Identification of niclosamide as a novel antiviral agent against porcine epidemic diarrhea virus infection by targeting viral internalization

Porcine epidemic diarrhea virus(PEDV),an enteropathogenic coronavirus,has catastrophic impacts on the global pig industry.However,there remain no effective drugs against PEDV infection.In this study,we utilized a recombinant PEDV expressing renilla luciferase(PEDV-Rluc)to screen potential anti-PEDV agents from an FDAapproved drug library in Vero cells.Four compounds were identified that significantly decreased luciferase activity of PEDV-Rluc.Among them,niclosamide was further characterized because it exhibited the most potent antiviral activity with the highest selectivity index.It can efficiently inhibit viral RNA synthesis,protein expression and viral progeny production of classical and variant PEDV strains in a dose-dependent manner.Time of addition assay showed that niclosamide exhibited potent anti-PEDV activity when added simultaneously with or after virus infection.Furthermore,niclosamide significantly inhibited the entry stage of PEDV infection by affecting viral internalization rather than viral attachment to cells.In addition,a combination with other small molecule inhibitors of endosomal acidification enhanced the anti-PEDV effect of niclosamide in vitro.Taken together,these findings suggested that niclosamide is a novel antiviral agent that might provide a basis for the development of novel drug therapies against PEDV and other related pathogenic coronavirus infections.

Recent advances in small-molecular therapeutics for COVID-19

The COVID-19 pandemic poses a fundamental challenge to global health.Since the outbreak of SARS-CoV-2,great efforts have been made to identify antiviral strategies and develop therapeutic drugs to combat the disease.There are different strategies for developing small molecular anti-SARS-CoV-2 drugs,including targeting coronavirus structural proteins(e.g.spike protein),non-structural proteins(nsp)(e.g.RdRp,Mpro,PLpro,helicase,nsp14,and nsp16),host proteases(e.g.TMPRSS2,cathepsin,and furin)and the pivotal proteins mediating endocytosis(e.g.PIKfyve),as well as developing endosome acidification agents and immune response modulators.Favipiravir and chloroquine are the anti-SARS-CoV-2 agents that were identified earlier in this epidemic and repurposed for COVID-19 clinical therapy based on these strategies.However,their efficacies are controversial.Currently,three small molecular anti-SARSCoV-2 agents,remdesivir,molnupiravir,and Paxlovid(PF-07321332 plus ritonavir),have been granted emergency use authorization or approved for COVID-19 therapy in many countries due to their significant curative effects in phase Ⅲ trials.Meanwhile,a large number of promising anti-SARS-CoV-2 drug candidates have entered clinical evaluation.The development of these drugs brings hope for us to finally conquer COVID-19.In this account,we conducted a comprehensive review of the recent advances in small molecule anti-SARS-CoV-2 agents according to the target classification.Here we present all the approved drugs and most of the important drug candidates for each target,and discuss the challenges and perspectives for the future research and development of anti-SARS-CoV-2 drugs.

Targeting host cofactors to inhibit viral infection

The majority of FDA-approved drugs indicated for the treatment of viral infections are inhibitors of viral proteins, of which the emergence of resistant strains is a major concern. This issue is exacerbated as most developed antiviral therapies are indicated for the treatment of viruses with error-prone replication. These problems may be addressed by the development of drugs that modulate the function of host factors involved in various aspects of a viral life cycle. Targeting host factors uncouples the mutation of a druggable protein gene from the replication and survival selection pressure exerted on a virus. Currently, a host-targeting antiviral （HTA）, maraviroc, is approved for the treatment of human immunodeficiency virus （HIV） infection. In addition, several HTAs indicated for the treatment of hepatitis C virus （HCV） or HIV infection are at various stages of clinical evaluation. Targeting host factors is an attractive complement to therapies directly targeting a viral protein because of the expected higher genetic barrier for resistance and an overall increase in the diversity of treatment options. We examine how the integrated roles of emerging host cofactor screening approaches and drug development strategies may advance current treatment options.