Identification of novel small-molecule inhibitors of SARS-CoV-2 by chemical genetics

There are only eight approved small molecule antiviral drugs for treating COVID-19.Among them,four are nucleotide analogues(remdesivir,JT001,molnupiravir,and azvudine),while the other four are protease inhibitors(nirmatrelvir,ensitrelvir,leritrelvir,and simnotrelvir-ritonavir).Antiviral resistance,unfavourable drug‒drug interaction,and toxicity have been reported in previous studies.Thus there is a dearth of new treatment options for SARS-CoV-2.In this work,a three-tier cell-based screening was employed to identify novel compounds with anti-SARS-CoV-2 activity.One compound,designated 172,demonstrated broad-spectrum antiviral activity against multiple human pathogenic coronaviruses and different SARS-CoV-2 variants of concern.Mechanistic studies validated by reverse genetics showed that compound 172 inhibits the 3-chymotrypsin-like protease(3CLpro)by binding to an allosteric site and reduces 3CLpro dimerization.A drug synergistic checkerboard assay demonstrated that compound 172 can achieve drug synergy with nirmatrelvir in vitro.In vivo studies confirmed the antiviral activity of compound 172 in both Golden Syrian Hamsters and K18 humanized ACE2 mice.Overall,this study identified an alternative druggable site on the SARS-CoV-23CLpro,proposed a potential combination therapy with nirmatrelvir to reduce the risk of antiviral resistance and shed light on the development of allosteric protease inhibitors for treating a range of coronavirus diseases.

A new generation M^(pro)inhibitor with potent activity against SARS-CoV-2 Omicron variants

Emerging SARS-CoV-2 variants, particularly the Omicron variant and its sublineages, continually threaten the global public health.Small molecule antivirals are an effective treatment strategy to fight against the virus. However, the first-generation antivirals eithershow limited clinical efficacy and/or have some defects in pharmacokinetic (PK) properties. Moreover, with increased use of thesedrugs across the globe, they face great pressure of drug resistance. We herein present the discovery and characterization of a newgeneration antiviral drug candidate (SY110), which is a potent and selective inhibitor of SARS-CoV-2 main protease (Mpro). Thiscompound displayed potent in vitro antiviral activity against not only the predominant SARS-CoV-2 Omicron sublineage BA.5, butalso other highly pathogenic human coronaviruses including SARS-CoV-1 and MERS-CoV. In the Omicron-infected K18-hACE2mouse model, oral treatment with SY110 significantly lowered the viral burdens in lung and alleviated the virus-induced pathology.Importantly, SY110 possesses favorable PK properties with high oral drug exposure and oral bioavailability, and also an outstandingsafety profile. Furthermore, SY110 exhibited sensitivity to several drug-resistance Mpro mutations. Collectively, this investigationprovides a promising new drug candidate against Omicron and other variants of SARS-CoV-2.

Analogous comparison unravels heightened antiviral defense and boosted viral infection upon immunosuppression in bat organoids

Horseshoe bats host numerous SARS-related coronaviruses without overt disease signs.Bat intestinal organoids,a unique model of bat intestinal epithelium,allow direct comparison with human intestinal organoids.We sought to unravel the cellular mechanism(s)underlying bat tolerance of coronaviruses by comparing the innate immunity in bat and human organoids.We optimized the culture medium,which enabled a consecutive passage of bat intestinal organoids for over one year.Basal expression levels of IFNs and IFN-stimulated genes were higher in bat organoids than in their human counterparts.Notably,bat organoids mounted a more rapid,robust and prolonged antiviral defense than human organoids upon Poly(I:C)stimulation.TLR3 and RLR might be the conserved pathways mediating antiviral response in bat and human intestinal organoids.The susceptibility of bat organoids to a bat coronavirus CoV-HKU4,but resistance to EV-71,an enterovirus of exclusive human origin,indicated that bat organoids adequately recapitulated the authentic susceptibility of bats to certain viruses.Importantly,TLR3/RLR inhibition in bat organoids significantly boosted viral growth in the early phase after SARS-CoV-2 or CoV-HKU4 infection.Collectively,the higher basal expression of antiviral genes,especially more rapid and robust induction of innate immune response,empowered bat cells to curtail virus propagation in the early phase of infection.

Intranasal administration of a single dose of a candidate live attenuated vaccine derived from an NSP16-deficient SARS-CoV-2 strain confers sterilizing immunity in animals

Live attenuated vaccines might elicit mucosal and sterilizing immunity against SARS-CoV-2 that the existing mRNA,adenoviral vector and inactivated vaccines fail to induce.Here,we describe a candidate live attenuated vaccine strain of SARS-CoV-2 in which the NSP16 gene,which encodes 2′-O-methyltransferase,is catalytically disrupted by a point mutation.This virus,designated d16,was severely attenuated in hamsters and transgenic mice,causing only asymptomatic and nonpathogenic infection.A single dose of d16 administered intranasally resulted in sterilizing immunity in both the upper and lower respiratory tracts of hamsters,thus preventing viral spread in a contact-based transmission model.It also robustly stimulated humoral and cell-mediated immune responses,thus conferring full protection against lethal challenge with SARS-CoV-2 in a transgenic mouse model.The neutralizing antibodies elicited by d16 effectively cross-reacted with several SARS-CoV-2 variants.Secretory immunoglobulin A was detected in the blood and nasal wash of vaccinated mice.Our work provides proof-of-principle evidence for harnessing NSP16-deficient SARS-CoV-2 for the development of live attenuated vaccines and paves the way for further preclinical studies of d16 as a prototypic vaccine strain,to which new features might be introduced to improve safety,transmissibility,immunogenicity and efficacy.

Berbamine inhibits SARS-CoV-2 infection by compromising TRPMLs-mediated endolysosomal trafficking of ACE2

Dear Editor,Middle East respiratory syndrome-related coronavirus(MERS-CoV)is the pathogen responsible for the outbreak of MERS,and we are currently being affected by coronavirus disease 2019(COVID-19)due to infection by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).The S protein of SARS-CoV-2 or MERS-CoV binds angiotensin-converting enzyme 2(ACE2)or dipeptidyl peptidase-4(DPP4),respectively,to facilitate viral particles entry into cells1.The COVID-19 pandemic has caused major socioeconomic disruptions globally.

Targeting papain-like protease for broad-spectrum coronavirus inhibition

The emergence of SARS-CoV-2 variants of concern and repeated outbreaks of coronavirus epidemics in the past two decades emphasize the need for next-generation pan-coronaviral therapeutics.Drugging the multi-functional papain-like protease(PLpro)domain of the viral nsp3 holds promise.However,none of the known coronavirus PLpro inhibitors has been shown to be in vivo active.Herein,we screened a structurally diverse library of 50,080 compounds for potential coronavirus PLpro inhibitors and identified a noncovalent lead inhibitor F0213 that has broad-spectrum anti-coronaviral activity,including against the Sarbecoviruses(SARSCoV-1 and SARS-CoV-2),Merbecovirus(MERS-CoV),as well as the Alphacoronavirus(hCoV-229E and hCoVOC43).Importantly,F0213 confers protection in both SARS-CoV-2-infected hamsters and MERS-CoV-infected human DPP4-knockin mice.F0213 possesses a dual therapeutic functionality that suppresses coronavirus replication via blocking viral polyprotein cleavage,as well as promoting antiviral immunity by antagonizing the PLpro deubiquitinase activity.Despite the significant difference of substrate recognition,mode of inhibition studies suggest that F0213 is a competitive inhibitor against SARS2-PLpro via binding with the 157K amino acid residue,whereas an allosteric inhibitor of MERSPLpro interacting with its 271E position.Our proof-ofconcept findings demonstrated that PLpro is a valid target for the development of broad-spectrum anticoronavirus agents.The orally administered F0213 may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and future coronavirus outbreaks.

Enhanced replication of SARS-CoV-2 Omicron BA.2 in human forebrain and midbrain organoids

Dear Editor,Coronavirus Disease 2019(COVID-19)is associated with a variety of neurological complications,including encephalopathy,encephalitis,dementia,and others.1 The pathogenic mechanism of these neurological manifestations remains incompletely understood but may be due to factors such as coagulation problem,immune-mediated response,or direct viral invasion into the central nervous system(CNS).2 We and others previously reported that ancestral SARS-CoV-2 could infect and replicate in human brain organoids.3,4 More recently,SARS-CoV-2 Omicron BA.1 emerged in late 2021 and demonstrated altered virological features including increased immunoevasion and attenuated pathogenicity comparing to SARS-CoV-2 wildtype(WT)and previous variants.5 However,the neuroinvasiveness of Omicron sublineages remain unexplored.Here,we investigated the neuroinvasion and neurotoxicity of Omicron BA.1 and BA.2,and compared the findings with those of SARS-CoV-2 WT and Delta in human forebrain and midbrain organoids.Our results demonstrated that BA.2 replicated more efficiently while triggered lower levels of type I interferon response than that of SARS-CoV-2 WT,Delta,and BA.1 in both human forebrain and midbrain organoids.In addition,BA.2 triggered substantially higher levels of apoptosis in the infected human forebrain and midbrain organoids.Together,these findings suggest that BA.2 may be different from SARS-CoV-2 WT and previous variants in its capacity in targeting and causing diseases in the human brain.