The coronavirus nsp15 endoribonuclease: A puzzling protein and pertinent antiviral drug target.

The SARS-CoV-2 pandemic has bolstered unprecedented research efforts to better understand the pathogenesis of coronavirus (CoV) infections and develop effective therapeutics. We here focus on non-structural protein nsp15, a hexameric component of the viral replication-transcription complex (RTC). Nsp15 possesses uridine-specific endoribonuclease (EndoU) activity for which some specific cleavage sites were recently identified in viral RNA. By preventing accumulation of viral dsRNA, EndoU helps the virus to evade RNA sensors of the innate immune response. The immune-evading property of nsp15 was firmly established in several CoV animal models and makes it a pertinent target for antiviral therapy. The search for nsp15 inhibitors typically proceeds via compound screenings and is aided by the rapidly evolving insight in the protein structure of nsp15. In this overview, we broadly cover this fascinating protein, starting with its structure, biochemical properties and functions in CoV immune evasion. Next, we summarize the reported studies in which compound screening or a more rational method was used to identify suitable leads for nsp15 inhibitor development. In this way, we hope to raise awareness on the relevance and druggability of this unique CoV protein.

Exploration of 1,2,3-triazolo fused triterpenoids as inhibitors of human coronavirus 229E targeting the viral nsp15 protein.

The coronavirus disease-19 (COVID-19) pandemic has raised major interest in innovative drug concepts to suppress human coronavirus (HCoV) infections. We previously reported on a class of 1,2,3-triazolo fused betulonic acid derivatives causing strong inhibition of HCoV-229E replication via the viral nsp15 protein, which is proposedly related to compound binding at an intermonomer interface in hexameric nsp15. In the present study, we further explored the structure-activity relationship (SAR), by varying the substituent at the 1,2,3-triazolo ring as well as the triterpenoid skeleton. The 1,2,3-triazolo fused triterpenoids were synthesized by a multicomponent triazolization reaction, which has been developed in-house. Several analogs possessing a betulin, oleanolic acid, or ursolic acid core displayed favorable activity and selectivity (EC50 values for HCoV-229E: 1.6-3.5 µM), but neither of them proved as effective as the lead compound containing betulonic acid. The 18ß-glycyrrhetinic acid-containing analogs had low selectivity. The antiviral findings were rationalized by in silico docking in the available structure of the HCoV-229E nsp15 protein. The new SAR insights will aid the further development of these 1,2,3-triazolo fused triterpenoid compounds as a unique type of coronavirus inhibitors.

PRO-2000 exhibits SARS-CoV-2 antiviral activity by interfering with spike-heparin binding.

Here, we report on the anti-SARS-CoV-2 activity of PRO-2000, a sulfonated polyanionic compound. In Vero cells infected with the Wuhan, alpha, beta, delta or omicron variant, PRO-2000 displayed EC50 values of 1.1 µM, 2.4 µM, 1.3 µM, 2.1 µM and 0.11 µM, respectively, and an average selectivity index (i.e. ratio of cytotoxic versus antiviral concentration) of 172. Its anti-SARS-CoV-2 activity was confirmed by virus yield assays in Vero cells, Caco2 cells and A549 cells overexpressing ACE2 and TMPRSS2 (A549-AT). Using pseudoviruses bearing the SARS-CoV-2 spike (S), PRO-2000 was shown to block the S-mediated pseudovirus entry in Vero cells and A549-AT cells, with EC50 values of 0.091 µM and 1.6 µM, respectively. This entry process is initiated by interaction of the S glycoprotein with angiotensin-converting enzyme 2 (ACE2) and heparan sulfate proteoglycans. Surface Plasmon Resonance (SPR) studies showed that PRO-2000 binds to the receptor-binding domain (RBD) of S with a KD of 1.6 nM. Similar KD values (range: 1.2 nM-2.1 nM) were obtained with the RBDs of the alpha, beta, delta and omicron variants. In an SPR neutralization assay, PRO-2000 had no effect on the interaction between the RBD and ACE2. Instead, PRO-2000 was proven to inhibit binding of the RBD to a heparin-coated sensor chip, yielding an IC50 of 1.1 nM. To conclude, PRO-2000 has the potential to inhibit a broad range of SARS-CoV-2 variants by blocking the heparin-binding site on the S protein.

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.

Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants.

Despite the vaccination campaigns for COVID-19, we still cannot control the spread of SARS-CoV-2, as evidenced by the ongoing circulation of the Omicron variants of concern. This highlights the need for broad-spectrum antivirals to further combat COVID-19 and to be prepared for a new pandemic with a (re-)emerging coronavirus. An interesting target for antiviral drug development is the fusion of the viral envelope with host cell membranes, a crucial early step in the replication cycle of coronaviruses. In this study, we explored the use of cellular electrical impedance (CEI) to quantitatively monitor morphological changes in real time, resulting from cell-cell fusion elicited by SARS-CoV-2 spike. The impedance signal in CEI-quantified cell-cell fusion correlated with the expression level of SARS-CoV-2 spike in transfected HEK293T cells. For antiviral assessment, we validated the CEI assay with the fusion inhibitor EK1 and measured a concentration-dependent inhibition of SARS-CoV-2 spike mediated cell-cell fusion (IC50 value of 0.13 µM). In addition, CEI was used to confirm the fusion inhibitory activity of the carbohydrate-binding plant lectin UDA against SARS-CoV-2 (IC50 value of 0.55 µM), which complements prior in-house profiling activities. Finally, we explored the utility of CEI in quantifying the fusogenic potential of mutant spike proteins and in comparing the fusion efficiency of SARS-CoV-2 variants of concern. In summary, we demonstrate that CEI is a powerful and sensitive technology that can be applied to studying the fusion process of SARS-CoV-2 and to screening and characterizing fusion inhibitors in a label-free and non-invasive manner.

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.

Carbohydrate-binding protein from stinging nettle as fusion inhibitor for SARS-CoV-2 variants of concern.

Urtica dioica agglutinin (UDA) is a carbohydrate-binding small monomeric protein isolated from stinging nettle rhizomes. It inhibits replication of a broad range of viruses, including coronaviruses, in multiple cell types, with appealing selectivity. In this work, we investigated the potential of UDA as a broad-spectrum antiviral agent against SARS-CoV-2. UDA potently blocks transduction of pseudotyped SARS-CoV-2 in A549.ACE2+-TMPRSS2 cells, with IC50 values ranging from 0.32 to 1.22 µM. Furthermore, UDA prevents viral replication of the early Wuhan-Hu-1 strain in Vero E6 cells (IC50 = 225 nM), but also the replication of SARS-CoV-2 variants of concern, including Alpha, Beta and Gamma (IC50 ranging from 115 to 171 nM). In addition, UDA exerts antiviral activity against the latest circulating Delta and Omicron variant in U87.ACE2+ cells (IC50 values are 1.6 and 0.9 µM, respectively). Importantly, when tested in Air-Liquid Interface (ALI) primary lung epithelial cell cultures, UDA preserves antiviral activity against SARS-CoV-2 (20A.EU2 variant) in the nanomolar range. Surface plasmon resonance (SPR) studies demonstrated a concentration-dependent binding of UDA to the viral spike protein of SARS-CoV-2, suggesting interference of UDA with cell attachment or subsequent virus entry. Moreover, in additional mechanistic studies with cell-cell fusion assays, UDA inhibited SARS-CoV-2 spike protein-mediated membrane fusion. Finally, pseudotyped SARS-CoV-2 mutants with N-glycosylation deletions in the S2 subunit of the spike protein remained sensitive to the antiviral activity of UDA. In conclusion, our data establish UDA as a potent fusion inhibitor for the current variants of SARS-CoV-2.

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.

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.

Exploration of the 2,3-dihydroisoindole pharmacophore for inhibition of the influenza virus PA endonuclease.

Seasonal influenza A and B viruses represent a global concern. Antiviral drugs are crucial to treat severe influenza in high-risk patients and prevent virus spread in case of a pandemic. The emergence of viruses showing drug resistance, in particular for the recently licensed polymerase inhibitor baloxavir marboxil, drives the need for developing alternative antivirals. The endonuclease activity residing in the N-terminal domain of the polymerase acidic protein (PAN) is crucial for viral RNA synthesis and a validated target for drug design. Its function can be impaired by molecules bearing a metal-binding pharmacophore (MBP) able to coordinate the two divalent metal ions in the active site. In the present work, the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one scaffold is explored for the inhibition of influenza virus PA endonuclease. The structure-activity relationship was analysed by modifying the substituents on the lipophilic moiety linked to the MBP. The new compounds exhibited nanomolar inhibitory activity in a FRET-based enzymatic assay, and a few compounds (15-17, 21) offered inhibition in the micromolar range, in a cell-based influenza virus polymerase assay. When investigated against a panel of PA-mutant forms, compound 17 was shown to retain full activity against the baloxavir-resistant I38T mutant. This was corroborated by docking studies providing insight into the binding mode of this novel class of PA inhibitors.

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.

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.

Functional Analysis of Human and Feline Coronavirus Cross-Reactive Antibodies Directed Against the SARS-CoV-2 Fusion Peptide.

To face the continuous emergence of SARS-CoV-2 variants, broadly protective therapeutic antibodies are highly needed. We here focused on the fusion peptide (FP) region of the viral spike antigen since it is highly conserved among alpha- and betacoronaviruses. First, we found that coronavirus cross-reactive antibodies are commonly formed during infection, being omnipresent in sera from COVID-19 patients, in ~50% of pre-pandemic human sera (rich in antibodies against endemic human coronaviruses), and even in feline coronavirus-infected cats. Pepscan analyses demonstrated that a confined N-terminal region of the FP is strongly immunogenic across diverse coronaviruses. Peptide-purified human antibodies targeting this conserved FP epitope exhibited broad binding of alpha- and betacoronaviruses, besides weak and transient SARS-CoV-2 neutralizing activity. Being frequently elicited by coronavirus infection, these FP-binding antibodies might potentially exhibit Fc-mediated effector functions and influence the kinetics or severity of coronavirus infection and disease.

Reprogramming of the Antibacterial Drug Vancomycin Results in Potent Antiviral Agents Devoid of Antibacterial Activity.

Influenza A and B viruses are a global threat to human health and increasing resistance to the existing antiviral drugs necessitates new concepts to expand the therapeutic options. Glycopeptide derivatives have emerged as a promising new class of antiviral agents. To avoid potential antibiotic resistance, these antiviral glycopeptides are preferably devoid of antibiotic activity. We prepared six vancomycin aglycone hexapeptide derivatives with the aim of obtaining compounds having anti-influenza virus but no antibacterial activity. Two of them exerted strong and selective inhibition of influenza A and B virus replication, while antibacterial activity was successfully eliminated by removing the critical N-terminal moiety. In addition, these two molecules offered protection against several other viruses, such as herpes simplex virus, yellow fever virus, Zika virus, and human coronavirus, classifying these glycopeptides as broad antiviral molecules with a favorable therapeutic index.

N-benzyl 4,4-disubstituted piperidines as a potent class of influenza H1N1 virus inhibitors showing a novel mechanism of hemagglutinin fusion peptide interaction.

The influenza virus hemagglutinin (HA) is an attractive target for antiviral therapy due to its essential role in mediating virus entry into the host cell. We here report the identification of a class of N-benzyl-4,4,-disubstituted piperidines as influenza A virus fusion inhibitors with specific activity against the H1N1 subtype. Using the highly efficient one-step Ugi four-component reaction, diverse library of piperidine-based analogues was synthesized and evaluated to explore the structure-activity relationships (SAR). Mechanistic studies, including resistance selection with the most active compound (2) demonstrated that it acts as an inhibitor of the low pH-induced HA-mediated membrane fusion process. Computational studies identified an as yet unrecognized fusion inhibitor binding site, which is located at the bottom of the HA2 stem in close proximity to the fusion peptide. A direct π-stacking interaction between the N-benzylpiperidine moiety of 2 and F9HA2 of the fusion peptide, reinforced with an additional π-stacking interaction with Y119HA2, and a salt bridge of the protonated piperidine nitrogen with E120HA2, were identified as important interactions to mediate ligand binding. This site rationalized the observed SAR and provided a structural explanation for the H1N1-specific activity of our inhibitors. Furthermore, the HA1-S326V mutation resulting in resistance to 2 is close to the proposed new binding pocket. Our findings point to the N-benzyl-4,4,-disubstituted piperidines as an interesting class of influenza virus inhibitors, representing the first example of fusion peptide binders with great potential for anti-influenza drug development.

Hemagglutinin Cleavability, Acid Stability, and Temperature Dependence Optimize Influenza B Virus for Replication in Human Airways.

Influenza A virus (IAV) and influenza B virus (IBV) cause yearly epidemics with significant morbidity and mortality. When zoonotic IAVs enter the human population, the viral hemagglutinin (HA) requires adaptation to achieve sustained virus transmission. In contrast, IBV has been circulating in humans, its only host, for a long period of time. Whether this entailed adaptation of IBV HA to the human airways is unknown. To address this question, we compared two seasonal IAVs (A/H1N1 and A/H3N2) and two IBVs (B/Victoria and B/Yamagata lineages) with regard to host-dependent activity of HA as the mediator of membrane fusion during viral entry. We first investigated proteolytic activation of HA by covering all type II transmembrane serine protease (TTSP) and kallikrein enzymes, many of which proved to be present in human respiratory epithelium. The IBV HA0 precursor is cleaved by a broader panel of TTSPs and activated with much higher efficiency than IAV HA0. Accordingly, knockdown of a single protease, TMPRSS2, abrogated spread of IAV but not IBV in human respiratory epithelial cells. Second, the HA fusion pH values proved similar for IBV and human-adapted IAVs (with one exception being the HA of 1918 IAV). Third, IBV HA exhibited higher expression at 33°C, a temperature required for membrane fusion by B/Victoria HA. This indicates pronounced adaptation of IBV HA to the mildly acidic pH and cooler temperature of human upper airways. These distinct and intrinsic features of IBV HA are compatible with extensive host adaptation during prolonged circulation of this respiratory virus in the human population.IMPORTANCE Influenza epidemics are caused by influenza A and influenza B viruses (IAV and IBV, respectively). IBV causes substantial disease; however, it is far less studied than IAV. While IAV originates from animal reservoirs, IBV circulates in humans only. Virus spread requires that the viral hemagglutinin (HA) is active and sufficiently stable in human airways. We resolve here how these mechanisms differ between IBV and IAV. Whereas human IAVs rely on one particular protease for HA activation, this is not the case for IBV. Superior activation of IBV by several proteases should enhance shedding of infectious particles. IBV HA exhibits acid stability and a preference for 33°C, indicating pronounced adaptation to the human upper airways, where the pH is mildly acidic and a cooler temperature exists. These adaptive features are rationalized by the long existence of IBV in humans and may have broader relevance for understanding the biology and evolution of respiratory viruses.

Synthesis and anti-coronavirus activity of a series of 1-thia-4-azaspiro[4.5]decan-3-one derivatives.

A series of 1-thia-4-azaspiro[4.5]decan-3-ones bearing an amide group at C-4 and various substitutions at C-2 and C-8 were synthesized and evaluated against human coronavirus and influenza virus. Compounds 7m, 7n, 8k, 8l, 8m, 8n, and 8p were found to inhibit human coronavirus 229E replication. The most active compound was N-(2-methyl-8-tert-butyl-3-oxo-1-thia-4-azaspiro[4.5]decan-4-yl)-3-phenylpropanamide (8n), with an EC50 value of 5.5 µM, comparable to the known coronavirus inhibitor, (Z)-N-[3-[4-(4-bromophenyl)-4-hydroxypiperidin-1-yl]-3-oxo-1-phenylprop-1-en-2-yl]benzamide (K22). Compound 8n and structural analogs were devoid of anti-influenza virus activity, although their scaffold is shared with a previously discovered class of H3 hemagglutinin-specific influenza virus fusion inhibitors. These findings point to the 1-thia-4-azaspiro[4.5]decan-3-one scaffold as a versatile chemical structure with high relevance for antiviral drug development.

Influenza virus entry via the GM3 ganglioside-mediated platelet-derived growth factor receptor ß signalling pathway.

The possible resistance of influenza virus against existing antiviral drugs calls for new therapeutic concepts. One appealing strategy is to inhibit virus entry, in particular at the stage of internalization. This requires a better understanding of virus-host interactions during the entry process, including the role of receptor tyrosine kinases (RTKs). To search for cellular targets, we evaluated a panel of 276 protein kinase inhibitors in a multicycle antiviral assay in Madin-Darby canine kidney cells. The RTK inhibitor Ki8751 displayed robust anti-influenza A and B virus activity and was selected for mechanistic investigations. Ki8751 efficiently disrupted the endocytic process of influenza virus in different cell lines carrying platelet-derived growth factor receptor ß (PDGFRß), an RTK that is known to act at GM3 ganglioside-positive lipid rafts. The more efficient virus entry in CHO-K1 cells compared to the wild-type ancestor (CHO-wt) cells indicated a positive effect of GM3, which is abundant in CHO-K1 but not in CHO-wt cells. Entering virus localized to GM3-positive lipid rafts and the PDGFRß-containing endosomal compartment. PDGFRß/GM3-dependent virus internalization involved PDGFRß phosphorylation, which was potently inhibited by Ki8751, and desialylation of activated PDGFRß by the viral neuraminidase. Virus uptake coincided with strong activation of the Raf/MEK/Erk cascade, but not of PI3K/Akt or phospholipase C-γ. We conclude that influenza virus efficiently hijacks the GM3-enhanced PDGFRß signalling pathway for cell penetration, providing an opportunity for host cell-targeting antiviral intervention.

Structure-activity relationship studies of lipophilic teicoplanin pseudoaglycon derivatives as new anti-influenza virus agents.

Six series of semisynthetic lipophilic glycopeptide antibiotic derivatives were evaluated for in vitro activity against influenza A and B viruses. The new teicoplanin pseudoaglycon-derived lipoglycopeptides were prepared by coupling one or two side chains to the N-terminus of the glycopeptide core, using various conjugation methods. Three series of derivatives bearing two lipophilic groups were synthesized by attaching bis-alkylthio maleimides directly or through linkers of different lengths to the glycopeptide. Access to the fourth and fifth series of compounds was achieved by click chemistry, introducing single alkyl/aryl chains directly or through a tetraethylene glycol linker to the same position. A sixth group of semisynthetic derivatives was obtained by sulfonylation of the N-terminus. Of the 42 lipophilic teicoplanin pseudoaglycon derivatives tested, about half showed broad activity against influenza A and B viruses, with some of them having reasonable or no cytotoxicity. Minor differences in the side chain length as well as lipophilicity appeared to have significant impact on antiviral activity and cytotoxicity. Several lipoglycopeptides were also found to be active against human coronavirus.

Identification of influenza PA-Nter endonuclease inhibitors using pharmacophore- and docking-based virtual screening.

Searching for new antiviral agents, we focused our interest on the influenza PA-Nter endonuclease. Therefore, we developed a three-dimensional pharmacophore model which contains the binding features addressed to the metal-chelating active site. The obtained hypothesis has been fruitfully employed to select three "hit compounds" through an in silico screening campaign on our in-house database of small molecules. We studied the binding poses of these hit compounds using molecular docking, and subjected them to an enzymatic assay with recombinant PA-Nter endonuclease. Compound 20 proved the most active inhibitor of the endonucleolytic cleavage reaction, with an IC50 value of 12 µM.