Identification of pyrrolo[3,2-c]pyridin-4-amine compounds as a new class of entry inhibitors against influenza viruses in vitro

Various influenza virus entry inhibitors are being developed as therapeutic antiviral agents in ongoing preparation for emerging influenza viruses, particularly those that may possess drug resistance to the current FDA-approved neuraminidase inhibitors. In this study, small molecules having the pyrrolopyridinamine (PPA), aminothiadiazole (ATD), dihydrofuropyridine carboxamide (HPC), or imidazopyridinamine (IPA) moiety were selected from a target-focused chemical library for their inhibitory activity against influenza A virus by high-throughput screening using the PR8GFP assay. Activity was evaluated by measuring changes the proportion of GFP-expressing cells as a reflection of influenza virus infection. Among them, PPA showed broad-spectrum activity against multiple influenza A viruses and influenza B virus. PPA was found to block the early stages of influenza virus infection using a time-of-addition assay. Using additional phenotypic assays that dissect the virus entry process, it appears that the antiviral activity of PPA against influenza virus can be attributed to interference of the post-fusion process: namely, virus uncoating and nuclear import of viral nucleoprotein complexes. Based on these results, PPA is an attractive chemical moiety that can be used to develop new antiviral drug candidates against influenza viruses. © 2016 Elsevier In

Comparative Pharmacodynamics of Three Different Botulinum Toxin Type A Preparations following Repeated Intramuscular Administration in Mice

Botulinum neurotoxin type A (BoNT/A) causes muscle paralysis by blocking cholinergic signaling at neuromuscular junctions and is widely used to temporarily correct spasticity-related disorders and deformities. The paralytic effects of BoNT/A are time-limited and require repeated injections at regular intervals to achieve long-term therapeutic benefits. Differences in the level and duration of effectivity among various BoNT/A products can be attributed to their unique manufacturing processes, formulation, and noninterchangeable potency units. Herein, we compared the pharmacodynamics of three BoNT/A formulations, i.e., Botox® (onabotulinumtoxinA), Xeomin® (incobotulinumtoxinA), and Coretox®, following repeated intramuscular (IM) injections in mice. Three IM injections of BoNT/A formulations (12 U/kg per dose), 12-weeks apart, were administered at the right gastrocnemius. Local paresis and chemodenervation efficacy were evaluated over 36 weeks using the digit abduction score (DAS) and compound muscle action potential (CMAP), respectively. One week after administration, all three BoNT/A formulations induced peak DAS and maximal reduction of CMAP amplitudes. Among the three BoNT/A formulations, only Coretox® afforded a significant increase in paretic effects and chemodenervation with a prolonged duration of action after repeated injections. These findings suggest that Coretox® may offer a better overall therapeutic performance in clinical settings

Identification of novel compounds inhibiting chikungunya virus-induced cell death by high throughput screening of a kinase inhibitor library.

International audienceChikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity--inhibition of virus-induced CPE--likely by targeting kinases involved in apoptosis

Kinome siRNA screen identifies novel cell-type specific dengue host target genes

Submitted by Luciane Willcox ([email protected]) on 2016-10-03T17:59:53Z No. of bitstreams: 1 Kinome siRNA screen.pdf: 4048808 bytes, checksum: bf050acf9f684cf1d04218d8e7424b31 (MD5)Approved for entry into archive by Luciane Willcox ([email protected]) on 2016-10-03T18:30:17Z (GMT) No. of bitstreams: 1 Kinome siRNA screen.pdf: 4048808 bytes, checksum: bf050acf9f684cf1d04218d8e7424b31 (MD5)Made available in DSpace on 2016-10-03T18:30:17Z (GMT). No. of bitstreams: 1 Kinome siRNA screen.pdf: 4048808 bytes, checksum: bf050acf9f684cf1d04218d8e7424b31 (MD5) Previous issue date: 2014-07-18Institut Pasteur Korea. Discovery Biology Group. Seongnam-si, South Korea.Institut Pasteur Korea. Discovery Biology Group. Seongnam-si, South Korea.Singapore Immunology Network. Agency for Science, Technology and Research. Buona Vista, Singapore.Institut Pasteur Korea. Center for Neglected Diseases Drug Discovery. Seongnam-si, South Korea.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Singapore Immunology Network. Agency for Science, Technology and Research. Buona Vista, Singapore.Institut Pasteur Korea. Center for Neglected Diseases Drug Discovery. Seongnam-si, South Korea.Institut Pasteur Korea. Center for Neglected Diseases Drug Discovery. Seongnam-si, South Korea.Singapore Immunology Network. Agency for Science, Technology and Research. Buona Vista, Singapore.Dengue is a global emerging infectious disease, with no specific treatment available. To identify novel human host cell targets important for dengue virus infection and replication, an image-based high-throughput siRNA assay screening of a human kinome siRNA library was conducted using human hepatocyte cell line Huh7 infected with a recent dengue serotype 2 virus isolate BR DEN2 01-01. In the primary siRNA screening of 779 kinase-related genes, knockdown of 22 genes showed a reduction in DENV-2 infection. Conversely, knockdown of 8 genes enhanced viral infection. To assess host cell specificity, the confirmed hits were tested in the DENV-infected monocytic cell line U937. While the expression of EIF2AK3, ETNK2 and SMAD7 was regulated in both cell lines after infection, most kinases were hepatocyte-specific. Monocytic cells represent initial targets of infection and an antiviral treatment targeting these cells is probably most effective to reduce initial viral load. In turn, infection of the liver could contribute to pathogenesis, and the novel hepatocyte-specific human targets identified here could be important for dengue infection and pathogenesis

Comparison of clinical features and hematologic abnormalities between dengue fever and dengue hemorrhagic fever among children in the Philippines.

To demonstrate the differences of clinical features and hematologic abnormalities between dengue fever (DF) and dengue hemorrhagic fever (DHF), 359 pediatric patients admitted St. Luke\u27s Medical Center in Quezon City, between 1999 and 2001 in Metro Manila, and adjoining provinces the Philippines, with a laboratory-confirmed dengue virus infection were evaluated. One third of the patients had DHF, and most of these patients were without shock. Restlessness, epistaxis, and abdominal pain were more associated with DHF. The platelet count was significantly lower in the DHF group than in the DF group before and after defervescence. In the DHF patients, the hematocrit was significantly increased before defervescence, and decreased the day after due to administration of intravenous fluid. Coagulation abnormalities associated with most DHF patients were thrombocytopenia and an increased fibrinolysis, but not disseminated intravascular coagulation. We present recent data on readily obtained clinical and laboratory data that can be used for early diagnosis and consequently earlier appropriate treatment of dengue virus infections

Comparison of clinical features and hematologic abnormalities between dengue fever and dengue hemorrhagic fever among children in the Philippines.

To demonstrate the differences of clinical features and hematologic abnormalities between dengue fever (DF) and dengue hemorrhagic fever (DHF), 359 pediatric patients admitted St. Luke's Medical Center in Quezon City, between 1999 and 2001 in Metro Manila, and adjoining provinces the Philippines, with a laboratory-confirmed dengue virus infection were evaluated. One third of the patients had DHF, and most of these patients were without shock. Restlessness, epistaxis, and abdominal pain were more associated with DHF. The platelet count was significantly lower in the DHF group than in the DF group before and after defervescence. In the DHF patients, the hematocrit was significantly increased before defervescence, and decreased the day after due to administration of intravenous fluid. Coagulation abnormalities associated with most DHF patients were thrombocytopenia and an increased fibrinolysis, but not disseminated intravascular coagulation. We present recent data on readily obtained clinical and laboratory data that can be used for early diagnosis and consequently earlier appropriate treatment of dengue virus infections

Inhibition of CHIKV-associated apoptotic bodies.

<p>HuH-7 cells infected with CHIKV-118-GFP expressing GFP at 24 hpi. The number of observed apoptotic blebs associated with CHIKV infection (marked by white arrows) were significantly lower when treated with the reference compound MPA and hit compounds CND0364 and CND0545. In contrast, apoptotic blebs were still prominent in CHIKV-infected HuH-7 cells in the presence of CQ. (false-color imaging were applied).</p