Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins
Viral diseases remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral diseases, new treatments are urgently needed. Here we show that small-molecules, which inhibit cellular anti-apoptotic Bcl-2 proteins (Bcl-2i), induced the premature death of cells infected with different RNA or DNA viruses, whereas, at the same concentrations, no toxicity was observed in mock-infected cells. Moreover, these compounds limited viral replication and spread. Surprisingly, Bcl-2i also induced the premature apoptosis of cells transfected with viral RNA or plasmid DNA but not of mock-transfected cells. These results suggest that Bcl-2i sensitizes cells containing foreign RNA or DNA to apoptosis. A comparison of the toxicity, antiviral activity, and side effects of six Bcl-2i allowed us to select A-1155463 as an antiviral lead candidate. Thus, our results pave the way for the further development of Bcl-2i for the prevention and treatment of viral diseases.
Repurposing drugs for treatment of SARS-CoV-2 infection: computational design insights into mechanisms of action
The COVID-19 pandemic has negatively affected human life globally. It has led to economic crises and health emergencies across the world, spreading rapidly among the human population and has caused many deaths. Currently, there are no treatments available for COVID-19 so there is an urgent need to develop therapeutic interventions that could be used against the novel coronavirus infection. In this research, we used computational drug design technologies to repurpose existing drugs as inhibitors of SARS-CoV-2 viral proteins. The Broad Institute’s Drug Repurposing Hub consists of in-development/approved drugs and was computationally screened to identify potential hits which could inhibit protein targets encoded by the SARS-CoV-2 genome. By virtually screening the Broad collection, using rationally designed pharmacophore features, we identified molecules which may be repurposed against viral nucleocapsid and non-structural proteins. The pharmacophore features were generated after careful visualisation of the interactions between co-crystalised ligands and the protein binding site. The ChEMBL database was used to determine the compound’s level of inhibition of SARS-CoV-2 and correlate the predicted viral protein target with whole virus in vitro data. The results from this study may help to accelerate drug development against COVID-19 and the hit compounds should be progressed through further in vitro and in vivo studies on SARS-CoV-2.
Gossypol, a disequiterpene obtained from cottonseed oil, and a series of peri-acylated gossylic nitriles were compared for their antiviral activities against HSV-II and for their toxicities to the host Vero cells. All of the peri-acylated gossylic nitriles exhibited lower cytotoxicities to the host cell than did the parent compound gossypol. Both gossypol and the series of derivatives exhibited antiviral activities against HSV-II when the virus was treated with drug at concentrations as low as 5 X 10(-7) M. Two of the derivatives, gossylic nitrile-1,1′-diacetate and gossylic nitrile-1,1′-divalerate, were capable of inhibiting viral multiplication in Vero cells that were infected with virus before administration of the drug. The results of this study indicate that modification of the aldehyde functional groups on gossypol lowers the toxicity of this drug but does not abolish its antiviral properties. Derivatives of gossypol may be useful antiviral agents.
Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings
We have used a combination of virtual screening (VS) and high-throughput screening (HTS) techniques to identify novel, non-peptidic small molecule inhibitors against human SARS-CoV 3CLpro. A structure-based VS approach integrating docking and pharmacophore based methods was employed to computationally screen 621,000 compounds from the ZINC library. The screening protocol was validated using known 3CLpro inhibitors and was optimized for speed, improved selectivity, and for accommodating receptor flexibility. Subsequently, a fluorescence-based enzymatic HTS assay was developed and optimized to experimentally screen approximately 41,000 compounds from four structurally diverse libraries chosen mainly based on the VS results. False positives from initial HTS hits were eliminated by a secondary orthogonal binding analysis using surface plasmon resonance (SPR). The campaign identified a reversible small molecule inhibitor exhibiting mixed-type inhibition with a Ki value of 11.1 μM. Together, these results validate our protocols as suitable approaches to screen virtual and chemical libraries, and the newly identified compound reported in our study represents a promising structural scaffold to pursue for further SARS-CoV 3CLpro inhibitor development.
BCL2 family proteins including pro-survival proteins, BH3-only proteins and BAX/BAK proteins control mitochondria-mediated apoptosis to maintain cell homeostasis via the removal of damaged cells and pathogen-infected cells. In this study, we examined the roles of BCL2 proteins in the induction of apoptosis in cells upon infection with flaviviruses, such as Japanese encephalitis virus, Dengue virus and Zika virus. We showed that survival of the infected cells depends on BCLXL, a pro-survival BCL2 protein due to suppression of the expression of another pro-survival protein, MCL1. Treatment with BCLXL inhibitors, as well as deficient BCLXL gene expression, induced BAX/BAK-dependent apoptosis upon infection with flaviviruses. Flavivirus infection attenuates cellular protein synthesis, which confers reduction of short-half-life proteins like MCL1. Inhibition of BCLXL increased phagocytosis of virus-infected cells by macrophages, thereby suppressing viral dissemination and chemokine production. Furthermore, we examined the roles of BCLXL in the death of JEV-infected cells during in vivo infection. Haploinsufficiency of the BCLXL gene, as well as administration of BH3 mimetic compounds, increased survival rate after challenge of JEV infection and suppressed inflammation. These results suggest that BCLXL plays a crucial role in the survival of cells infected with flaviviruses, and that BCLXL may provide a novel antiviral target to suppress propagation of the family of Flaviviridae viruses.
Repurposing Therapeutics to Identify Novel Inhibitors Targeting 2′-O-Ribose Methyltransferase Nsp16 of SARS-CoV-2
Three coronaviruses (CoVs) severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the recently identified SARS-CoV-2 in December 2019, have caused deadly pneumonia in humans since the beginning of the 21st century The SARS-CoV-2 causes coronavirus disease-19 (COVID-19) with influenza-like symptoms ranging from mild discomfort to severe lung injury and multi-organ failure, eventually leading to death As of April 30, 2020, more than three million (3,175,207) COVID-19 cases were reported worldwide, and more than 220,000 (224,172) patients have died (https//www who int/emergencies/diseases/novel-coronavirus-2019) Effective treatments and vaccines for SARS-CoV-2 infection do not currently exist Thus, it will be of great benefit to identify and repurpose already well-characterized compounds and approved drugs for use in combating COVID-19 /CoVs are positive-sense RNA viruses that replicate in the cytoplasm of infected cells Replication and transcription of the CoV RNA genome are achieved by a complex RNA replication/transcription machinery, consisting of at least 16 viral nonstructural proteins (nsp) Previous studies demonstrated that nsp16 proteins of SARS-CoV-1 and MERS-CoV have methyltransferase (MTase) activities that catalyze methylation of the first transcribed nucleotide at the ribose 2â€™-O position (2â€™-O-Me) The 2â€™-O-Me of virus caRNAs protects itself from degradation by 5â€²-3â€² exoribonucleases, ensures efficient translation, and helps to prevent recognition by the host innate immune system The importance of nsp16 2′-O-MTase activity for CoV infection and pathogenesis was previously documented by in vitro and in vivo studies For SARS-CoV-1, the absence of nsp16 2â€²-O-MTase activity results in significant attenuation characterized by decreased viral replication, reduced weight loss, and limited breathing dysfunction in mice In addition, nsp16 down-regulates the activities of innate immune sensing factors retinoic acid-inducible gene (RIG-I) and melanoma differentiation-associated gene 5 protein (MDA5) Thus, inhibition of nsp16 2â€™-O-MTase activities should restrain viral replication and enable recognition by the host innate immune system, making the nsp16-MTase a promising target for the identification of new anti-SARS-CoV-2 drugs /In the present study, we employed structural analysis, virtual screening, and systematic drug repurposing approaches to identify â€œapprovedâ€ drugs which can act as promising inhibitors against nsp16 2â€²-O-MTase of SARS-CoV-2 We first performed comparative analysis of primary amino acid sequences and crystal structures of seven human CoVs and defined the key residues for nsp16 2-Oâ€™-MTase functions From the virtual screening against nsp16 2â€²-O-MTase of SARS-CoV-2, we provide a ranking of the predicted binding affinities of 1,380 tohit compounds corresponding to 967 â€œapprovedâ€ drugs Furthermore, we have calculated various structural parameters of our top-ranking drugs Our studies provided the foundation to further test and repurpose these candidate drugs experimentally and clinically for COVID-19 treatment /br
At a concentration of 10 mum, camptothecin inhibited vaccinia deoxyribonucleic acid (DNA) synthesis in HeLa cells. Inhibition of viral DNA synthesis was observed when the drug was added before infection or at 1 or 2 hr after infection. Inhibitory effects of camptothecin on vaccinia DNA synthesis could be reversed, even after exposure to the alkaloid for 2 hr. Viral DNA, isolated from vaccinia-infected, camptothecin-treated cells, displayed an altered sedimentation constant after alkaline sucrose density gradient centrifugation. Incorporation of uridine into vaccinia messenger ribonucleic acid was inhibited by camptothecin, but the activity of ribonucleic acid polymerase, as tested in isolated vaccinia cores, was not affected by the drug. Camptothecin had essentially no effect on replication of poliovirus in HeLa cells.
Screening of drugs by FRET analysis identifies inhibitors of SARS-CoV 3CL protease
SARS-CoV 3CL protease is essential for viral protein processing and is regarded as a good drug target to prevent SARS-CoV replication. In the present study, we established a high-throughput FRET technique for screening for anti-SARS-CoV 3CL protease drugs. Of a thousand existing drugs examined, hexachlorophene was identified as the most potent in inhibiting SARS-CoV 3CL protease. Further characterization showed that it was effective at micromolar concentrations (Ki = 4 μM). The binding mode was competitive, and the inhibitory effect was dependent on preincubation time. Two other drugs, triclosan and nelfinavir, were about 10 times less potent. The structure-based search and biological evaluation of various hexachlorophene analogues were described. These analogues gave optimal inhibitory activity against SARS-CoV 3CL protease with IC50 values ranging from 7.6 to 84.5 μM. Optimization of hexachlorophene analogues was shown to provide several active 3CL protease inhibitors that function as potential anti-SARS agents.
Biflavonoids from Torreya nucifera displaying SARS-CoV 3CLpro inhibition
As part of our search for botanical sources of SARS-CoV 3CLpro inhibitors, we selected Torreya nucifera, which is traditionally used as a medicinal plant in Asia. The ethanol extract of T. nucifera leaves exhibited good SARS-CoV 3CLpro inhibitory activity (62% at 100 μg/mL). Following bioactivity-guided fractionation, eight diterpenoids (1–8) and four biflavonoids (9–12) were isolated and evaluated for SARS-CoV 3CLpro inhibition using fluorescence resonance energy transfer analysis. Of these compounds, the biflavone amentoflavone (9) (IC50 = 8.3 μM) showed most potent 3CLpro inhibitory effect. Three additional authentic flavones (apigenin, luteolin and quercetin) were tested to establish the basic structure–activity relationship of biflavones. Apigenin, luteolin, and quercetin inhibited 3CLpro activity with IC50 values of 280.8, 20.2, and 23.8 μM, respectively. Values of binding energy obtained in a molecular docking study supported the results of enzymatic assays. More potent activity appeared to be associated with the presence of an apigenin moiety at position C-3′ of flavones, as biflavone had an effect on 3CLpro inhibitory activity.
Advances in Developing Therapies to Combat Zika Virus: Current Knowledge and Future Perspectives
Zika virus (ZIKV) remained largely quiescent for nearly six decades after its first appearance in 1947. ZIKV reappeared after 2007, resulting in a declaration of an international “public health emergency” in 2016 by the World Health Organization (WHO). Until this time, ZIKV was considered to induce only mild illness, but it has now been established as the cause of severe clinical manifestations, including fetal anomalies, neurological problems, and autoimmune disorders. Infection during pregnancy can cause congenital brain abnormalities, including microcephaly and neurological degeneration, and in other cases, Guillain-Barré syndrome, making infections with ZIKV a substantial public health concern. Genomic and molecular investigations are underway to investigate ZIKV pathology and its recent enhanced pathogenicity, as well as to design safe and potent vaccines, drugs, and therapeutics. This review describes progress in the design and development of various anti-ZIKV therapeutics, including drugs targeting virus entry into cells and the helicase protein, nucleosides, inhibitors of NS3 protein, small molecules, methyltransferase inhibitors, interferons, repurposed drugs, drugs designed with the aid of computers, neutralizing antibodies, convalescent serum, antibodies that limit antibody-dependent enhancement, and herbal medicines. Additionally, covalent inhibitors of viral protein expression and anti-Toll-like receptor molecules are discussed. To counter ZIKV-associated disease, we need to make rapid progress in developing novel therapies that work effectually to inhibit ZIKV.
Certain compounds such as QL-XII-47 and QL-XII-54 are quinolines (covalent inhibitors of DENV) that act by inhibiting viral E and NS5 protein expression without significantly affecting the host housekeeping protein GAPDH. Due to structural similarities between flaviviral E and NS5 proteins, similar compounds can be employed to combat ZIKV infection (de Wispelaere et al., 2016). Host caspases have been found to mediate the lethality of multiple pathogenic agents, based on the HapMap Project of B lymphoblastoid cells from a cohort of persons of African, European, and Asian ancestry. Bithionol (an FDA-approved drug) inhibits caspases and has been found to be effective in reducing the negative effects of ZIKV and bacterial and plant toxins. Thus, elucidation of such host proteins that further disease can be used to design drugs for ZIKV (Leonardi et al., 2016). Nordihydroguaiaretic acid (NDGA) alters the lipid metabolism of a host by intervening in the sterol regulatory element binding protein (SREBP) pathway. More recently, inhibitors of the SREBP pathway, including NDGA and its methylated derivative tetra-O-methyl nordihydroguaiaretic (M4N), PF-429242, and fatostatin, have also been found to reduce WNV and ZIKV replication. These drug candidates may serve as effective anti-viral agents against ZIKV (Merino-Ramos et al., 2017).