Visualizing intracellular sialidase activity of influenza A virus neuraminidase using a fluorescence imaging probe.

In influenza A virus-infected cells, newly synthesized viral neuraminidases (NAs) transiently localize at the host cell Golgi due to glycosylation, before their expression on the cell surface. It remains unproven whether Golgi-localized intracellular NAs exhibit sialidase activity. We have developed a sialidase imaging probe, [2-(benzothiazol-2-yl)-5-(non-1-yn-1-yl) phenyl]-α-D-N-acetylneuraminic acid (BTP9-Neu5Ac). This probe is designed to be cleaved by sialidase activity, resulting in the release of a hydrophobic fluorescent compound, 2-(benzothiazol-2-yl)-5-(non-1-yn-1-yl) phenol (BTP9). BTP9-Neu5Ac makes the location of sialidase activity visually detectable by the BTP9 fluorescence that results from the action of sialidase activity. In this study, we established a protocol to visualize the sialidase activity of intracellular NA at the Golgi of influenza A virus-infected cells using BTP9-Neu5Ac. Furthermore, we employed this fluorescence imaging protocol to elucidate the intracellular inhibition of laninamivir octanoate, an anti-influenza drug. At approximately 7 h after infection, newly synthesized viral NAs localized at the Golgi. Using our developed protocol, we successfully histochemically stained the sialidase activity of intracellular viral NAs localized at the Golgi. Importantly, we observed that laninamivir octanoate effectively inhibited the intracellular viral NA, in contrast to drugs like zanamivir or laninamivir. Our study establishes a visualization protocol for intracellular viral NA sialidase activity and visualizes the inhibitory effect of laninamivir octanoate on Golgi-localized intracellular viral NA in infected cells.

Ubiquitin E3 ligase SPOP is a host negative regulator of enterovirus 71-encoded 2A protease.

IMPORTANCE: EV71 poses a significant health threat to children aged 5 and below. The process of EV71 infection and replication is predominantly influenced by ubiquitination modifications. Our previous findings indicate that EV71 prompts the activation of host deubiquitinating enzymes, thereby impeding the host interferon signaling pathway as a means of evading the immune response. Nevertheless, the precise mechanisms by which the host employs ubiquitination modifications to hinder EV71 infection remain unclear. The present study demonstrated that the nonstructural protein 2Apro, which is encoded by EV71, exhibits ubiquitination and degradation mediated by the host E3 ubiquitin ligase SPOP. In addition, it is the first report, to our knowledge, that SPOP is involved in the host antiviral response.

Neuraminidase-dependent entry of influenza A virus is determined by hemagglutinin receptor-binding specificity.

IMPORTANCE: Influenza A viruses (IAVs) contain hemagglutinin (HA) proteins involved in sialoglycan receptor binding and neuraminidase (NA) proteins that cleave sialic acids. While the importance of the NA protein in virion egress is well established, its role in virus entry remains to be fully elucidated. NA activity is needed for the release of virions from mucus decoy receptors, but conflicting results have been reported on the importance of NA activity in virus entry in the absence of decoy receptors. We now show that inhibition of NA activity affects virus entry depending on the receptor-binding properties of HA and the receptor repertoire present on cells. Inhibition of entry by the presence of mucus correlated with the importance of NA activity for virus entry, with the strongest inhibition being observed when mucus and OsC were combined. These results shed light on the importance in virus entry of the NA protein, an important antiviral drug target.

Preparation of eicosavalent triazolylsialoside-conjugated human serum albumin as a dual hemagglutinin/neuraminidase inhibitor and virion adsorbent for the prevention of influenza infection.

A triazolylsialoside-human serum albumin conjugate was prepared as a multivalent hemagglutinin and neuraminidase inhibitor using a di-(N-succinimidyl) adipate strategy. Matrix-Assisted Laser Desorption/Ionization-Time of Flight-Mass Spectrometry (MALDI-TOF-MS) indicated that five tetravalent sialyl galactosides were grafted onto the protein backbone resulting in an eicosavalent triazolylsialoside-protein complex. Compared with monomeric sialic acid, molecular interaction studies showed that the synthetic pseudo-glycoprotein bound tightly not only to hemagglutinin (HA)/neuraminidase (NA) but also to mutated drug-resistant NA on the surface of the influenza virus with a dissociation constant (KD) in the 1 µM range, attributed to the cluster effect. Moreover, this glycoconjugate exhibited potent antiviral activity against a broad spectrum of virus strains and showed no cytotoxicity towards Human Umbilical Vein Endothelial Cells (HUVECs) and Madin-Darby canine kidney (MDCK) cells at high concentrations. Further mechanistic studies demonstrated this multivalent sialyl conjugate showed strong capture and trapping of influenza virions, thus disrupting the ability of the influenza virus to infect host cells. This research lays the experimental foundation for the development of new antiviral agents based on multivalent sialic acid-protein conjugates.

Identification of a leucine-zipper motif in pUL51 essential for HCMV replication and potential target for antiviral development.

Human cytomegalovirus (HCMV) can cause serious diseases in immunocompromised patients. Use of current antivirals is limited by their adverse effects and emergence of drug resistance mutations. Thus, new drugs are an urgent need. The terminase complex (pUL56-pUL89-pUL51) represents a target of choice for new antivirals development. pUL51 was shown to be crucial for the cleavage of concatemeric HCMV DNA and viral replication. Its C-terminal part plays a critical role for the terminase complex assembly. However, no interaction domain is clearly identified. Sequence comparison of herpesvirus homologs and protein modelling were performed on pUL51. Importance of a putative interaction domain is validated by the generation of recombinant viruses with specific alanine substitutions of amino acids implicated in the domain. We identified a Leucine-Zipper (LZ) domain involving the leucine residues L126-X6-L133-X6-L140-X6-L147 in C-terminal part of pUL51. These leucines are crucial for viral replication, suggesting the significance for pUL51 structure and function. A mimetic-peptide approach has been used and tested in antiviral assays to validate the interaction domain as a new therapeutic target. Cytotoxicity was evaluated by LDH release measurement. The peptide TAT-HK29, homologous to the pUL51-LZ domain, inhibits HCMV replication by 27% ± 9% at 1.25 µM concentration without cytotoxicity. Our results highlight the importance of a leucine zipper domain in the C-terminal part of pUL51 involving leucines L126, L133, L140 and L147. We also confirm the potential of mimetic peptides to inhibit HCMV replication and the importance to target interaction domains to develop antiviral agents.

Advanced simulations and screening to repurposing a 3C protease inhibitor against the rupintrivir-resistant human norovirus-induced gastroenteritis.

Human norovirus (HuNoV) causes acute viral gastroenteritis in all age groups, and dehydration and severe diarrhea in the elderly. The World Health Organization reports ∼1.45 million deaths from acute gastroenteritis annually in the world. Rupintrivir, an inhibitory medicine against the human rhinovirus C3 protease, has been reported to inhibit HuNoV 3C protease. However, several HuNoV 3C protease mutations have been revealed to reduce the susceptibility of HuNoV to rupintrivir. The structural details behind rupintrivir-resistance of these single-point mutations (A105V and I109V) are not still clear. Hence, in this study, a combination of computational techniques were used to determine the rupintrivir-resistance mechanism and to propose an inhibitor against wild-type and mutant HuNoV 3C protease through structure-based virtual screening. Dynamic structural results indicated the unstable binding of rupintrivir at the cleft binding site of the wild-type and mutant 3C proteases, leading to its detachment. Our findings presented that the domain II of the HuNoV 3C protease had a critical role in binding of inhibitory molecules. Binding energy computations, steered molecular dynamics and umbrella sampling simulations confirmed that amentoflavone, the novel suggested inhibitor, strongly binds to the cleft site of all protease models and has a good structural stability in the complex system along the molecular dynamic simulations. Our in silico study proposed the selected compound as a potential inhibitor against the HuNoV 3C protease. However, additional experimental and clinical studies are required to corroborate the therapeutic efficacy of the compound.

Repurposing N-hydroxy thienopyrimidine-2,4-diones (HtPD) as inhibitors of human cytomegalovirus pUL89 endonuclease: Synthesis and biological characterization.

The terminase complex of human cytomegalovirus (HCMV) is required for viral genome packaging and cleavage. Critical to the terminase functions is a metal-dependent endonuclease at the C-terminus of pUL89 (pUL89-C). We have previously reported metal-chelating N-hydroxy thienopyrimidine-2,4-diones (HtPD) as inhibitors of human immunodeficiency virus 1 (HIV-1) RNase H. In the current work, we have synthesized new analogs and resynthesized known analogs of two isomeric HtPD subtypes, anti-HtPD (13), and syn-HtPD (14), and characterized them as inhibitors of pUL89-C. Remarkably, the vast majority of analogs strongly inhibited pUL89-C in the biochemical endonuclease assay, with IC50 values in the nM range. In the cell-based antiviral assay, a few analogs inhibited HCMV in low µM concentrations. Selected analogs were further characterized in a biophysical thermal shift assay (TSA) and in silico molecular docking, and the results support pUL89-C as the protein target of these inhibitors. Collectively, the biochemical, antiviral, biophysical, and in silico data reported herein indicate that the isomeric HtPD chemotypes 13-14 can serve as valuable chemical platforms for designing improved inhibitors of HCMV pUL89-C.

Alerta monitoramento do horizonte tecnológico: Tecovirimat para o tratamento da monkeypox / Technological horizon monitoring alert: Tecovirimat for the treatment of monkeypox

A TECNOLOGIA: Descrição da tecnologia: Tecovirimat é um antiviral sintético inibidor da atividade da proteína viral VP37, indicado para o tratamento da infecção por monkeypox. A proteína VP37, codificada por um gene altamente conservado do gênero orthopoxvirus, está presente na superfície do vírus e envolvida no processo de encapsulação e secreção de suas formas extracelulares. O fármaco inibe o último estágio de replicação viral, a partir do bloqueio da interação de VP37 com proteínas celulares do hospedeiro (Rab9 GTPase e TIP47), o que impede a formação de viriões encapsulados competentes, necessários para a disseminação sanguínea, célula a célula e de longo alcance do vírus. A atividade antiviral de tecovirimat é específica para os orthopoxvirus, ou seja, não inibe a ação de outros tipos de DNA ou RNA-vírus e a proliferação celular. Em estudos pré-clínicos com modelos animais, o fármaco demonstrou boa eficácia contra a monkeypox quando administrado no início da infecção, aumentando as taxas de sobrevivência. A segurança e a tolerabilidade do medicamento também foram apontadas em ensaios clínicos com humanos, sendo raros os eventos adversos (náusea e cefaleia). Entretanto, a eficácia de tecovirimat em humanos ainda não foi bem estabelecida. O medicamento pode afetar a imunogenicidade quando administrado concomitantemente com a vacina ACAM2000. INFORMAÇÕES REGULATÓRIAS: Informações sobre registro: No dia 26 de agosto de 2022, a Agência Nacional de Vigilância Sanitária (Anvisa) aprovou a dispensa de registro para importação e uso exclusivo pelo Ministério da Saúde do medicamento tecovirimat no tratamento da monkeypox no Brasil (RDC 747/2022). Essa dispensa é temporária, com validade de seis meses, desde que não seja expressamente revogada pela Anvisa. O medicamento aprovado está disponível na forma de cápsulas duras com concentração de 200 mg, destinadas à administração oral, para o tratamento de doenças causadas pelo orthopoxvirus em adultos, adolescentes e crianças com peso corporal mínimo de 13 kg12. PANORAMA DE DESENVOLVIMENTO: Estratégia de busca: Foi realizada uma busca no ClinicalTrials.gov14 e no Cortellis1 em 30 de agosto de 2022 para a identificação de ensaios clínicos (em qualquer fase em andamento ou finalizados em até cinco anos, registrados no ClinicalTrials.gov) que avaliassem o uso de tecovirimat no tratamento da monkeypox. A busca identificou seis ensaios clínicos, sendo que cinco avaliavam a tecnologia em questão para smallpox; o outro avaliava o uso de tecorivimat para orthopoxvirus, entretanto, foi finalizado em 2010. Portanto, nenhum ensaio clínico atendeu aos critérios de inclusão. CONSIDERAÇÕES FINAIS: A monkeypox é uma doença zoonótica viral causada por um orthopoxvirus, também denominado monkeypox, endêmica das regiões de floresta tropical da África, principalmente na África Ocidental e Central. Devido ao rápido aumento de incidência de casos da doença em países não endêmicos, a partir da transmissão humana, em julho de 2022, a OMS decretou Emergência de Saúde Pública de Importância Internacional. Atualmente, a única tecnologia disponível para o tratamento de monkeypox é o tecovirimat, cuja dispensa de registro foi aprovada pela Anvisa em agosto de 2022. Devido à raridade da doença, a efetividade do medicamento foi avaliada apenas em estudos pré-clínicos conduzidos com animais infectados. Segundo os resultados desses estudos, o medicamento foi capaz de reduzir em cerca de 80% o risco de morte quando o tratamento era iniciado entre o quarto e quinto dia da infecção. Em estudos de segurança com humanos não infectados, o medicamento apontou para adequada segurança e tolerabilidade, sendo os eventos adversos mais comuns náusea e cefaleia. Diante disso, essa tecnologia tem potencial de auxiliar no controle dos casos de monkeypox no Brasil. Para que ocorra a oferta desse medicamento no SUS, é necessária sua análise pela Comissão Nacional de Incorporação de Tecnologias no Sistema Único de Saúde (Conitec), conforme disposto na Lei nº 12.401/2011, que alterou a Lei nº 8.080/1990. Os relatórios de recomendação da Conitec levam em consideração as evidências científicas sobre eficácia, a acurácia, a efetividade e a segurança do medicamento, e, também, a avaliação econômica comparativa dos benefícios e dos custos em relação às tecnologias já incorporadas e o impacto da incorporação da tecnologia no SUS. Ressalta-se a excepcionalidade prevista no art. 29 do Decreto 7.646/2011, que prevê que o Ministro de Estado da Saúde poderá, em caso de relevante interesse público, mediante processo administrativo simplificado, determinar a incorporação ou alteração pelo SUS de tecnologias em saúde. O comando é regulamentado pelos arts. 25-A e 25-B do anexo XVI da Portaria de Consolidação GM/MS nº 1/201716. Conforme o normativo, considera-se caso de relevante interesse público a situação de emergência em saúde pública de importância nacional.

4,5-Dihydroxypyrimidine Methyl Carboxylates, Carboxylic Acids, and Carboxamides as Inhibitors of Human Cytomegalovirus pUL89 Endonuclease.

Human cytomegalovirus (HCMV) terminase complex entails a metal-dependent endonuclease at the C-terminus of pUL89 (pUL89-C). We report herein the design, synthesis, and characterization of dihydroxypyrimidine (DHP) acid (14), methyl ester (13), and amide (15) subtypes as inhibitors of HCMV pUL89-C. All analogs synthesized were tested in an endonuclease assay and a thermal shift assay (TSA) and subjected to molecular docking to predict binding affinity. Although analogs inhibiting pUL89-C in the sub-µM range were identified from all three subtypes, acids (14) showed better overall potency, substantially larger thermal shift, and considerably better docking scores than esters (13) and amides (15). In the cell-based antiviral assay, six analogs inhibited HCMV with moderate activities (EC50 = 14.4-22.8 µM). The acid subtype (14) showed good in vitro ADME properties, except for poor permeability. Overall, our data support the DHP acid subtype (14) as a valuable scaffold for developing antivirals targeting HCMV pUL89-C.

Identifying Inhibitors of -1 Programmed Ribosomal Frameshifting in a Broad Spectrum of Coronaviruses.

Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases in recent years have highlighted the importance of developing therapeutics with broad-spectrum activity against CoVs. Because all CoVs use -1 programmed ribosomal frameshifting (-1 PRF) to control expression of key viral proteins, the frameshift signal in viral mRNA that stimulates -1 PRF provides a promising potential target for such therapeutics. To test the viability of this strategy, we explored whether small-molecule inhibitors of -1 PRF in SARS-CoV-2 also inhibited -1 PRF in a range of bat CoVs-the most likely source of future zoonoses. Six inhibitors identified in new and previous screens against SARS-CoV-2 were evaluated against the frameshift signals from a panel of representative bat CoVs as well as MERS-CoV. Some drugs had strong activity against subsets of these CoV-derived frameshift signals, while having limited to no effect on -1 PRF caused by frameshift signals from other viruses used as negative controls. Notably, the serine protease inhibitor nafamostat suppressed -1 PRF significantly for multiple CoV-derived frameshift signals. These results suggest it is possible to find small-molecule ligands that inhibit -1 PRF specifically in a broad spectrum of CoVs, establishing frameshift signals as a viable target for developing pan-coronaviral therapeutics.

Collection of Monoclonal Antibodies Targeting SARS-CoV-2 Proteins.

In early 2020, the COVID-19 pandemic sparked a global crisis that continues to pose a serious threat to human health and the economy. Further advancement in research is necessary and requires the availability of quality molecular tools, including monoclonal antibodies. Here, we present the development and characterization of a collection of over 40 new monoclonal antibodies directed against different SARS-CoV-2 proteins. Recombinant SARS-CoV-2 proteins were expressed, purified, and used as immunogens. Upon development of specific hybridomas, the obtained monoclonal antibody (mAb) clones were tested for binding to recombinant proteins and infected cells. We generated mAbs against structural proteins, the Spike and Nucleocapsid protein, several non-structural proteins (nsp1, nsp7, nsp8, nsp9, nsp10, nsp16) and accessory factors (ORF3a, ORF9b) applicable in flow cytometry, immunofluorescence, or Western blot. Our collection of mAbs provides a set of novel, highly specific tools that will allow a comprehensive analysis of the viral proteome, which will allow further understanding of SARS-CoV-2 pathogenesis and the design of therapeutic strategies.

A dual mechanism of action of AT-527 against SARS-CoV-2 polymerase.

The guanosine analog AT-527 represents a promising candidate against Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2). AT-527 recently entered phase III clinical trials for the treatment of COVID-19. Once in cells, AT-527 is converted into its triphosphate form, AT-9010, that presumably targets the viral RNA-dependent RNA polymerase (RdRp, nsp12), for incorporation into viral RNA. Here we report a 2.98 Å cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-nsp82-RNA complex, showing AT-9010 bound at three sites of nsp12. In the RdRp active-site, one AT-9010 is incorporated at the 3' end of the RNA product strand. Its modified ribose group (2'-fluoro, 2'-methyl) prevents correct alignment of the incoming NTP, in this case a second AT-9010, causing immediate termination of RNA synthesis. The third AT-9010 is bound to the N-terminal domain of nsp12 - known as the NiRAN. In contrast to native NTPs, AT-9010 is in a flipped orientation in the active-site, with its guanine base unexpectedly occupying a previously unnoticed cavity. AT-9010 outcompetes all native nucleotides for NiRAN binding, inhibiting its nucleotidyltransferase activity. The dual mechanism of action of AT-527 at both RdRp and NiRAN active sites represents a promising research avenue against COVID-19.

Neuraminidase Inhibitor of Garcinia atroviridis L. Fruits and Leaves Using Partial Purification and Molecular Characterization.

Neuraminidase (NA) is an enzyme that prevents virions from aggregating within the host cell and promotes cell-to-cell spread by cleaving glycosidic linkages to sialic acid. The best-known neuraminidase is the viral neuraminidase, which present in the influenza virus. Thus, the development of anti-influenza drugs that inhibit NA has emerged as an important and intriguing approach in the treatment of influenza. Garcinia atroviridis L. (GA) dried fruits (GAF) are used commercially as seasoning and in beverages. The main objective of this study was to identify a new potential neuraminidase inhibitor from GA. A bioassay-guided fractionation method was applied to obtain the bioactive compounds leading to the identification of garcinia acid and naringenin. In an enzyme inhibition study, garcinia acid demonstrated the highest activity when compared to naringenin. Garcinia acid had the highest activity, with an IC50 of 17.34-17.53 µg/mL or 91.22-92.21 µM against Clostridium perfringens-NA, and 56.71-57.85 µg/mL or 298.32-304.31 µM against H1N1-NA. Based on molecular docking results, garcinia acid interacted with the triad arginine residues (Arg118, Arg292, and Arg371) of the viral neuraminidase, implying that this compound has the potential to act as a NA enzyme inhibitor.

Structure-based inhibitor design and repurposing clinical drugs to target SARS-CoV-2 proteases.

SARS-CoV-2, the coronavirus responsible for the current COVID-19 pandemic, encodes two proteases, 3CLpro and PLpro, two of the main antiviral research targets. Here we provide an overview of the structures and functions of 3CLpro and PLpro and examine strategies of structure-based drug designing and drug repurposing against these proteases. Rational structure-based drug design enables the generation of potent and target-specific antivirals. Drug repurposing offers an attractive prospect with an accelerated turnaround. Thus far, several protease inhibitors have been identified, and some candidates are undergoing trials that may well prove to be effective antivirals against SARS-CoV-2.

Human Endogenous Retrovirus Type K Promotes Proliferation and Confers Sensitivity to Antiretroviral Drugs in Merlin-Negative Schwannoma and Meningioma.

Deficiency of the tumor suppressor Merlin causes development of schwannoma, meningioma, and ependymoma tumors, which can occur spontaneously or in the hereditary disease neurofibromatosis type 2 (NF2). Merlin mutations are also relevant in a variety of other tumors. Surgery and radiotherapy are current first-line treatments; however, tumors frequently recur with limited treatment options. Here, we use human Merlin-negative schwannoma and meningioma primary cells to investigate the involvement of the endogenous retrovirus HERV-K in tumor development. HERV-K proteins previously implicated in tumorigenesis were overexpressed in schwannoma and all meningioma grades, and disease-associated CRL4DCAF1 and YAP/TEAD pathways were implicated in this overexpression. In normal Schwann cells, ectopic overexpression of HERV-K Env increased proliferation and upregulated expression of c-Jun and pERK1/2, which are key components of known tumorigenic pathways in schwannoma, JNK/c-Jun, and RAS/RAF/MEK/ERK. Furthermore, FDA-approved retroviral protease inhibitors ritonavir, atazanavir, and lopinavir reduced proliferation of schwannoma and grade I meningioma cells. These results identify HERV-K as a critical regulator of progression in Merlin-deficient tumors and offer potential strategies for therapeutic intervention. SIGNIFICANCE: The endogenous retrovirus HERV-K activates oncogenic signaling pathways and promotes proliferation of Merlin-deficient schwannomas and meningiomas, which can be targeted with antiretroviral drugs and TEAD inhibitors.

Broad-spectrum prodrugs with anti-SARS-CoV-2 activities: Strategies, benefits, and challenges.

In this era, broad-spectrum prodrugs with anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activities are gaining considerable attention owing to their potential clinical benefits and role in combating the fast-spreading coronavirus disease 2019 (COVID-19) pandemic. The last 2 years have seen a surge of reports on various broad-spectrum prodrugs against SARS-CoV-2, and in in vitro studies, animal models, and clinical practice. Currently, only remdesivir (with many controversies and limitations) has been approved by the U.S. FDA for the treatment of SARS-CoV-2 infection, and additional potent anti-SARS-CoV-2 drugs are urgently required to enrich the defense arsenals. The world has ubiquitously grappled with the COVID-19 pandemic, and the availability of broad-spectrum prodrugs provides great hope for us to subdue this global threat. This article reviews promising treatment strategies, antiviral mechanisms, potential benefits, and daunting clinical challenges of anti-SARS-CoV-2 agents to provide some important guidance for future clinical treatment.

Bifunctional Inhibitors of Influenza Virus Neuraminidase: Molecular Design of a Sulfonamide Linker.

The growing resistance of the influenza virus to widely used competitive neuraminidase inhibitors occupying the active site of the enzyme requires the development of bifunctional compounds that can simultaneously interact with other regulatory sites on the protein surface. When developing such an inhibitor and combining structural fragments that could be located in the sialic acid cavity of the active site and the adjacent 430-cavity, it is necessary to select a suitable linker not only for connecting the fragments, but also to ensure effective interactions with the unique arginine triad Arg118-Arg292-Arg371 of neuraminidase. Using molecular modeling, we have demonstrated the usefulness of the sulfonamide group in the linker design and the potential advantage of this functional group over other isosteric analogues.

Drug Repurposing for Influenza Virus Polymerase Acidic (PA) Endonuclease Inhibitor.

Drug repurposing can quickly and effectively identify novel drug repurposing opportunities. The PA endonuclease catalytic site has recently become regarded as an attractive target for the screening of anti-influenza drugs. PA N-terminal (PAN) inhibitor can inhibit the entire PA endonuclease activity. In this study, we screened the effectivity of PAN inhibitors from the FDA database through in silico methods and in vitro experiments. PAN and mutant PAN-I38T were chosen as virtual screening targets for overcoming drug resistance. Gel-based PA endonuclease analysis determined that the drug lifitegrast can effectively inhibit PAN and PAN-I38T, when the IC50 is 32.82 ± 1.34 µM and 26.81 ± 1.2 µM, respectively. Molecular docking calculation showed that lifitegrast interacted with the residues around PA or PA-I38 T's active site, occupying the catalytic site pocket. Both PAN/PAN-I38T and lifitegrast can acquire good equilibrium in 100 ns molecular dynamic simulation. Because of these properties, lifitegrast, which can effectively inhibit PA endonuclease activity, was screened through in silico and in vitro research. This new research will be of significance in developing more effective and selective drugs for anti-influenza therapy.

Coronavirus Infection-Associated Cell Death Signaling and Potential Therapeutic Targets.

COVID-19 is the name of the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that occurred in 2019. The virus-host-specific interactions, molecular targets on host cell deaths, and the involved signaling are crucial issues, which become potential targets for treatment. Spike protein, angiotensin-converting enzyme 2 (ACE2), cathepsin L-cysteine peptidase, transmembrane protease serine 2 (TMPRSS2), nonstructural protein 1 (Nsp1), open reading frame 7a (ORF7a), viral main protease (3C-like protease (3CLpro) or Mpro), RNA dependent RNA polymerase (RdRp) (Nsp12), non-structural protein 13 (Nsp13) helicase, and papain-like proteinase (PLpro) are molecules associated with SARS-CoV infection and propagation. SARS-CoV-2 can induce host cell death via five kinds of regulated cell death, i.e., apoptosis, necroptosis, pyroptosis, autophagy, and PANoptosis. The mechanisms of these cell deaths are well established and can be disrupted by synthetic small molecules or natural products. There are a variety of compounds proven to play roles in the cell death inhibition, such as pan-caspase inhibitor (z-VAD-fmk) for apoptosis, necrostatin-1 for necroptosis, MCC950, a potent and specific inhibitor of the NLRP3 inflammasome in pyroptosis, and chloroquine/hydroxychloroquine, which can mitigate the corresponding cell death pathways. However, NF-κB signaling is another critical anti-apoptotic or survival route mediated by SARS-CoV-2. Such signaling promotes viral survival, proliferation, and inflammation by inducing the expression of apoptosis inhibitors such as Bcl-2 and XIAP, as well as cytokines, e.g., TNF. As a result, tiny natural compounds functioning as proteasome inhibitors such as celastrol and curcumin can be used to modify NF-κB signaling, providing a responsible method for treating SARS-CoV-2-infected patients. The natural constituents that aid in inhibiting viral infection, progression, and amplification of coronaviruses are also emphasized, which are in the groups of alkaloids, flavonoids, terpenoids, diarylheptanoids, and anthraquinones. Natural constituents derived from medicinal herbs have anti-inflammatory and antiviral properties, as well as inhibitory effects, on the viral life cycle, including viral entry, replication, assembly, and release of COVID-19 virions. The phytochemicals contain a high potential for COVID-19 treatment. As a result, SARS-CoV-2-infected cell death processes and signaling might be of high efficacy for therapeutic targeting effects and yielding encouraging outcomes.

2-((1H-indol-3-yl)thio)-N-phenyl-acetamides: SARS-CoV-2 RNA-dependent RNA polymerase inhibitors.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of Coronavirus Disease 2019 (COVID-19) pandemic. Despite intensive and global efforts to discover and develop novel antiviral therapies, only Remdesivir has been approved as a treatment for COVID-19. Therefore, effective antiviral therapeutics are still urgently needed to combat and halt the pandemic. Viral RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 demonstrates high potential as a reliable target for the development of antivirals. We previously developed a cell-based assay to assess the efficiency of compounds that target SARS-CoV-2 RdRp, as well as their tolerance to viral exoribonuclease-mediated proof-reading. In our previous study, we discovered that 2-((1H-indol-3-yl)thio)-N-phenyl-acetamides specifically targets the RdRp of both respiratory syncytial virus (RSV) and influenza A virus. Thus, we hypothesize that 2-((1H-indol-3-yl)thio)-N-phenyl-acetamides may also have the ability to inhibit SARS-CoV-2 replication by targeting its RdRp activity. In this research, we test a compound library containing 103 of 2-((1H-indol-3-yl)thio)-N-phenyl-acetamides against SARS-CoV-2 RdRp, using our cell-based assay. Among these compounds, the top five candidates strongly inhibit SARS-CoV-2 RdRp activity while exhibiting low cytotoxicity and resistance to viral exoribonuclease. Compound 6-72-2a is the most promising candidate with the lowest EC50 value of 1.41 µM and highest selectivity index (CC50/EC50) (above 70.92). Furthermore, our data suggests that 4-46b and 6-72-2a also inhibit the replication of HCoV-OC43 and HCoV-NL63 virus in a dose-dependent manner. Compounds 4-46b and 6-72-2a exhibit EC50 values of 1.13 µM and 0.94 µM, respectively, on HCoV-OC43 viral replication. However, higher concentrations of these compounds are needed to effectively block HCoV-NL63 replication. Together, our findings successfully identified 4-46b and 6-72-2a as promising inhibitors against SARS-CoV-2 RdRp.