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Discovery of safe-in-man broad-spectrum Anti viral agents










Review discovery anddevelopment of BSAAs and summarize the information on 120 safe-in-man agents in a freely accessibledatabase (https://drugvirus.info/). Future and ongoing pre-clinical and clinical studies will increase thenumber of BSAAs, expand the spectrum of their indications, and identify drug combinations fortreatment of emerging and re-emerging viral infections as well as co-infections.© 2020 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).



The discovery of novel activities of BSAAs starts with exposingcells to the candidate antiviral agent at different concentrationsand infecting the cells with a virus or mock. Immortalizedcancerous cell cultures and co-cultures, which express appropriateviral receptors, are most commonly used in thisfirst step. The halfmaximalcytotoxic concentrations (CC50) for a compound arecalculated based on their dose-response curves obtained on mock-infected cells. The half-maximal effective concentrations (EC50) arecalculated based on the analysis of curves obtained on infectedcells. Statistical analyses can help to determine if the differencesbetween CC50 and EC50 are significant, given the inherentvariability of the experiment (Meneghini and Hamasaki, 1967).A relative effectiveness of a drug is defined as selectivity index(SI = CC50/EC50).Cell viability assays and cell death assays are commonly used toassess the cytotoxicity and efficacy of BSAAs (Figure 2A). Cellviability assays include MTT, MTS, resazurin or similar assays,mitochondrial membrane potential-dependent dyes-based assays,esterase cleaved dye-based assays, ATP-ADP assays, and assays thatmeasure glycolyticflux and oxygen consumption. Other cell deathassays include LDH enzyme leakage assays, membrane impermeabledye-based assays, and apoptosis assays, such as Annexin V,TUNEL, and caspase assays (Shen et al., 2019). For example, the CellTiter Glo (CTG) assay quantifies ATP, an indicator of metabolicallyactive living cells, whereas Cell Tox Green assay usesfluorescentasymmetric cyanine dye that stains the DNA of dead cells (Boslet al., 2019; Bulanova et al., 2017; Ianevski et al., 2018; Muller et al.,2014).Viral strains or cell lines expressing reporter proteins are alsoused to assess the efficacy of BSAAs in infected cells. For example,TZM-bl cells expressingfirefly luciferase under control of HIV-1LTR promoter allowed quantitation of BSAA action on HIV-1infection (tat-protein expression by integrated HIV-1 provirus)usingfirefly luciferase assay (Sarzotti-Kelsoe et al., 2014; Xinget al., 2016). RFP-expressing RVFV, nanoLuc-expressing CHIKV andRRV, as well as GFP-expressing FLUAV, HCV and HMPV also allowedidentification of novel activities of several BSAAs (Andersen et al.,2019b; Bosl et al., 2019; de Graaf et al., 2007; Habjan et al., 2008;Ianevski et al., 2018; Jupille et al., 2011; Kittel et al., 2004; Lee et al.,2017; Utt et al., 2016). In addition, qPCR/RT-qPCR, RNA/DNAsequencing, RNA/DNA hybridization, immuno- and plaque assaysas well as CRISPR-Cas9 systems could be used for detection ofinhibitory effects of BSAAs (Boonham et al., 2014; Fischer et al.,2017; Konig et al., 2019; Laamiri et al., 2018; Landry, 1990; Perezet al., 2013; Sashital, 2018; Zhou et al., 2018). Interestingly, CRISPR-Cas9, siRNA and shRNA approaches were used for identification ofBSAA targets (Deans et al., 2016; Puschnik et al., 2017).Novel anti-HSV-2 and anti-EV1 activities of emetine werediscovered recently using CTG/plaque assays in human nonmalignantRPE cells. Moreover, novel antiviral activities of the drugwere identified using RFP-expressing RVFV, and GFP-expressingHMPV or FLUAV strains in RPE cells (Andersen et al., 2019a). Giventhat emetine also inhibits ZIKV, EBOV, RABV, CMV, HCoV-OC43 andHIV-1 infections (Chaves Valadao et al., 2015; MacGibeny et al.,2018; Mukhopadhyay et al., 2016; Shen et al., 2019; Yang et al.,2018), and that it is an FDA-approved anti-protozoal drug, it mayrepresent a promising safe-in-man BSAA candidate.Evaluation of BSAAs in human primary cell culture and co-culturesImmortalized cell cultures/co-cultures and reporter viralstrains represent excellent model systems for the discovery ofnovel activities of safe-in-man BSAAs. However, these geneticallymodified systems have certain limitations (attenuated or incompletevirus replication cycle, accumulation of mutations duringrepeated cell and virus passaging, defective innate immuneresponses and viral counter-responses, etc.) (Carter and Shieh,2015). Thereby, novel antiviral activities of BSAAs should be furthervalidated in primary human cells using different viral strains(including wild-type viruses), different viral loads, different timesof compound addition, different endpoint measurements andcompound concentration range. Primary cell cultures give moreaccurate images of drug responses (Alves et al., 2018; Denisovaet al., 2012; Koban et al., 2018; Postnikova et al., 2018). They have alow population doubling level and therefore more closelyrecapitulate the physiological conditions observed in vivo.Primary cells are cells isolated directly from tissues or bloodusing enzymatic or mechanical methods. The cells are character-ized by their high degrees of specialization, are often fullydifferentiated and thus require defined culture conditions (serum-free media) in order to preserve their original phenotype.Peripheral blood mononuclear (PBMC), placental, amniotic andfetal primary cultures as well as vaginal/cervical epithelial andmale germ cells have been used intensively to validate BSAAactivity (Barrows et al., 2016; Denisova et al., 2012; Fink et al., 2018;Rausch et al., 2017; Robinson et al., 2018). Although primary cellcultures are relevant systems for validation of BSAAs, there aretechnical difficulties limiting their use, such as ethical issues,purity of population of primary cells, and limited shelf life of thecells. In addition, age, race, sex and other genetic and epigeneticfactors of donor cells should be considered to determine commonbiological effect across a significant number of donors therebyavoiding minor variants (Lee et al., 2014; Zhang et al., 2013).The obstacles associated with use of human primary cellcultures can be bypassed using human embryonic stem cells (ESCs)and human induced pluripotent stem cells (iPSCs). ESCs areisolated from surplus human embryos, whereas iPSCs are obtained



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