A diagnostic host response biosignature for COVID-19 from RNA profiling of nasal swabs and blood.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease-19 (COVID-19), has emerged as the cause of a global pandemic. We used RNA sequencing to analyze 286 nasopharyngeal (NP) swab and 53 whole-blood (WB) samples from 333 patients with COVID-19 and controls. Overall, a muted immune response was observed in COVID-19 relative to other infections (influenza, other seasonal coronaviruses, and bacterial sepsis), with paradoxical down-regulation of several key differentially expressed genes. Hospitalized patients and outpatients exhibited up-regulation of interferon-associated pathways, although heightened and more robust inflammatory responses were observed in hospitalized patients with more clinically severe illness. Two-layer machine learning-based host classifiers consisting of complete (>1000 genes), medium (<100), and small (<20) gene biomarker panels identified COVID-19 disease with 85.1-86.5% accuracy when benchmarked using an independent test set. SARS-CoV-2 infection has a distinct biosignature that differs between NP swabs and WB and can be leveraged for COVID-19 diagnosis.

SARS-CoV-2 seroprevalence and neutralizing activity in donor and patient blood.

Given the limited availability of serological testing to date, the seroprevalence of SARS-CoV-2-specific antibodies in different populations has remained unclear. Here, we report very low SARS-CoV-2 seroprevalence in two San Francisco Bay Area populations. Seroreactivity was 0.26% in 387 hospitalized patients admitted for non-respiratory indications and 0.1% in 1,000 blood donors in early April 2020. We additionally describe the longitudinal dynamics of immunoglobulin-G (IgG), immunoglobulin-M (IgM), and in vitro neutralizing antibody titers in COVID-19 patients. The median time to seroconversion ranged from 10.3-11.0 days for these 3 assays. Neutralizing antibodies rose in tandem with immunoglobulin titers following symptom onset, and positive percent agreement between detection of IgG and neutralizing titers was >93%. These findings emphasize the importance of using highly accurate tests for surveillance studies in low-prevalence populations, and provide evidence that seroreactivity using SARS-CoV-2 anti-nucleocapsid protein IgG and anti-spike IgM assays are generally predictive of in vitro neutralizing capacity.

SARS-CoV-2 seroprevalence and neutralizing activity in donor and patient blood from the San Francisco Bay Area.

We report very low SARS-CoV-2 seroprevalence in two San Francisco Bay Area populations. Seropositivity was 0.26% in 387 hospitalized patients admitted for non-respiratory indications and 0.1% in 1,000 blood donors. We additionally describe the longitudinal dynamics of immunoglobulin-G, immunoglobulin-M, and in vitro neutralizing antibody titers in COVID-19 patients. Neutralizing antibodies rise in tandem with immunoglobulin levels following symptom onset, exhibiting median time to seroconversion within one day of each other, and there is >93% positive percent agreement between detection of immunoglobulin-G and neutralizing titers.

Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis.

The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP(+) during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.

Structural and enzymatic analyses reveal the binding mode of a novel series of Francisella tularensis enoyl reductase (FabI) inhibitors.

Because of structural and mechanistic differences between eukaryotic and prokaryotic fatty acid synthesis enzymes, the bacterial pathway, FAS-II, is an attractive target for the design of antimicrobial agents. We have previously reported the identification of a novel series of benzimidazole compounds with particularly good antibacterial effect against Francisella tularensis, a Category A biowarfare pathogen. Herein we report the crystal structure of the F. tularensis FabI enzyme in complex with our most active benzimidazole compound bound with NADH. The structure reveals that the benzimidazole compounds bind to the substrate site in a unique conformation that is distinct from the binding motif of other known FabI inhibitors. Detailed inhibition kinetics have confirmed that the compounds possess a novel inhibitory mechanism that is unique among known FabI inhibitors. These studies could have a strong impact on future antimicrobial design efforts and may reveal new avenues for the design of FAS-II active antibacterial compounds.

Discovery of a novel and potent class of F. tularensis enoyl-reductase (FabI) inhibitors by molecular shape and electrostatic matching.

Enoyl-acyl carrier protein (ACP) reductase, FabI, is a key enzyme in the bacterial fatty acid biosynthesis pathway (FAS II). FabI is an NADH-dependent oxidoreductase that acts to reduce enoyl-ACP substrates in a final step of the pathway. The absence of this enzyme in humans makes it an attractive target for the development of new antibacterial agents. FabI is known to be unresponsive to structure-based design efforts due to a high degree of induced fit and a mobile flexible loop encompassing the active site. Here we discuss the development, validation, and careful application of a ligand-based virtual screen used for the identification of novel inhibitors of the Francisella tularensis FabI target. In this study, four known classes of FabI inhibitors were used as templates for virtual screens that involved molecular shape and electrostatic matching. The program ROCS was used to search a high-throughput screening library for compounds that matched any of the four molecular shape queries. Matching compounds were further refined using the program EON, which compares and scores compounds by matching electrostatic properties. Using these techniques, 50 compounds were selected, ordered, and tested. The tested compounds possessed novel chemical scaffolds when compared to the input query compounds. Several hits with low micromolar activity were identified and follow-up scaffold-based searches resulted in the identification of a lead series with submicromolar enzyme inhibition, high ligand efficiency, and a novel scaffold. Additionally, one of the most active compounds showed promising whole-cell antibacterial activity against several Gram-positive and Gram-negative species, including the target pathogen. The results of a preliminary structure-activity relationship analysis are presented.

Structure of dihydroorotase from Bacillus anthracis at 2.6†Šresolution.

Dihydroorotase (EC 3.5.2.3) catalyzes the reversible cyclization of N-carbamoyl-L-aspartate to L-dihydroorotate in the third step of the pyrimidine-biosynthesis pathway in Bacillus anthracis. A comparison is made between the structures of dihydroorotase from four different organisms, including B. anthracis dihydroorotase, and reveals substantial variations in the active site, dimer interface and overall tertiary structure. These differences demonstrate the utility of exploring multiple structures of a molecular target as expressed from different organisms and how these differences can be exploited for structure-based drug discovery.

Structure of the Francisella tularensis enoyl-acyl carrier protein reductase (FabI) in complex with NAD(+) and triclosan.

Enoyl-acyl carrier protein reductase (FabI) catalyzes the last rate-limiting step in the elongation cycle of the fatty-acid biosynthesis pathway and has been validated as a potential antimicrobial drug target in Francisella tularensis. The development of new antibiotic therapies is important both to combat potential drug-resistant bioweapons and to address the broader societal problem of increasing antibiotic resistance among many pathogenic bacteria. The crystal structure of FabI from F. tularensis (FtuFabI) in complex with the inhibitor triclosan and the cofactor NAD(+) has been solved to a resolution of 2.1 Å. Triclosan is known to effectively inhibit FabI from different organisms. Precise characterization of the mode of triclosan binding is required to develop highly specific inhibitors. Comparison of our structure with the previously determined FtuFabI structure (PDB code 2jjy) which is bound to only NAD(+) reveals the conformation of the substrate-binding loop, electron density for which was missing in the earlier structure, and demonstrates a shift in the conformation of the NAD(+) cofactor. This shift in the position of the phosphate groups allows more room in the active site for substrate or inhibitor to bind and be better accommodated. This information will be crucial for virtual screening studies to identify novel scaffolds for development into new active inhibitors.

Glutamate racemase dimerization inhibits dynamic conformational flexibility and reduces catalytic rates.

Glutamate racemase (RacE) is a bacterial enzyme that converts l-glutamate to d-glutamate, an essential precursor for peptidoglycan synthesis. In prior work, we have shown that both isoforms cocrystallize with d-glutamate as dimers, and the enzyme is in a closed conformation with limited access to the active site [May, M., et al. (2007) J. Mol. Biol. 371, 1219-1237]. The active site of RacE2 is especially restricted. We utilize several computational and experimental approaches to understand the overall conformational dynamics involved during catalysis when the ligand enters and the product exits the active site. Our steered molecular dynamics simulations and normal-mode analysis results indicate that the monomeric form of the enzyme is more flexible than the native dimeric form. These results suggest that the monomeric enzyme might be more active than the dimeric form. We thus generated site-specific mutations that disrupt dimerization and find that the mutants exhibit significantly higher catalytic rates in the d-Glu to l-Glu reaction direction than the native enzyme. Low-resolution models restored from solution X-ray scattering studies correlate well with the first six normal modes of the dimeric form of the enzyme, obtained from NMA. Thus, along with the local active site residues, global domain motions appear to be implicated in the catalytically relevant structural dynamics of this enzyme and suggest that increased flexibility may accelerate catalysis. This is a novel observation that residues distant from the catalytic site restrain catalytic activity through formation of the dimer structure.