Novel Polymyxin-Inspired Peptidomimetics Targeting the SARS-CoV-2 Spike:hACE2 Interface.

Though the bulk of the COVID-19 pandemic is behind, the search for effective and safe anti-SARS-CoV-2 drugs continues to be relevant. A highly pursued approach for antiviral drug development involves targeting the viral spike (S) protein of SARS-CoV-2 to prevent its attachment to the cellular receptor ACE2. Here, we exploited the core structure of polymyxin B, a naturally occurring antibiotic, to design and synthesize unprecedented peptidomimetics (PMs), intended to target contemporarily two defined, non-overlapping regions of the S receptor-binding domain (RBD). Monomers 1, 2, and 8, and heterodimers 7 and 10 bound to the S-RBD with micromolar affinity in cell-free surface plasmon resonance assays (KD ranging from 2.31 µM to 2.78 µM for dimers and 8.56 µM to 10.12 µM for monomers). Although the PMs were not able to fully protect cell cultures from infection with authentic live SARS-CoV-2, dimer 10 exerted a minimal but detectable inhibition of SARS-CoV-2 entry in U87.ACE2+ and A549.ACE2.TMPRSS2+ cells. These results validated a previous modeling study and provided the first proof-of-feasibility of using medium-sized heterodimeric PMs for targeting the S-RBD. Thus, heterodimers 7 and 10 may serve as a lead for the development of optimized compounds, which are structurally related to polymyxin, with improved S-RBD affinity and anti-SARS-CoV-2 potential.

Nucleoside analogs for management of respiratory virus infections: mechanism of action and clinical efficacy.

The COVID-19 pandemic has accelerated the development of nucleoside analogs to treat respiratory virus infections, with remdesivir being the first compound to receive worldwide authorization and three other nucleoside analogs (i.e. favipiravir, molnupiravir, and bemnifosbuvir) in the pipeline. Here, we summarize the current knowledge concerning their clinical efficacy in suppressing the virus and reducing the need for hospitalization or respiratory support. We also mention trials of favipiravir and lumicitabine, for influenza and respiratory syncytial virus, respectively. Besides, we outline how nucleoside analogs interact with the polymerases of respiratory viruses, to cause lethal virus mutagenesis or disturbance of viral RNA synthesis. In this way, we aim to convey the key findings on this rapidly evolving class of respiratory virus medication.

Synthesis and Anticancer and Antiviral Activities of C-2'-Branched Arabinonucleosides.

d-Arabinofuranosyl-pyrimidine and -purine nucleoside analogues containing alkylthio-, acetylthio- or 1-thiosugar substituents at the C2' position were prepared from the corresponding 3',5'-O-silylene acetal-protected nucleoside 2'-exomethylenes by photoinitiated, radical-mediated hydrothiolation reactions. Although the stereochemical outcome of the hydrothiolation depended on the structure of both the thiol and the furanoside aglycone, in general, high d-arabino selectivity was obtained. The cytotoxic effect of the arabinonucleosides was studied on tumorous SCC (mouse squamous cell) and immortalized control HaCaT (human keratinocyte) cell lines by MTT assay. Three pyrimidine nucleosides containing C2'-butylsulfanylmethyl or -acetylthiomethyl groups showed promising cytotoxicity at low micromolar concentrations with good selectivity towards tumor cells. SAR analysis using a methyl ß-d-arabinofuranoside reference compound showed that the silyl-protecting group, the nucleobase and the corresponding C2' substituent are crucial for the cell growth inhibitory activity. The effects of the three most active nucleoside analogues on parameters indicative of cytotoxicity, such as cell size, division time and cell generation time, were investigated by near-infrared live cell imaging, which showed that the 2'-acetylthiomethyluridine derivative induced the most significant functional and morphological changes. Some nucleoside analogues also exerted anti-SARS-CoV-2 and/or anti-HCoV-229E activity with low micromolar EC50 values; however, the antiviral activity was always accompanied by significant cytotoxicity.

Semisynthetic teicoplanin derivatives with dual antimicrobial activity against SARS-CoV-2 and multiresistant bacteria.

Patients infected with SARS-CoV-2 risk co-infection with Gram-positive bacteria, which severely affects their prognosis. Antimicrobial drugs with dual antiviral and antibacterial activity would be very useful in this setting. Although glycopeptide antibiotics are well-known as strong antibacterial drugs, some of them are also active against RNA viruses like SARS-CoV-2. It has been shown that the antiviral and antibacterial efficacy can be enhanced by synthetic modifications. We here report the synthesis and biological evaluation of seven derivatives of teicoplanin bearing hydrophobic or superbasic side chain. All but one teicoplanin derivatives were effective in inhibiting SARS-CoV-2 replication in VeroE6 cells. One lipophilic and three perfluoroalkyl conjugates showed activity against SARS-CoV-2 in human Calu-3 cells and against HCoV-229E, an endemic human coronavirus, in HEL cells. Pseudovirus entry and enzyme inhibition assays established that the teicoplanin derivatives efficiently prevent the cathepsin-mediated endosomal entry of SARS-CoV-2, with some compounds inhibiting also the TMPRSS2-mediated surface entry route. The teicoplanin derivatives showed good to excellent activity against Gram-positive bacteria resistant to all approved glycopeptide antibiotics, due to their ability to dually bind to the bacterial membrane and cell-wall. To conclude, we identified three perfluoralkyl and one monoguanidine analog of teicoplanin as dual inhibitors of Gram-positive bacteria and SARS-CoV-2.

A Versatile Class of 1,4,4-Trisubstituted Piperidines Block Coronavirus Replication In Vitro.

There is a clear need for novel antiviral concepts to control SARS-CoV-2 infection. Based on the promising anti-coronavirus activity observed for a class of 1,4,4-trisubstituted piperidines, we here conducted a detailed analysis of the structure-activity relationship of these structurally unique inhibitors. Despite the presence of five points of diversity, the synthesis of an extensive series of analogues was readily achieved by Ugi four-component reaction from commercially available reagents. After evaluating 63 analogues against human coronavirus 229E, four of the best molecules were selected and shown to have micromolar activity against SARS-CoV-2. Since the action point was situated post virus entry and lying at the stage of viral polyprotein processing and the start of RNA synthesis, enzymatic assays were performed with CoV proteins involved in these processes. While no inhibition was observed for SARS-CoV-2 nsp12-nsp7-nsp8 polymerase, nsp14 N7-methyltransferase and nsp16/nsp10 2'-O-methyltransferase, nor the nsp3 papain-like protease, the compounds clearly inhibited the nsp5 main protease (Mpro). Although the inhibitory activity was quite modest, the plausibility of binding to the catalytic site of Mpro was established by in silico studies. Therefore, the 1,4,4-trisubstituted piperidines appear to represent a novel class of non-covalent CoV Mpro inhibitors that warrants further optimization and development.

Impact of SARS-CoV-2 Spike Mutations on Its Activation by TMPRSS2 and the Alternative TMPRSS13 Protease.

The continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urges better understanding of the functional motifs in the spike (S) protein and their tolerance to mutations. Here, we focused on the S2' motif, which, during virus entry, requires cleavage by a host cell protease to release the fusion peptide. Though belonging to an immunogenic region, the SARS-CoV-2 S2' motif (811-KPSKR-815) has shown hardly any variation, with its three basic (K/R) residues being >99.99% conserved thus far. By creating a series of mutant pseudoviruses bearing the spikes of Wuhan-Hu-1, its G614 mutant or the Delta and Omicron variants, we show that residue K814 (preceding the scissile R815) is dispensable for TMPRSS2 yet favored by the alternative TMPRSS13 protease. Activation by TMPRSS13 was drastically reduced when the SARS-CoV-2 S2' motif was swapped with that of the low pathogenic 229E coronavirus (685-RVAGR-689), and also, the reverse effect was seen. This swap had no impact on recognition by TMPRSS2. In the Middle East respiratory syndrome coronavirus (MERS-CoV) spike, introducing a dibasic scissile motif was easily accepted by TMPRSS13 but less so by TMPRSS2, confirming that TMPRSS13 favors a sequence rich in K/R residues. Pseudovirus entry experiments in Calu-3 cells confirmed that the S2' mutations have minor impact on TMPRSS2. Our findings are the first to demonstrate which S2' residues are important for SARS-CoV-2 spike activation by these two airway proteases, with TMPRSS2 being more tolerant to variation than TMPRSS13. This preemptive insight will help to estimate the impact of S2' motif changes as they appear in new SARS-CoV-2 variants. IMPORTANCE Since its introduction in humans, SARS-CoV-2 is evolving with frequent appearance of new variants. The surveillance would benefit from proactive characterization of the functional motifs in the spike (S) protein, the most variable viral factor. This is linked to immune evasion but also influences spike functioning. Remarkably, though located in a strongly immunogenic region, the S2' cleavage motif has, thus far, remained highly conserved. This suggests that its sequence is critical for spike activation by airway proteases. To investigate this, we assessed how pseudovirus entry is affected by changes in the S2' motif. We demonstrate that TMPRSS2 readily accepts variations in this motif, whereas the alternative TMPRSS13 protease is more fastidious. The Wuhan-Hu-1, G614, Delta and Omicron spikes showed no difference in this regard. Being the first in its kind, our study will help to assess the impact of S2' variations as soon as they are detected during variant surveillance.

Overcome Double Trouble: Baloxavir Marboxil Suppresses Influenza Thereby Mitigating Secondary Invasive Pulmonary Aspergillosis.

Influenza-associated pulmonary aspergillosis (IAPA) is a global recognized superinfection in critically ill influenza patients. Baloxavir marboxil, a cap-dependent endonuclease inhibitor, is a newly approved anti-influenza therapeutic. Although the benefits as a treatment for influenza are clear, its efficacy against an influenza-A. fumigatus co-infection has yet to be determined. We investigated the therapeutic effect of baloxavir marboxil in a murine model for IAPA. Immunocompetent mice received intranasal instillation of influenza A followed by orotracheal inoculation with Aspergillus fumigatus 4 days later. Administration of baloxavir marboxil or sham was started at day 0, day 2 or day 4. Mice were monitored daily for overall health status, lung pathology with micro-computed tomography (µCT) and fungal burden with bioluminescence imaging (BLI). In vivo imaging was supplemented with virological, mycological and biochemical endpoint investigations. We observed an improved body weight, survival and viral clearance in baloxavir marboxil treated mice. µCT showed less pulmonary lesions and bronchial dilation after influenza and after Aspergillus co-infection in a treatment-dependent pattern. Furthermore, baloxavir marboxil was associated with effective inhibition of fungal invasion. Hence, our results provide evidence that baloxavir marboxil mitigates severe influenza thereby decreasing the susceptibility to a lethal invasive Aspergillus superinfection.

Facile synthesis, antimicrobial and antiviral evaluation of novel substituted phenyl 1,3-thiazolidin-4-one sulfonyl derivatives.

A series of novel substituted phenyl 1, 3-thiazolidin-4-one sulfonyl derivatives 5 (a-t) were synthesized and screened for their in-vitro anti-microbial and anti-viral activity. The result of the anti-microbial assay demonstrated compounds 5d, 5f, 5g, 5h, 5i, 5j showed prominent inhibitory activity against all the tested Gram-positive and Gram-negative bacterial strains, while compounds 5g, 5j, 5o, 5p, 5q showed significant activity against the entire set of fungal strains as compared to standard drug Ampicillin and Clotrimazole, respectively. The antimicrobial study revealed that compounds having electron-withdrawing groups showed significant antimicrobial potency. The most active antibacterial compound 5j showed potent inhibition of S. aureus DNA Gyrase enzyme as a possible mechanism of action for antimicrobial activity. Moreover, the antiviral testing of selected compounds showed considerable activity against Herpes simplex virus-1(KOS), Herpes simplex virus-2 (G), Herpes simplex virus-1(TK- KOS ACVr), Vaccinia virus, Human Coronavirus (229E), Reovirus-1, Sindbis virus, Coxsackie virus B4, Yellow Fever virus and Influenza A, B virus. Compounds 5h exhibited low anti-viral activity against HIV-1(strain IIIB) and HIV-2 (strain ROD). The study clearly outlined that synthesized compounds endowed with good antimicrobial property together with considerable antiviral activity.

Favipiravir Does Not Inhibit Chikungunya Virus Replication in Mosquito Cells and Aedes aegypti Mosquitoes.

Favipiravir (T-705) is a broad-spectrum antiviral drug that inhibits RNA viruses after intracellular conversion into its active form, T-705 ribofuranosyl 5'-triphosphate. We previously showed that T-705 is able to significantly inhibit the replication of chikungunya virus (CHIKV), an arbovirus transmitted by Aedes mosquitoes, in mammalian cells and in mouse models. In contrast, the effect of T-705 on CHIKV infection and replication in the mosquito vector is unknown. Since the antiviral activity of T-705 has been shown to be cell line-dependent, we studied here its antiviral efficacy in Aedes-derived mosquito cells and in Aedes aegypti mosquitoes. Interestingly, T-705 was devoid of anti-CHIKV activity in mosquito cells, despite being effective against CHIKV in Vero cells. By investigating the metabolic activation profile, we showed that, unlike Vero cells, mosquito cells were not able to convert T-705 into its active form. To explore whether alternative metabolization pathways might exist in vivo, Aedes aegypti mosquitoes were infected with CHIKV and administered T-705 via an artificial blood meal. Virus titrations of whole mosquitoes showed that T-705 was not able to reduce CHIKV infection in mosquitoes. Combined, these in vitro and in vivo data indicate that T-705 lacks antiviral activity in mosquitoes due to inadequate metabolic activation in this animal species.

The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways.

The high transmissibility of SARS-CoV-2 is related to abundant replication in the upper airways, which is not observed for the other highly pathogenic coronaviruses SARS-CoV and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards the human respiratory tract. First, the S proteins exhibit an intrinsic temperature preference, corresponding with the temperature of the upper or lower airways. Pseudoviruses bearing the SARS-CoV-2 spike (SARS-2-S) were more infectious when produced at 33°C instead of 37°C, a property shared with the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV and MERS-CoV favored 37°C, in accordance with virus preference for the lower airways. Next, SARS-2-S-driven entry was efficiently activated by not only TMPRSS2, but also the TMPRSS13 protease, thus broadening the cell tropism of SARS-CoV-2. Both proteases proved relevant in the context of authentic virus replication. TMPRSS13 appeared an effective spike activator for the virulent coronaviruses but not the low pathogenic HCoV-229E virus. Activation of SARS-2-S by these surface proteases requires processing of the S1/S2 cleavage loop, in which both the furin recognition motif and extended loop length proved critical. Conversely, entry of loop deletion mutants is significantly increased in cathepsin-rich cells. Finally, we demonstrate that the D614G mutation increases SARS-CoV-2 stability, particularly at 37°C, and, enhances its use of the cathepsin L pathway. This indicates a link between S protein stability and usage of this alternative route for virus entry. Since these spike properties may promote virus spread, they potentially explain why the spike-G614 variant has replaced the early D614 variant to become globally predominant. Collectively, our findings reveal adaptive mechanisms whereby the coronavirus spike protein is adjusted to match the temperature and protease conditions of the airways, to enhance virus transmission and pathology.

Betulonic Acid Derivatives Interfering with Human Coronavirus 229E Replication via the nsp15 Endoribonuclease.

To develop antiviral therapeutics against human coronavirus (HCoV) infections, suitable coronavirus drug targets and corresponding lead molecules must be urgently identified. Here, we describe the discovery of a class of HCoV inhibitors acting on nsp15, a hexameric protein component of the viral replication-transcription complexes, endowed with immune evasion-associated endoribonuclease activity. Structure-activity relationship exploration of these 1,2,3-triazolo-fused betulonic acid derivatives yielded lead molecule 5h as a strong inhibitor (antiviral EC50: 0.6 µM) of HCoV-229E replication. An nsp15 endoribonuclease active site mutant virus was markedly less sensitive to 5h, and selected resistance to the compound mapped to mutations in the N-terminal part of HCoV-229E nsp15, at an interface between two nsp15 monomers. The biological findings were substantiated by the nsp15 binding mode for 5h, predicted by docking. Hence, besides delivering a distinct class of inhibitors, our study revealed a druggable pocket in the nsp15 hexamer with relevance for anti-coronavirus drug development.

Synthesis and biological evaluation of substituted phenyl azetidine-2-one sulphonyl derivatives as potential antimicrobial and antiviral agents.

In the present study, we intend to synthesize a series of novel substituted phenyl azetidine-2-one sulphonyl derivatives. The entire set of derivatives 5 (a-t) were screened for in-vitro antibacterial, and antifungal activity, and among them eleven compounds were further screened for the antiviral activity to predict their efficacy against pathogenic viruses. Interestingly, compound 5d, 5e, 5f, 5h, 5i, and 5j showed similar or better antibacterial activity as compared to ampicillin (standard). Moreover, compounds 5h, 5i, 5j, and 5q showed good inhibitory activity against fungal strains whereas other derivatives had mild or diminished activity in comparison with standard drug clotrimazole. The antimicrobial study indicated that compounds having electron-withdrawing groups showed the highest activity. Interestingly, these tested compounds showed weak antiviral activity against Vaccinia virus, Human Coronavirus (229E), Reovirus-1, Herpes simplex virus, Sindbis virus, Coxsackievirus B4, Yellow Fever virus, and Influenza B virus in HEL cell, Vero cell, and MDCK cell cultures. The findings of the present study might open new avenues to target human disease-causing deadly microbes and viruses.