

This is the first report of two near-complete genome sequences of foot-and-mouth disease virus (FMDV) serotype O from Kenya. The viruses were isolated from bovine epithelium collected in 2014 and 2016 from local FMD outbreaks. These full-genome sequences are critical for improving the understanding of regional FMDV molecular epidemiology.

Foot-and-mouth disease (FMD) is a highly contagious viral disease that severely impacts global food security and is one of the greatest constraints on international trade of animal products. Extensive viral population diversity and rapid, continuous mutation of circulating FMD viruses (FMDVs) pose significant obstacles to the control and ultimate eradication of this important transboundary pathogen. The current study investigated mechanisms contributing to within-host evolution of FMDV in a natural host species (cattle). Specifically, vaccinated and non-vaccinated cattle were infected with FMDV under controlled, experimental conditions and subsequently sampled for up to 35 days to monitor viral genomic changes as related to phases of disease and experimental cohorts. Consensus-level genomic changes across the entire FMDV coding region were characterized through three previously defined stages of infection: early, transitional, and persistent. The overall conclusion was that viral evolution occurred via a combination of two mechanisms: emergence of full-genomic minority haplotypes from within the inoculum super-swarm, and concurrent continuous point mutations. Phylogenetic analysis indicated that individuals were infected with multiple distinct haplogroups that were pre-existent within the ancestral inoculum used to infect all animals. Multiple shifts of dominant viral haplotype took place during the early and transitional phases of infection, whereas few shifts occurred during persistent infection. Overall, this work suggests that the establishment of the carrier state is not associated with specific viral genomic characteristics. These insights into FMDV population dynamics have important implications for virus sampling methodology and molecular epidemiology., Competing Interests: The authors have declared that no competing interests exist.

Drugs that treat chronic obstructive pulmonary disease by antagonizing the M3 muscarinic acetylcholine receptor (M3R) have had a significant effect on health, but can suffer from their lack of selectivity against the M2R subtype, which modulates heart rate. Beginning with the crystal structures of M2R and M3R, we exploited a single amino acid difference in their orthosteric binding pockets using molecular docking and structure-based design. The resulting M3R antagonists had up to 100-fold selectivity over M2R in affinity and over 1,000-fold selectivity in vivo. The crystal structure of the M3R-selective antagonist in complex with M3R corresponded closely to the docking-predicted geometry, providing a template for further optimization., Competing Interests: Conflict of interest statement: B.K.K. is a cofounder of and consultant for ConfometRx, Inc., (Copyright © 2018 the Author(s). Published by PNAS.)

Introduction : Depression is associated with negative social, economic, and family outcomes and the majority of individuals with depression in low and middle income countries (LMICs) are untreated. A critical first step in bridging the treatment gap is accurate, feasible, and culturally appropriate screening to identify those who need treatment. The WHO's Perceived Well-Being Index (WHO-5) well-being instrument can potentially meet the screening needs of LMICs in primary care and community-based settings. This study tested the feasibility and validity of this tool to identify depression among adult parents of young children in Addis Ababa, Ethiopia. Successful identification and treatment of depression in parents extends benefits to children and families. Method : The WHO-5 was translated to Amharic and administered to 849 adults and compared with simultaneous administration of the well-established PHQ-9 instrument. Feasibility was assessed and analyses evaluated frequency of positive screens for depression, internal consistency, sensitivity and specificity of the WHO-5, and sociodemographic correlates of depression. Results : The prevalence of probable depression was similar as assessed by the PHQ-9 (17.3%) and the WHO-5 (18.5%). The internal consistency of the WHO-5 was strong (Cronbach's alpha = .83). WHO-5 agreement with the PHQ-9 was moderate; sensitivity and specificity were strong. Correlates of depression included unemployment and financial status. Discussion : The study provides promising evidence to support use of the WHO-5 to identify depression in Ethiopia. Feasibility was good, and it was culturally and linguistically acceptable. The results suggest that minimally trained community health and education workers in countries like Ethiopia could use the WHO-5 effectively in primary health and education settings. (PsycINFO Database Record, ((c) 2018 APA, all rights reserved).)

The seven transmembrane protein Smoothened is required for Hedgehog signaling during embryonic development and adult tissue homeostasis. Inappropriate activation of the Hedgehog signalling pathway leads to cancers such as basal cell carcinoma and medulloblastoma, and Smoothened inhibitors are now available clinically to treat these diseases. However, resistance to these inhibitors rapidly develops thereby limiting their efficacy. The determination of Smoothened crystal structures enables structure-based discovery of new ligands with new chemotypes that will be critical to combat resistance. In this study, we docked 3.2 million available, lead-like molecules against Smoothened, looking for those with high physical complementarity to its structure; this represents the first such campaign against the class Frizzled G-protein coupled receptor family. Twenty-one high-ranking compounds were selected for experimental testing, and four, representing three different chemotypes, were identified to antagonize Smoothened with IC50 values better than 50 μM. A screen for analogs revealed another six molecules, with IC50 values in the low micromolar range. Importantly, one of the most active of the new antagonists continued to be efficacious at the D473H mutant of Smoothened, which confers clinical resistance to the antagonist vismodegib in cancer treatment.

A key challenge in structure-based discovery is accounting for modulation of protein-ligand interactions by ordered and bulk solvent. To investigate this, we compared ligand binding to a buried cavity in Cytochrome c Peroxidase (CcP), where affinity is dominated by a single ionic interaction, versus a cavity variant partly opened to solvent by loop deletion. This opening had unexpected effects on ligand orientation, affinity, and ordered water structure. Some ligands lost over ten-fold in affinity and reoriented in the cavity, while others retained their geometries, formed new interactions with water networks, and improved affinity. To test our ability to discover new ligands against this opened site prospectively, a 534,000 fragment library was docked against the open cavity using two models of ligand solvation. Using an older solvation model that prioritized many neutral molecules, three such uncharged docking hits were tested, none of which was observed to bind; these molecules were not highly ranked by the new, context-dependent solvation score. Using this new method, another 15 highly-ranked molecules were tested for binding. In contrast to the previous result, 14 of these bound detectably, with affinities ranging from 8 µM to 2 mM. In crystal structures, four of these new ligands superposed well with the docking predictions but two did not, reflecting unanticipated interactions with newly ordered waters molecules. Comparing recognition between this open cavity and its buried analog begins to isolate the roles of ordered solvent in a system that lends itself readily to prospective testing and that may be broadly useful to the community.

Rates of tyrosine and lysine transport and incorporation into protein were measured in control and undernourished weanling rats. Undernutrition was induced by feeding lactating dams a low protein diet (12 percent casein) from birth to day 21. At weaning, body and brain weights of undernourished rats were 50 percent and 88 percent, respectively, of control values. Lysine and tyrosine transport rates into skeletal muscle were reduced by over 75 percent, more than twice the reduction seen in brain. Rates of amino acid incorporation into muscle protein were reduced by approximately 50 percent; the change in rate of incorporation into brain protein was not statistically significant. These data indicate that, in spite of marked retardation of amino acid transport into brain, the brain seems fully capable of maintaining normal rates of protein synthesis.