An open repository of real-time COVID-19 indicators.

The COVID-19 pandemic presented enormous data challenges in the United States. Policy makers, epidemiological modelers, and health researchers all require up-to-date data on the pandemic and relevant public behavior, ideally at fine spatial and temporal resolution. The COVIDcast API is our attempt to fill this need: Operational since April 2020, it provides open access to both traditional public health surveillance signals (cases, deaths, and hospitalizations) and many auxiliary indicators of COVID-19 activity, such as signals extracted from deidentified medical claims data, massive online surveys, cell phone mobility data, and internet search trends. These are available at a fine geographic resolution (mostly at the county level) and are updated daily. The COVIDcast API also tracks all revisions to historical data, allowing modelers to account for the frequent revisions and backfill that are common for many public health data sources. All of the data are available in a common format through the API and accompanying R and Python software packages. This paper describes the data sources and signals, and provides examples demonstrating that the auxiliary signals in the COVIDcast API present information relevant to tracking COVID activity, augmenting traditional public health reporting and empowering research and decision-making.

Effectiveness and adverse effects of the use of mirtazapine as compared to duloxetine for fibromyalgia: real-life data from a retrospective cohort.

On the background of a restricted armamentarium of drugs available for the management of fibromyalgia (FM), we aimed to compare the real-world effectiveness of two serotonin-norepinephrine reuptake inhibitors (SNRIs), mirtazapine (MTZ) and duloxetine (DLX) in FM. A medical records review was done to identify patients diagnosed with FM and prescribed a stable dose of either MTZ or DLX for more than 6 months. Their present status was determined by a telephonic interview which included a subjective assessment of improvement (Likert scale), FIQR (Revised Fibromyalgia Impact Questionnaire), adverse drug effects and compliance. One-fifty-eight patients were screened to include 81 patients [mean age 46.7 (± 13.0) years, 64 (79%) females]. Sixty (79%) had primary fibromyalgia and 66 (81.5%) were on DLX (20-40 mg) while 15(18.5%) were on MTZ (7.5 mg). In addition to the drugs, lifestyle modification was followed by 57 (70.3%). A moderate-to-good improvement was seen in 66 (81.5%), while 15 (18.5%) reported poor to no improvement overall. In the DLX group, a majority (59, 89.4%) showed moderate-to-good improvement compared to 7(46.7%) on MTZ [p = 0.001, 9.6(2.6-34)]. However, FIQR was similar for those on DLX (3.6 ± 0.9) and MTZ (3.8 ± 0.7). Adverse effects were reported for 51 (77%) of patients on DLX and all (100%) on MTZ with a poorer compliance with MTZ 5 (33.3%) compared to DLX 47 (71.2%) [p = 0.008, OR 0.1(0.03-0.4)]. On multivariate analysis, DLX use [OR 16.7 (95% CI 2.7-100); p = 0.008] and lifestyle modification [p = 0.002; OR 11.2(1.5-83.3)] were associated with better subjective outcomes. Low-dose MTZ appears to be inferior to DLX in the management of FM in this real-world cohort.

Nonstructural protein 5B promotes degradation of the NORE1A tumor suppressor to facilitate hepatitis C virus replication.

Hepatitis C virus (HCV) infection is a common risk factor for the development of liver cancer. The molecular mechanisms underlying this effect are only partially understood. Here, we show that the HCV protein, nonstructural protein (NS) 5B, directly binds to the tumor suppressor, NORE1A (RASSF5), and promotes its proteosomal degradation. In addition, we show that NORE1A colocalizes to sites of HCV viral replication and suppresses the replication process. Thus, NORE1A has antiviral activity, which is specifically antagonized by NS5B. Moreover, the suppression of NORE1A protein levels correlated almost perfectly with elevation of Ras activity in primary human samples. Therefore, NORE1A inactivation by NS5B may be essential for maximal HCV replication and may make a major contribution to HCV-induced liver cancer by shifting Ras signaling away from prosenescent/proapoptotic signaling pathways. CONCLUSION: HCV uses NS5B to specifically suppress NORE1A, facilitating viral replication and elevated Ras signaling. (Hepatology 2017;65:1462-1477).

Anti-cancer and anti-hepatitis C virus NS5B polymerase activity of etodolac 1,2,4-triazoles.

Arachidonic acid is an unsaturated fatty acid liberated from phospholipids of cell membranes. NSAIDs are known as targets of cyclooxygenase enzyme (COX-1, COX-2 and COX-3) in arachidonic acid metabolism. This mechanism of COX-2 in carcinogenesis causes cancer. In addition, COX-2 plays a role in the early stages of hepatocarcinogenesis. Hepatitis C virus (HCV) infection is cause of liver cirrhosis and hepatocellular carcinoma (HCC). The aim of our study was to improve effective agents against HCV. A novel series of new etodolac 1,2,4-triazoles derivatives (4a-h) have been synthesized and investigated for their activity against HCV NS5B polymerase. Compound 4a was found to be the most active with IC(50) value of 14.8 µM. In accordance with these results, compound 4a was screened for anti-cancer activity on liver cancer cell lines (Huh7, Mahlavu, HepG2, FOCUS). Compound 4a showed anti-cancer activity against Huh7 human hepatoma cell line with IC(50) value of 4.29 µM. Therefore, compound 4a could be considered as a new anti-cancer and anti-HCV lead compound.

Novel 4-thiazolidinones as non-nucleoside inhibitors of hepatitis C virus NS5B RNA-dependent RNA polymerase.

In continuation of our efforts to develop new derivatives as hepatitis C virus (HCV) NS5B inhibitors, we synthesized novel 5-arylidene-4-thiazolidinones. The novel compounds 29-42, together with their synthetic precursors 22-28, were tested for HCV NS5B inhibitory activity; 12 of these compounds displayed IC50 values between 25.3 and 54.1 µM. Compound 33, an arylidene derivative, was found to be the most active compound in this series with an IC50 value of 25.3 µM. Molecular docking studies were performed on the thumb pocket-II of NS5B to postulate the binding mode for these compounds.

Multiple e-pharmacophore modeling, 3D-QSAR, and high-throughput virtual screening of hepatitis C virus NS5B polymerase inhibitors.

The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRP) is a crucial and unique component of the HCV RNA replication machinery and a validated target for drug discovery. Multiple crystal structures of NS5B inhibitor complexes have facilitated the identification of novel compound scaffolds through in silico analysis. With the goal of discovering new NS5B inhibitor leads, HCV NS5B crystal structures bound with inhibitors in the palm and thumb allosteric pockets in combination with ligands with known inhibitory potential were explored for a comparative pharmacophore analyses. The energy-based and 3D-QSAR-based pharmacophore models were validated using enrichment analysis, and the six models thus developed were employed for high-throughput virtual screening and docking to identify nonpeptidic leads. The hits derived at each stage were analyzed for diversity based on the six pharmacophore models, followed by molecular docking and filtering based on their interaction with amino acids in the NS5B allosteric pocket and 3D-QSAR predictions. The resulting 10 hits displaying diverse scaffold were then screened employing biochemical and cell-based NS5B and anti-HCV inhibition assays. Of these, two molecules H-5 and H-6 were the most promising, exhibiting IC50 values of 28.8 and 47.3 µM against NS5B polymerase and anti-HCV inhibition of 96% and 86% at 50 µM, respectively. The identified leads comprised of benzimidazole (H-5) and pyridine (H-6) scaffolds thus constitute prototypical molecules for further optimization and development as NS5B inhibitors.

Design and synthesis of L- and D-phenylalanine derived rhodanines with novel C5-arylidenes as inhibitors of HCV NS5B polymerase.

Hepatitis C virus (HCV) NS5B polymerase is a key target for anti-HCV therapeutics development. Herein, we report the synthesis and in vitro evaluation of anti-NS5B polymerase activity of a molecular hybrid of our previously reported lead compounds 1 (IC50=7.7 µM) and 2 (IC50=10.6 µM) as represented by hybrid compound 27 (IC50=6.7 µM). We have explored the optimal substituents on the terminal phenyl ring of the 3-phenoxybenzylidene moiety in 27, by generating a set of six analogs. This resulted in the identification of compound 34 with an IC50 of 2.6 µM. To probe the role of stereochemistry towards the observed biological activity, we synthesized and evaluated the D-isomers 41 (IC50=19.3 µM) and 45 (IC50=5.4 µM) as enantiomers of the l-isomers 27 and 34, respectively. The binding site of compounds 32 and 34 was mapped to palm pocket-I (PP-I) of NS5B. The docking models of 34 and 45 within the PP-I of NS5B were investigated to envisage the molecular mechanism of inhibition.

Synthesis and characterization of celecoxib derivatives as possible anti-inflammatory, analgesic, antioxidant, anticancer and anti-HCV agents.

A series of novel N-(3-substituted aryl/alkyl-4-oxo-1,3-thiazolidin-2-ylidene)-4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamides 2a-e were synthesized by the addition of ethyl a-bromoacetate and anhydrous sodium acetate in dry ethanol to N-(substituted aryl/alkylcarbamothioyl)-4-[5-(4-methylphenyl)-3-(trifluoro-methyl)-1H-pyrazol-1-yl]benzene sulfonamides 1a-e, which were synthesized by the reaction of alkyl/aryl isothiocyanates with celecoxib. The structures of the isolated products were determined by spectral methods and their anti-inflammatory, analgesic, antioxidant, anticancer and anti-HCV NS5B RNA-dependent RNA polymerase (RdRp) activities evaluated. The compounds were also tested for gastric toxicity and selected compound 1a was screened for its anticancer activity against 60 human tumor cell lines. These investigations revealed that compound 1a exhibited anti-inflammatory and analgesic activities and further did not cause tissue damage in liver, kidney, colon and brain compared to untreated controls or celecoxib. Compounds 1c and 1d displayed modest inhibition of HCV NS5B RdRp activity. In conclusion, N-(ethylcarbamothioyl)-4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (1a) may have the potential to be developed into a therapeutic agent.

Identification and characterization of coumestans as novel HCV NS5B polymerase inhibitors.

The hepatitis C virus (HCV) NS5B is essential for viral RNA replication and is therefore a prime target for development of HCV replication inhibitors. Here, we report the identification of a new class of HCV NS5B inhibitors belonging to the coumestan family of phytoestrogens. Based on the in vitro NS5B RNA-dependent RNA polymerase (RdRp) inhibition in the low micromolar range by wedelolactone, a naturally occurring coumestan, we evaluated the anti-NS5B activity of four synthetic coumestan analogues bearing different patterns of substitutions in their A and D rings, and observed a good structure-activity correlation. Kinetic characterization of coumestans revealed a noncompetitive mode of inhibition with respect to nucleoside triphosphate (rNTP) substrate and a mixed mode of inhibition towards the nucleic acid template, with a major competitive component. The modified order of addition experiments with coumestans and nucleic acid substrates affected the potencies of the coumestan inhibitors. Coumestan interference at the step of NS5B-RNA binary complex formation was confirmed by cross-linking experiments. Molecular docking of coumestans within the allosteric site of NS5B yielded significant correlation between their calculated binding energies and IC(50) values. Coumestans thus add to the diversifying pool of anti-NS5B agents and provide a novel scaffold for structural refinement and development of potent NS5B inhibitors.

4-Thiazolidinones: a novel class of hepatitis C virus NS5B polymerase inhibitors.

In a quest to identify novel compounds targeting HCV viral replicase, we evaluated a new series of 4-thiazolidinone derivatives (18 compounds). Our in vitro NS5B RdRp inhibition analysis with a series of 2',4'-difluoro-4-hydroxybiphenyl-3-carboxylic acid (2-(5-nitro-2-furyl/substituted phenyl)-4-thiazolidinone-3-yl) amides (1-7) yielded IC50 values ranging between 45-75 microM. Of these, lead compound 6: 2',4'-difluoro-4-hydroxybiphenyl-3-carboxylic acid(2-(2-fluorophenyl)-4-thiazolidinone-3-yl)amide exhibited an IC50 value of 48 microM and inhibited NS5B non-competitively with respect to UTP and exhibited a mixed mode of inhibition with respect to RNA. Molecular docking of thiazolidinone derivatives within the allosteric site of NS5B yielded significant correlation between their calculated binding affinity and IC50 values. Taken together, these data suggest that the 4-thiazolidinone scaffold may be optimized for generating new analogues with improved anti-NS5B potency.

Peptide inhibition of HIV-1: current status and future potential.

More than 2 decades of intensive research has focused on defining replication mechanisms of HIV type 1 (HIV-1), the etiologic agent of AIDS. The delineation of strategies for combating this viral infection has yielded many innovative approaches toward this end. HIV-1 is a lentivirus in the family retroviridae that is relatively small with regard to both structure and genome size, having a diploid RNA genome of approximately 9 kb, with only three major genes and several gene products resulting from alternate splicing and translational frameshifting. Most marketed drugs for treating AIDS are inhibitors of HIV-1 reverse transcriptase or protease enzymes, but new targets include the integrase enzyme, cell surface interactions that facilitate viral entry, and also virus particle maturation and assembly. The emergence of drug-resistant variants of HIV-1 has been the main impediment to successful treatment of AIDS. Thus, there is a pressing need to develop novel treatment strategies targeting multiple stages of the virus life-cycle. Research efforts aimed at developing successful means for combating HIV-1 infection have included development of peptide inhibitors of HIV-1. This article summarizes past and current endeavors in the development of peptides that inhibit replication of HIV-1 and the role of peptide inhibitors in the search for new anti-HIV drugs.

A highly stable polyethylene glycol-conjugated human single-chain antibody neutralizing granulocyte-macrophage colony stimulating factor at low nanomolar concentration.

GM-CSF (granulocyte-macrophage colony stimulating factor) plays a central role in inflammatory processes. Treatment with antibodies neutralizing murine GM-CSF showed significant therapeutic effects in mouse models of inflammatory diseases. We constructed by phage display technology a human scFv, which could potently neutralize human GM-CSF. At first, a human V(L) repertoire was combined with the V(H) domain of a parental GM-CSF-neutralizing rat antibody. One dominant rat/human scFv clone was selected, neutralizing human GM-CSF with an IC50 of 7.3 nM. The human V(L) of this clone was then combined with a human V(H) repertoire. The latter preserved the CDR 3 of the parental rat V(H) domain to retain binding specificity. Several human scFvs were selected, which neutralized human GM-CSF at low nanomolar concentrations (IC50 > or = 2.6 nM). To increase serum half-life, a branched 40 kDa PEG-polymer was coupled to the most potent GM-CSF-neutralizing scFv (3077) via an additional C-terminal cysteine. PEG conjugation had a negligible effect on the in vitro neutralizing potential of the scFv, although it caused a significant drop in binding affinity owing to a reduced on-rate. It also significantly increased the stability of the scFv at elevated temperatures. In mouse experiments, the PEGylated scFv 3077 showed a significantly prolonged elimination half-life of 59 h as compared with 2 h for the unconjugated scFv version. PEGylated scFv 3077 is a potential candidate for development of a novel antibody therapy to treat pro-inflammatory human diseases.

Synthesis of novel diflunisal hydrazide-hydrazones as anti-hepatitis C virus agents and hepatocellular carcinoma inhibitors.

Hepatitis C virus (HCV) infection is a main cause of chronic liver disease, leading to liver cirrhosis and hepatocellular carcinoma (HCC). The objective of our research was to develop effective agents against viral replication. We have previously identified the hydrazide-hydrazone scaffold as a promising hepatitis C virus (HCV) and hepatocelluler inhibitor. Herein we describe the design a number of 2',4'-difluoro-4-hydroxy-N'-(arylmethylidene) biphenyl-3-carbohydrazide (3a-t) as anti-HCV and anticancer agents. Results from evaluation of anti-HCV activity indicated that most of the synthesized hydrazone derivatives inhibited viral replication in the Huh7/Rep-Feo1b and Huh 7.5-FGR-JCI-Rluc2A reporter systems. Antiproliferative activities of increasing concentrations of 2',4'-difluoro-4-hydroxy-N'-(2-pyridyl methylidene)biphenyl-3-carbohydrazide 3b and diflunisal (2.5-40 µM) were assessed in liver cancer cell lines (Huh7, HepG2, Hep3B, Mahlavu, FOCUS and SNU-475) with sulforhodamine B assay for 72 h. Compound 3b with 2-pyridinyl group in the hydrazone part exhibited promising cytotoxic activity against all cell lines with IC50 values of 10, 10.34 16.21 4.74, 9.29 and 8.33 µM for Huh7, HepG2, Hep3B, Mahlavu, FOCUS and SNU-475 cells, respectively, and produced dramatic cell cycle arrest at SubG1/G0 phase as an indicator of apoptotic cell death induction.

5-Carba-pterocarpens: A new scaffold with anti-HCV activity.

The synthesis of a series of 5-carba-pterocarpens derivatives involving the cyclization of α-aryl-α-tetralones is described. Several compounds demonstrated potent activity and selectivity in vitro against HCV replicon reporter cells. The best profile in Huh7/Rep-Feo1b replicon reporter cells was observed with 2h (EC50 = 5.5 µM/SI = 20), while 2e was the most active in Huh7.5-FGR-JC1-Rluc2A replicon reporter cells (EC50 = 1.5 µM/SI = 70). Hydroxy groups at A- and D-rings are essential for anti-HCV activity, and substitutions in the A-ring at positions 3 and 4 resulted in enhanced activity of the compounds.

Bicyclic octahydrocyclohepta[b]pyrrol-4(1H)one derivatives as novel selective anti-hepatitis C virus agents.

We report the discovery of the bicyclic octahydrocyclohepta[b]pyrrol-4(1H)-one scaffold as a new chemotype with anti-HCV activity on genotype 1b and 2a subgenomic replicons. The most potent compound 34 displayed EC50 values of 1.8 µM and 4.5 µM in genotype 1b and 2a, respectively, coupled with the absence of any antimetabolic effect (gt 1b SI = 112.4; gt 2a SI = 44.2) in a cell-based assay. Compound 34 did not target HCV NS5B, IRES, NS3 helicase, or selected host factors, and thus future work will involve the unique mechanism of action of these new antiviral compounds.

Synthesis and biological evaluation of α-aryl-α-tetralone derivatives as hepatitis C virus inhibitors.

The synthesis of a novel series of 1-carba-isoflavanones through the α-arylation of α-tetralones is described. Several of these compounds demonstrated potent activity and selectivity in-vitro against HCV replicon reporter cells. Compound 10 (LQB-314) exhibited the best profile being active and selective in both replicon reporter cells (IC50 1.8 µM, SI > 111 and IC50 4.3 µM, SI > 46 in Huh7/Rep-Feo1b and Huh7.5-FGR-JC1-Rluc2A, respectively). Compound 3 (LQB-307) was the more potent and selective for Huh7.5-FGR-JC1-Rluc2A replicon reporter cells (IC50 1.5 µM, SI > 101.4).

New 1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides inhibit hepatitis C virus replication via suppression of cyclooxygenase-2.

We report here the synthesis and mechanism of inhibition of pyrazolecarboxamide derivatives as a new class of HCV inhibitors. Compounds 6, 7, 8 and 16 inhibited the subgenomic HCV replicon 1b genotype at EC50 values between 5 and 8 µM and displayed an even higher potency against the infectious Jc1 HCV 2a genotype. Compound 6 exhibited an EC50 of 6.7 µM and selectivity index of 23 against HCV 1b, and reduced the RNA copies of the infectious Jc1 chimeric 2a clone by 82% at 7 µM. Evaluation of the mode of anti-HCV activity of 6 revealed that it suppressed HCV-induced COX-2 mRNA and protein expression, displaying an IC50 of 3.2 µM in COX-2 promoter-linked luciferase reporter assay. Conversely, the anti-HCV activity of 6 was abrogated upon over-expression of COX-2. These findings suggest that 6 as a representative of these pyrazolecarboxamides function as anti-HCV agents via targeting COX-2 at both the transcription and translation levels.

Discovery of the 2-phenyl-4,5,6,7-Tetrahydro-1H-indole as a novel anti-hepatitis C virus targeting scaffold.

Although all-oral direct-acting antiviral (DAA) therapy for hepatitis C virus (HCV) treatment is now a reality, today's HCV drugs are expensive, and more affordable drugs are still urgently needed. In this work, we report the identification of the 2-phenyl-4,5,6,7-Tetrahydro-1H-indole chemical scaffold that inhibits cellular replication of HCV genotype 1b and 2a subgenomic replicons. The anti-HCV genotype 1b and 2a profiling and effects on cell viability of a selected representative set of derivatives as well as their chemical synthesis are described herein. The most potent compound 39 displayed EC50 values of 7.9 and 2.6 µM in genotype 1b and 2a, respectively. Biochemical assays showed that derivative 39 had no effect on HCV NS5B polymerase, NS3 helicase, IRES mediated translation and selected host factors. Thus, future work will involve both the chemical optimization and target identification of 2-phenyl-4,5,6,7-Tetrahydro-1H-indoles as new anti-HCV agents.

Inhibition of HIV-1 replication by anti-trans-activation responsive polyamide nucleotide analog.

Efficient replication and gene expression of human immunodeficiency virus-1 (HIV-1) involves specific interaction of the viral protein Tat, with its trans-activation responsive element (TAR) which forms a highly stable stem-loop structure. We have earlier shown that a 15-mer polyamide nucleotide analog (PNA) targeted to the loop and bulge region of TAR blocks Tat-mediated transactivation of the HIV-1 LTR both in vitro and in cell culture (Mayhood et al., Biochemistry 39 (2000) 11532). In this communication, we have designed four anti-TAR PNAs of different length such that they either complement the entire loop and bulge region (PNA(TAR-16) and PNA(TAR-15)) or are short of few sequences in the loop (PNA(TAR-13)) or in both the loop and bulge (PNA(TAR-12)), and examined their functional efficacy in vitro as well as in HIV-1 infected cell cultures. All four anti-TAR PNAs showed strong affinity for TAR RNA, while their ability to block in vitro reverse transcription was influenced by their length. In marked contrast to PNA(TAR-12) and PNA(TAR-13), the two longer PNA(TARs) were able to efficiently sequester the targeted site on TAR RNA, thereby substantially inhibiting Tat-mediated transactivation of the HIV-1 LTR. Further, a substantial inhibition of virus production was noted with all the four anti-TAR PNA, with PNA(TAR-16) exhibiting a dramatic reduction of HIV-1 production by nearly 99%. These results point to PNA(TAR-16) as a potential anti-HIV agent.

New pyrazolobenzothiazine derivatives as hepatitis C virus NS5B polymerase palm site I inhibitors.

We have previously identified the pyrazolobenzothiazine scaffold as a promising chemotype against hepatitis C virus (HCV) NS5B polymerase, a validated and promising anti-HCV target. Herein we describe the design, synthesis, enzymatic, and cellular characterization of new pyrazolobenzothiazines as anti-HCV inhibitors. The binding site for a representative derivative was mapped to NS5B palm site I employing a mutant counterscreen assay, thus validating our previous in silico predictions. Derivative 2b proved to be the best selective anti-HCV derivative within the new series, exhibiting a IC50 of 7.9 µM against NS5B polymerase and antiviral effect (EC50 = 8.1 µM; EC90 = 23.3 µM) coupled with the absence of any antimetabolic effect (CC50 > 224 µM; SI > 28) in a cell based HCV replicon system assay. Significantly, microscopic analysis showed that, unlike the parent compounds, derivative 2b did not show any significant cell morphological alterations. Furthermore, since most of the pyrazolobenzothiazines tested altered cell morphology, this undesired aspect was further investigated by exploring possible perturbation of lipid metabolism during compound treatment.