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RationaleCOVID-19 is an acute infectious disease caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Human surfactant protein D (SP-D) is known to interact with spike protein of SARS-CoV, but its immune-surveillance against SARS-CoV-2 is not known.ObjectiveThis study aimed to examine the potential of a recombinant fragment of human SP-D (rfhSP-D) as an inhibitor of replication and infection of SARS-CoV-2.MethodsrfhSP-D interaction with spike protein of SARS-CoV-2 and hACE-2 receptor was predicted via docking analysis. The inhibition of interaction between spike protein and ACE-2 by rfhSP-D was confirmed using direct and indirect ELISA. The effect of rfhSP-D on replication and infectivity of SARS-CoV-2 from clinical samples was studied by measuring the expression of RdRp gene of the virus using qPCR.Measurements and Main ResultsIn-silico interaction studies indicated that three amino acid residues in the RBD of spike of SARS-CoV-2 were commonly involved in interacting with rfhSP-D and ACE-2. Studies using clinical samples of SARS-CoV-2 positive cases (asymptomatic, n=7 and symptomatic, n=8 and negative controls n=15) demonstrated that treatment with 5μM rfhSP-D inhibited viral replication by ~5.5 fold and was more efficient than Remdesivir (100 μM). Approximately, a 2-fold reduction in viral infectivity was also observed after treatment with 5μM rfhSP-D.ConclusionsThese results conclusively demonstrate that the calcium independent rfhSP-D mediated inhibition of binding between the receptor binding domain of the S1 subunit of the SARS-CoV-2 spike protein and human ACE-2, its host cell receptor, and a significant reduction in SARS-CoV-2 infection and replication in-vitro.

Bovine viral diarrhea virus (BVDV), also known as Pestivirus A, causes severe infection mostly in cattle, but also in pigs, sheep and goats, causing huge economical losses on agricultural farms every year. The infections are actually controlled by isolation of persistently infected animals and vaccination, but no antivirals are currently available to control the spread of BVDV on farms. BVDV binds the host cell using envelope protein E2, which has only recently been targeted in the research of a potent and efficient antiviral. In contrast, RdRp has been successfully inhibited by several classes of compounds in the last few decades. As a part of an enduring antiviral research agenda, we designed a new series of derivatives that emerged from an isosteric substitution of the main scaffold in previously reported anti-BVDV compounds. Here, the new compounds were characterized and tested, where several turned out to be potent and selectively active against BVDV. The mechanism of action was thoroughly studied using a time-of-drug-addition assay and the results were validated using docking simulations.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the newly emergent causative agent of coronavirus disease-19 (COVID-19), has resulted in more than two million deaths worldwide since it was first detected in 2019. There is a critical global need for therapeutic intervention strategies that can be deployed to safely treat COVID-19 disease and reduce associated morbidity and mortality. Increasing evidence shows that both natural and synthetic antimicrobial peptides (AMPs), also referred to as Host Defense Proteins/Peptides (HDPs), can inhibit SARS-CoV-2, paving the way for the potential clinical use of these molecules as therapeutic options. In this manuscript, we describe the potent antiviral activity exerted by brilacidin—a de novo designed synthetic small molecule that captures the biological properties of HDPs—on SARS-CoV-2 in a human lung cell line (Calu-3) and a monkey cell line (Vero). These data suggest that SARS-CoV-2 inhibition in these cell culture models is likely to be a result of the impact of brilacidin on viral entry and its disruption of viral integrity. Brilacidin demonstrated synergistic antiviral activity when combined with remdesivir. Collectively, our data demonstrate that brilacidin exerts potent inhibition of SARS-CoV-2 against different strains of the virus in cell culture.

KR13, a peptide triazole thiol previously established to inhibit HIV-1 infection and cause virus lysis, was evaluated by flow cytometry against JRFL Env-presenting cells to characterize induced Env and membrane transformations leading to irreversible inactivation. Transiently transfected HEK293T cells were preloaded with calcein dye, treated with KR13 or its thiol-blocked analogue KR13b, fixed, and stained for gp120 (35O22), MPER (10E8), 6-helix-bundle (NC-1), immunodominant loop (50-69), and fusion peptide (VRC34.01). KR13 induced dose-dependent transformations of Env and membrane characterized by transient poration, MPER exposure, and 6-helix-bundle formation (analogous to native fusion events), but also reduced immunodominant loop and fusion peptide exposure. Using a fusion peptide mutant (V504E), we found that KR13 transformation does not require functional fusion peptide for poration. In contrast, simultaneous treatment with fusion inhibitor T20 alongside KR13 prevented membrane poration and MPER exposure, showing that these events require 6-helix-bundle formation. Based on these results, we formulated a model for PTT-induced Env transformation portraying how, in the absence of CD4/co-receptor signaling, PTT may provide alternate means of perturbing the metastable Env-membrane complex, and inducing fusion-like transformation. In turn, the results show that such transformations are intrinsic to Env and can be diverted for irreversible inactivation of the protein complex.

Human adenoviruses (HAdVs) display a wide range of tissue tropism and can cause an array of symptoms from mild respiratory illnesses to disseminated and life-threatening infections in immunocompromised individuals. However, no antiviral drug has been approved specifically for the treatment of HAdV infections. Herein, we report our continued efforts to optimize salicylamide derivatives and discover compound 16 (JMX0493) as a potent inhibitor of HAdV infection. Compound 16 displays submicromolar IC50 values, a higher selectivity index (SI &gt, 100) and 2.5-fold virus yield reduction compared to our hit compound niclosamide. Moreover, unlike niclosamide, our mechanistic studies suggest that the antiviral activity of compound 16 against HAdV is achieved through the inhibition of viral particle escape from the endosome, which bars subsequent uncoating and the presentation of lytic protein VI.

Hepatitis C Virus (HCV) remains an important public health threat with approximately 170 million carriers worldwide who are at risk of developing hepatitis C-associated end-stage liver diseases. Despite improvement of HCV treatment using the novel direct-acting antivirals (DAAs) targeting viral replication, there is a lack of prophylactic measures for protection against HCV infection. Identifying novel antivirals such as those that target viral entry could help broaden the therapeutic arsenal against HCV. Herein, we investigated the anti-HCV activity of the methanolic extract from Rhizoma coptidis (RC), a widely used traditional Chinese medicine documented by the WHO and experimentally reported to possess several pharmacological functions including antiviral effects. Using the cell culture-derived HCV system, we demonstrated that RC dose-dependently inhibited HCV infection of Huh-7.5 cells at non-cytotoxic concentrations. In particular, RC blocked HCV attachment and entry/fusion into the host cells without exerting any significant effect on the cell-free viral particles or modulating key host cell entry factors to HCV. Moreover, RC robustly suppressed HCV pseudoparticles infection of Huh-7.5 cells and impeded infection by several HCV genotypes. Collectively, our results identified RC as a potent antagonist to HCV entry with potential pan-genotypic properties, which deserves further evaluation for use as an anti-HCV agent.

International audience; We previously reported that Toll-like receptor 9 (TLR9)-CpG oligonucleo-tides could inhibit the establishment of hepatitis B virus (HBV) infections in hepato-cytes. Our aim was to uncover the underlying mechanisms of this inhibition. HepaRG cells, RPMI-B lymphoblastoma cells, and primary plasmacytoid dendritic cells (pDCs) exposed to HBV and TLR9 ligands/agonists in various configurations were used. We observed an inhibition of HBV infection upon TLR9 stimulations only when agonist was applied during inoculation. This inhibition was independent of interleukin-6 (IL-6)/interferon-inducible protein 10 (IP-10) production as well as of TLR9 expression in hepatocytes. We further demonstrated an entry inhibition mechanism by showing a noncovalent binding of TLR9 agonist to HBV particles. Besides inhibiting HBV entry into hepatocytes, this biophysical interaction between HBV virions and TLR9 agonist was responsible for a reduction of alpha interferon (IFN-) expression by pDCs. Interestingly , subviral particles composed of only HBsAg were able to genuinely inhibit the TLR9 pathway, without titrating TLR9 ligands. To conclude, our data suggest that synthetic TLR9-CpG oligonucleotides can strongly inhibit HBV entry by " coating " HBV virions and thereby preventing their interaction with cellular receptor. This titration effect of TLR9 agonist is also artifactually responsible for the inhibition of TLR9 engagement in pDCs, whereas a genuine inhibition of this innate pathway was confirmed with HBsAg subviral particles. H epatitis B virus (HBV) chronically infects 240 million people worldwide and represents one of the major etiologies for cirrhosis and hepatocellular carcinoma (1, 2). The progression rate of chronicity is tightly linked with the maturity of the immune system, since 90 to 95% of infected newborns become chronic carriers, whereas only 5 to 10% of adults do (3). As HBV long-term persistence is characterized by subversion of both innate and adaptive immunity (4, 5), a better understanding of the underlying cellular and molecular mechanisms of this escape should help in defining new therapeutic strategies to reactivate the host immune system and control viral replication. Current front-line therapies include the use of nucleoside analogues to specifically inhibit viral reverse transcription and/or pegylated interferon alpha 2a or 2b (Peg-IFN

Here, we report the anti-human immunodeficiency virus (HIV) potency and underlying mechanisms of a Keggin polyoxometalate (PT-1, K6HPTi2W10O40). Our findings showed that PT-1 exhibited highly potent effects against a diverse group of HIV type 1 (HIV-1) strains and displayed low cytotoxicity and genotoxicity. The time-addition assay revealed that PT-1 acted at an early stage of infection, and these findings were supported by the observation that PT-1 had more potency against Env-pseudotyped virus than vesicular stomatitis virus glycoprotein (VSVG) pseudotyped virus. Surface plasmon resonance binding assays and flow cytometry analysis showed that PT-1 blocked the gp120 binding site in the CD4 receptor. Moreover, PT-1 bound directly to gp41 NHR (N36 peptide), thereby interrupting the core bundle formation of gp41. In conclusion, our data suggested that PT-1 may be developed as a new anti-HIV-1 agent through its effects on entry inhibition.

The small molecule CCR5 inhibitors are a new class of drugs for treating infection by human immunodeficiency virus type 1 (HIV-1). They act by binding to the CCR5 co-receptor and preventing its use during HIV-1–cell fusion. Escape mutants can be raised against CCR5 inhibitors in vitro and will arise when these drugs are used clinically. Here, we have assessed the responses of CCR5 inhibitor-resistant viruses to other anti-retroviral drugs that act by different mechanisms, and their sensitivities to neutralizing antibodies (NAbs). The rationale for the latter study is that the resistance pathway for CCR5 inhibitors involves changes in the HIV-1 envelope glycoproteins (Env), which are also targets for NAbs. The escape mutants CC101.19 and D1/85.16 were selected for resistance to AD101 and vicriviroc (VVC), respectively, from the primary R5 HIV-1 isolate CC1/85. Each escape mutant was cross-resistant to other small molecule CCR5 inhibitors (aplaviroc, maraviroc, VVC, AD101 and CMPD 167), but sensitive to protein ligands of CCR5: the modified chemokine PSC-RANTES and the humanized MAb PRO-140. The resistant viruses also retained wild-type sensitivity to the nucleoside reverse transcriptase inhibitor (RTI) zidovudine, the non-nucleoside RTI nevirapine, the protease inhibitor atazanavir and other attachment and fusion inhibitors that act independently of CCR5 (BMS-806, PRO-542 and enfuvirtide). Of note is that the escape mutants were more sensitive than the parental CC1/85 isolate to a subset of neutralizing monoclonal antibodies and to some sera from HIV-1-infected people, implying that sequence changes in Env that confer resistance to CCR5 inhibitors can increase the accessibility of some NAb epitopes. The need to preserve NAb resistance may therefore be a constraint upon how escape from CCR5 inhibitors occurs in vivo.

Tuberculosis (TB) remains one of the most worrying infectious diseases affecting public health around the world; 8.7 million new TB cases were reported in 2011. The search for an Mycobacterium tuberculosis H37Rv protein sequence which is functionally important in host-pathogen interaction has been proposed for developing a new vaccine which will allow efficient and safe control of the spread of this disease. The present study thus reports the results obtained for the Rv1268c protein described in the M. tuberculosis H37Rv genome as a hypothetical unknown, probably secreted, protein based on a highly robust, specific, sensitive and functional approach to the search for potential epitopes to be included in an anti-tuberculosis vaccine. Rv1268c presence was determined by immunoblotting after obtaining polyclonal sera against mycobacterial total sonicate or subcellular fractions. Such sera were used in electron immunomicroscopy (EIM) for confirming protein localisation on the M. tuberculosis envelop by recognising colloidal gold-labelled immunoglobulin. Screening assays revealed the presence of two sequences having high binding activity: one binding A549 alveolar epithelial cells ( 141TGMAALEQYLGSGHAVIVSI160) and other binding U937 monocyte-derived macrophages (21AVALGLASPADAAAGTMYGD40). Such sequences' ability to inhibit mycobacterial entry during in vitro assays was analysed. The structure of synthetic peptides binding to target cells was also determined, bearing in mind the structure-function relationship. These results, together with those obtained for other proteins, have been involved in selecting peptides which might be included in a subunit-based anti-tuberculosis vaccine. © 2013 Elsevier Ltd. All rights reserved.

Polymer therapeutics is a class of (bio)hybrid materials that include a plethora of biologically active (synthetic) macromolecules that either consist of polymeric drugs, drug-polymer conjugates, peptide/protein-polymer conjugates or of drug, peptide or proteins covalently linked to self-assembled entities, such as polymer micelles and liposomes. Interest in polymer therapeutics has increased in the last decades particularly as anti-cancer therapeutics, influenza therapeutics, non-viral alternatives to gene delivery and in the advent of biological terrorism as anthrax inoculates. The human immunodeficiency virus (HIV) is one of the most deadly diseases known and despite improvement in education, preventative methods and drug therapies, the virus still affects approximately 33 million people worldwide. Classical therapeutics against HIV consist of low molecular weight drugs that inhibit various viral enzymes in the cell, including reverse transcriptase, integrase and protease. More recently, drugs that target the entry and fusion of the virus have formed a new class of inhibitors, some of which are effective (candidate) drugs. One of the fusion inhibitors that were approved is the drug T-20. T-20 is a 36-amino acid peptide derived from the heptad repeat 2 (HR2) of the HIV-1 glycoprotein gp41and acts as a competitive inhibitor by preventing the fusion of the viral and host cell membrane. Despite the effectiveness of T-20, one major drawback is associated with this drug namely low plasma lifetime. One of the goals of this Thesis is to design and synthesize effective HR2 derived fusion inhibitors through conjugation with synthetic polymers, while extending the lifetime. In addition, a novel concept for macromolecular HIV entry inhibition known as polyvalent inhibition is assessed in this Thesis. This Thesis consists of five chapters in which polymer therapeutics as carriers and polyvalent entities are explored against HIV-1. With the state of the art of HIV-1 therapeutics including polymer therapeutics summarized in Chapter 1, the remainder of the Thesis expands and defines the terrain with regard to novel (bio)hybrid materials as therapeutics against HIV-1. Very few polymer therapeutics that target HIV before cellular entry have been described. Even fewer polymer-based therapeutics inhibit HIV-1 entry via the specific interaction to the HIV-1 envelope proteins. In Chapter 2, novel polyvalent copolymers that specifically target the HIV-1 envelope protein gp120 were generated using a living polymerization technique known as ‘reversible addition-fragmentation chain transfer’ (RAFT), followed by a post-polymerization modification strategy. The strategy involves two synthetic steps, namely an ester/amide exchange followed by a thiol-ene addition of peptide ligands to generate a library of polyvalent copolymers, with varying peptide ligand, 2-hydroxypropyl methacrylamide and allyl methacrylamide content. These novel polyvalent polymers were shown to be effective HIV-1 entry inhibitors. Fusion inhibitors derived from the HR2 domain of gp41 such as T-20, act as competitive inhibitors by binding to the HR1 domain on the envelope glycoprotein gp41, which is involved in HIV-1 – host cell membrane fusion. However, the peptidic nature of these fusion inhibitors restricts their lifetime because they are prone to proteolytic degradation. Chapter 3 explores the effect of attaching synthetic polymer poly(ethylene glycol) (PEG) site-specifically to a HR2 derived peptide fusion inhibitor. The PEGylated peptides were assessed for their biophysical properties, efficacy as membrane fusion inhibitors and stability towards trypsin that was used as a model to show the effectiveness of the PEG to protect the peptide from proteolytic degradation. Although PEGylation causes a reduction in efficacy, this Chapter demonstrates that judicial PEGylation of the peptide can minimize the detrimental effects on inhibitory efficacy, while increasing lifetime against a protease. Conjugation of PEG and the site of PEG conjugation both have a profound effect on the efficacy of HR2 derived membrane fusion inhibitors. However, the effects on the mechanism of HIV-1 infectivity inhibition by polymer conjugated HR2 peptide inhibitors are not known. Chapter 4 investigates the effect of architecturally different synthetic linear PEG, v-shaped PEG or branched poly(glycerol) polymers conjugated to HR2 derived peptides. The peptide-polymer conjugates were then assessed for their inhibition efficacy, affinity and kinetics of binding towards a model of gp41, known as 5-Helix. The results indicate that attachment of a synthetic linear or branched polymer, despite causing a reduction in HIV-1 infectivity inhibition, does not significantly affect affinity towards 5-Helix. However, the rates of association and dissociation towards and from 5-Helix were reduced. Furthermore, synthetic polymer conjugation to HR2 derived peptides prolong their lifetime bound to gp41 and thus potentially reduces HIV-1 inhibition escape. Therefore, this Chapter demonstrates a previously unknown advantage of polymer conjugation to HIV-1 fusion peptide inhibitors. Chapter 5 systematically explores the modification of hydrophilic residues on the hydrophobic face of a HR2 derived peptide. The peptide mutants were assessed for their efficacy against membrane fusion inhibition as well as against fusion of HIV-1 wild type and T-20 resistant variants. Although the peptide mutants did not exhibit any improvement towards the inhibition of wild type membrane fusion, selected peptide mutants with point mutations were shown to be effective inhibitors towards T-20 resistant HIV-1 strains. In conclusion, peptide-polymer hybrid materials can make excellent candidates as long lasting therapeutics against wild type HIV-1 but also against emergent HIV-1 mutant variants. The knowledge from these Chapters will aid the design of innovative, next generation of peptide-polymer hybrid therapeutics.

Human immunodeficiency virus type 1 (HIV-1) infection can be inhibited by small molecules that target the CCR5 coreceptor. Here, we describe some properties of clonal viruses resistant to one such inhibitor, SCH-D, using both chimeric, infectious molecular clones and Env-pseudotypes. Studies using combinations of CCR5 ligands, including small molecule inhibitors, monoclonal antibodies (MAbs) and chemokine derivatives such as PSC-RANTES show that the fully SCH-D-resistant viruses enter target cells by using the SCH-D-bound form of CCR5. However, the way resistance to SCH-D and other small molecule CCR5 inhibitors is manifested depends on the target cell and the nature of the assay (single- vs multi-cycle). In multi-cycle assays using primary lymphocytes, SCH-D does not inhibit resistant molecular clones, and it can even enhance their infectivity modestly. In contrast, the same viruses (as Env-pseudotypes) are significantly inhibited by SCH-D in single-cycle entry assays using U87-CD4/CCR5 cells, resistance being manifested by incomplete inhibition at high SCH-D concentrations. When a single-cycle, Env-pseudotype entry assay was performed using either U87-CD4/CCR5 cells or PBMC under comparable conditions, entry was inhibited by up to 88% in the former cells but by only 28% in the PBMC. Hence, there are both cell- and assay-dependent influences on how resistance is manifested. We also take this opportunity to correct our previous report that SCH-D-resistant isolates are also substantially cross-resistant to PSC-RANTES (Marozsan, A. J., Kuhmann, S. E., Morgan, T., Herrera, C., Rivera-Troche, E., Xu, S., Baroudy, B. M., Strizki, J., and Moore, J. P. (2005). Generation and properties of a human immunodeficiency virus type 1 isolate resistant to the small molecule CCR5 inhibitor, vicriviroc (SCH-D; SCH-417690). Virology 338, 182-199). A substantial element of this resistance was attributable to the unappreciated carry-over of SCH-D from the selection cultures into analytical assays.

Background Hepatitis C virus (HCV) is a major cause of liver disease and a potential cause of substantial morbidity and mortality worldwide. The overall prevalence of HCV infection is 2%, representing 120 million people worldwide. Current standard treatment using pegylated interferon and ribavirin is effective in only 50% of the patients infected with HCV genotype 1, and is associated with significant side effects. Therefore, it is still of importance to develop new drugs for treatment of HCV. Antiviral substances obtained from natural products, including medicinal plants, are potentially good targets to study. In this study, we evaluated Indonesian medicinal plants for their anti-HCV activities. Methods Ethanol extracts of 21 samples derived from 17 species of medicinal plants explored in the East Java region were tested. Anti-HCV activities were determined by a cell culture method using Huh7.5 cells and HCV strains of 9 different genotypes (1a to 7a, 1b and 2b). Results Four of the 21 samples tested showed antiviral activities against HCV: Toona sureni leaves (TSL) with 50% inhibitory concentrations (IC50) of 13.9 and 2.0 μg/ml against the HCV J6/JFH1-P47 and -P1 strains, respectively, Melicope latifolia leaves (MLL) with IC50 of 3.5 and 2.1 μg/ml, respectively, Melanolepis multiglandulosa stem (MMS) with IC50 of 17.1 and 6.2 μg/ml, respectively, and Ficus fistulosa leaves (FFL) with IC50 of 15.0 and 5.7 μg/ml, respectively. Time-of-addition experiments revealed that TSL and MLL inhibited both at the entry and post-entry steps while MMS and FFL principally at the entry step. TSL and MLL inhibited all of 11 HCV strains of all the genotypes tested to the same extent. On the other hand, FFL showed significantly weaker inhibitory activities against the HCV genotype 1a strain, and MMS against the HCV strains of genotypes 2b and 7a to a lesser extent, compared to the other HCV genotypes. Conclusions Ethanol extracts of TSL, MLL, MMS and FFL showed antiviral activities against all the HCV genotypes tested with the exception that some genotype(s) showed significant resistance to FFL and to MMS to a lesser extent. These plant extracts may be good candidates for the development of anti-HCV drugs.