Curcumin-Poly(sodium 4-styrenesulfonate) Conjugates as Potent Zika Virus Entry Inhibitors.

Curcumin, a natural product with recognized antiviral properties, is limited in its application largely due to its poor solubility. This study presents the synthesis of water-soluble curcumin-poly(sodium 4-styrenesulfonate) (Cur-PSSNan) covalent conjugates. The antiflaviviral activity of conjugates was validated in vitro by using the Zika virus as a model. In the development of these water-soluble curcumin-containing derivatives, we used the macromolecules reported by us to also hamper viral infections. Mechanistic investigations indicated that the conjugates exhibited excellent stability and bioavailability. The curcumin and macromolecules in concerted action interact directly with virus particles and block their attachment to host cells, hampering the infection process.

Reversible rearrangement of the cellular cytoskeleton: A key to the broad-spectrum antiviral activity of novel amphiphilic polymers.

The battle against emerging viral infections has been uneven, as there is currently no broad-spectrum drug available to contain the spread of novel pathogens throughout the population. Consequently, the pandemic outbreak that occurred in early 2020 laid bare the almost empty state of the pandemic box. Therefore, the development of novel treatments with broad specificity has become a paramount concern in this post-pandemic era. Here, we propose copolymers of poly (sodium 2-(acrylamido)-2-methyl-1-propanesulfonate) (PAMPS) and poly (sodium 11-(acrylamido)undecanoate (AaU), both block (PAMPS75-b-PAaUn) and random (P(AMPSm-co-AaUn)) that show efficacy against a broad range of alpha and betacoronaviruses. Owing to their intricate architecture, these polymers exhibit a highly distinctive mode of action, modulating nano-mechanical properties of cells and thereby influencing viral replication. Through the employment of confocal and atomic force microscopy techniques, we discerned perturbations in actin and vimentin filaments, which correlated with modification of cellular elasticity and reduction of glycocalyx layer. Intriguingly, this process was reversible upon polymer removal from the cells. To ascertain the applicability of our findings, we assessed the efficacy and underlying mechanism of the inhibitors using fully differentiated human airway epithelial cultures, wherein near-complete abrogation of viral replication was documented. Given their mode of action, these polymers can be classified as biologically active nanomaterials that exploit a highly conserved molecular target-cellular plasticity-proffering the potential for truly broad-spectrum activity while concurrently for drug resistance development is minimal.

Poly(ethylene glycol)-block-poly(sodium 4-styrenesulfonate) Copolymers as Efficient Zika Virus Inhibitors: In Vitro Studies.

A series of poly(ethylene glycol)-block-poly(sodium 4-styrenesulfonate) (PEG-b-PSSNa) copolymers were synthesized, and their antiviral activity against Zika virus (ZIKV) was determined. The polymers inhibit ZIKV replication in vitro in mammalian cells at nontoxic concentrations. The mechanistic analysis revealed that the PEG-b-PSSNa copolymers interact directly with viral particles in a zipper-like mechanism, hindering their interaction with the permissive cell. The antiviral activity of the copolymers is well-correlated with the length of the PSSNa block, indicating that the copolymers' ionic blocks are biologically active. The blocks of PEG present in copolymers studied do not hinder that interaction. Considering the practical application of PEG-b-PSSNa and the electrostatic nature of the inhibition, the interaction between the copolymers and human serum albumin (HSA) was evaluated. The formation of PEG-b-PSSNa-HSA complexes in the form of negatively charged nanoparticles well-dispersed in buffer solution was observed. That observation is promising, given the possible practical application of the copolymers.

Self-Organized Nanoparticles of Random and Block Copolymers of Sodium 2-(Acrylamido)-2-methyl-1-propanesulfonate and Sodium 11-(Acrylamido)undecanoate as Safe and Effective Zika Virus Inhibitors.

A series of anionic homopolymers, poly(sodium 2-(acrylamido)-2-methyl-1-propanesulfonate) (PAMPS) and amphiphilic copolymers of AMPS and sodium 11-(acrylamido)undecanoate (AaU), both block (PAMPS75-b-PAaUn), and random (P(AMPSm-co-AaUn)), were synthesized and their antiviral activity against Zika virus (ZIKV) was evaluated. Interestingly, while the homopolymers showed limited antiviral activity, the copolymers are very efficient antivirals. This observation was explained considering that under the conditions relevant to the biological experiments (pH 7.4 PBS buffer) the macromolecules of these copolymers exist as negatively charged (zeta potential about -25 mV) nanoparticles (4-12 nm) due to their self-organization. They inhibit the ZIKV replication cycle by binding to the cell surface and thereby blocking virus attachment to host cells. Considering good solubility in aqueous media, low toxicity, and high selectivity index (SI) of the PAMPS-b-PAaU copolymers, they can be considered promising agents against ZIKV infections.

Acriflavine, a clinically approved drug, inhibits SARS-CoV-2 and other betacoronaviruses.

The COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort and available vaccines, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identify acriflavine (ACF) as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.

l-Arginine Improves Solubility and ANTI SARS-CoV-2 Mpro Activity of Rutin but Not the Antiviral Activity in Cells.

The COVID-19 pandemic outbreak prompts an urgent need for efficient therapeutics, and repurposing of known drugs has been extensively used in an attempt to get to anti-SARS-CoV-2 agents in the shortest possible time. The glycoside rutin shows manifold pharmacological activities and, despite its use being limited by its poor solubility in water, it is the active principle of many pharmaceutical preparations. We herein report our in silico and experimental investigations of rutin as a SARS-CoV-2 Mpro inhibitor and of its water solubility improvement obtained by mixing it with l-arginine. Tests of the rutin/l-arginine mixture in a cellular model of SARS-CoV-2 infection highlighted that the mixture still suffers from unfavorable pharmacokinetic properties, but nonetheless, the results of this study suggest that rutin might be a good starting point for hit optimization.

SARS-CoV-2 inhibition using a mucoadhesive, amphiphilic chitosan that may serve as an anti-viral nasal spray.

There are currently no cures for coronavirus infections, making the prevention of infections the only course open at the present time. The COVID-19 pandemic has been difficult to prevent, as the infection is spread by respiratory droplets and thus effective, scalable and safe preventive interventions are urgently needed. We hypothesise that preventing viral entry into mammalian nasal epithelial cells may be one way to limit the spread of COVID-19. Here we show that N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ), a positively charged polymer that has been through an extensive Good Laboratory Practice toxicology screen, is able to reduce the infectivity of SARS-COV-2 in A549ACE2+ and Vero E6 cells with a log removal value of - 3 to - 4 at a concentration of 10-100 µg/ mL (p < 0.05 compared to untreated controls) and to limit infectivity in human airway epithelial cells at a concentration of 500 µg/ mL (p < 0.05 compared to untreated controls). In vivo studies using transgenic mice expressing the ACE-2 receptor, dosed nasally with SARS-COV-2 (426,000 TCID50/mL) showed a trend for nasal GCPQ (20 mg/kg) to inhibit viral load in the respiratory tract and brain, although the study was not powered to detect statistical significance. GCPQ's electrostatic binding to the virus, preventing viral entry into the host cells, is the most likely mechanism of viral inhibition. Radiolabelled GCPQ studies in mice show that at a dose of 10 mg/kg, GCPQ has a long residence time in mouse nares, with 13.1% of the injected dose identified from SPECT/CT in the nares, 24 h after nasal dosing. With a no observed adverse effect level of 18 mg/kg in rats, following a 28-day repeat dose study, clinical testing of this polymer, as a COVID-19 prophylactic is warranted.

Ozone Treatment Is Insufficient to Inactivate SARS-CoV-2 Surrogate under Field Conditions.

COVID-19 caused by SARS-CoV-2 caused a worldwide crisis, highlighting the importance of preventive measures in infectious diseases control. SARS-CoV-2 can remain infectious on surfaces for up to several weeks; therefore, proper disinfection is required to mitigate the risk of indirect virus spreading. Gaseous ozone treatment has received particular attention as an easily accessible disinfection tool. In this study, we evaluated the virucidal effectiveness of gaseous ozone treatment (>7.3 ppm, 2 h) on murine hepatitis virus (MHV)-contaminated stainless-steel surface and PBS-suspended virus under field conditions at ambient (21.8%) and high (49.8-54.2%) relative humidity. Surficial virus was soiled with 0.3 g/L of BSA. Parallelly, a half-hour vaporization with 7.3% hydrogen peroxide was performed on contaminated carriers. The obtained results showed that gaseous ozone, whilst quite effective against suspended virus, was insufficient in sanitizing coronavirus contaminated surfaces, especially under low RH. Increased humidity created more favorable conditions for MHV inactivation, resulting in 2.1 log titre reduction. Vaporization with 7.3% hydrogen peroxide presented much better virucidal performance than ozonation in a similar experimental setup, indicating that its application may be more advantageous regarding gaseous disinfection of surfaces contaminated with other coronaviruses, including SARS-CoV-2.

Seleno-Functionalization of Quercetin Improves the Non-Covalent Inhibition of Mpro and Its Antiviral Activity in Cells against SARS-CoV-2.

The development of new antiviral drugs against SARS-CoV-2 is a valuable long-term strategy to protect the global population from the COVID-19 pandemic complementary to the vaccination. Considering this, the viral main protease (Mpro) is among the most promising molecular targets in light of its importance during the viral replication cycle. The natural flavonoid quercetin 1 has been recently reported to be a potent Mpro inhibitor in vitro, and we explored the effect produced by the introduction of organoselenium functionalities in this scaffold. In particular, we report here a new synthetic method to prepare previously inaccessible C-8 seleno-quercetin derivatives. By screening a small library of flavonols and flavone derivatives, we observed that some compounds inhibit the protease activity in vitro. For the first time, we demonstrate that quercetin (1) and 8-(p-tolylselenyl)quercetin (2d) block SARS-CoV-2 replication in infected cells at non-toxic concentrations, with an IC50 of 192 µM and 8 µM, respectively. Based on docking experiments driven by experimental evidence, we propose a non-covalent mechanism for Mpro inhibition in which a hydrogen bond between the selenium atom and Gln189 residue in the catalytic pocket could explain the higher Mpro activity of 2d and, as a result, its better antiviral profile.

HTCC as a Polymeric Inhibitor of SARS-CoV-2 and MERS-CoV.

Among seven coronaviruses that infect humans, three (severe acute respiratory syndrome coronavirus [SARS-CoV], Middle East respiratory syndrome coronavirus [MERS-CoV], and the newly identified severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) are associated with a severe, life-threatening respiratory infection and multiorgan failure. We previously proposed that the cationically modified chitosan N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC) is a potent inhibitor of human coronavirus NL63 (HCoV-NL63). Next, we demonstrated the broad-spectrum antiviral activity of the compound, as it inhibited all low-pathogenicity human coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1). Here, using in vitro and ex vivo models of human airway epithelia, we show that HTCC effectively blocks MERS-CoV and SARS-CoV-2 infection. We also confirmed the mechanism of action for these two viruses, showing that the polymer blocks the virus entry into the host cell by interaction with the S protein.IMPORTANCE The beginning of 2020 brought us information about the novel coronavirus emerging in China. Rapid research resulted in the characterization of the pathogen, which appeared to be a member of the SARS-like cluster, commonly seen in bats. Despite the global and local efforts, the virus escaped the health care measures and rapidly spread in China and later globally, officially causing a pandemic and global crisis in March 2020. At present, different scenarios are being written to contain the virus, but the development of novel anticoronavirals for all highly pathogenic coronaviruses remains the major challenge. Here, we describe the antiviral activity of an HTCC compound, previously developed by us, which may be used as a potential inhibitor of currently circulating highly pathogenic coronaviruses-SARS-CoV-2 and MERS-CoV.

In Vitro Inhibition of Zika Virus Replication with Poly(Sodium 4-Styrenesulfonate).

Zika virus (ZIKV) is an emerging mosquito-borne pathogen associated with microcephaly and other congenital abnormalities in newborns as well as neurologic complications in adults. The explosive transmission of the virus in the last ten years put it in the limelight and improved our understanding of its biology and pathology. Currently, no vaccine or drugs are available to prevent or treat ZIKV infections. Knowing the potential of flaviviruses to broaden their geographic distribution, as observed for the West Nile virus, it is of importance to develop novel antiviral strategies. In this work, we identified poly(sodium 4-styrenesulfonate) (PSSNa) as a new polymeric ZIKV inhibitor. We demonstrated that PSSNa inhibits ZIKV replication in vitro both in animal and human cells, while no cytotoxicity is observed. Our mechanistic studies indicated that PSSNa acts mostly through direct binding to ZIKV particle and blocking its attachment to the host cells.

Berberine Hampers Influenza A Replication through Inhibition of MAPK/ERK Pathway.

BACKGROUND: Berberine (BBR) is an isoquinoline alkaloid which exhibits a variety of biological and therapeutic properties, and has been reported by some to block replication of the influenza virus. However, contradictory results have also been presented, and the mechanistic explanation is lacking. METHODS: A panel of cell lines (Madin-Darby canine kidney (MDCK), adenocarcinoma human alveolar basal epithelial cells (A549), lung epithelial type I (LET1)) and primary human airway epithelial cells (HAE) susceptible to influenza virus infection were infected with a seasonal influenza A virus in the presence or absence of BBR. Cytotoxicity towards cell lines was measured using XTT assay. The yield of the virus was analyzed using RT-qPCR. To study the molecular mechanism of BBR, confocal microscopy and Western blot analyses of cellular fractions were applied. RESULTS AND CONCLUSIONS: Our results show cell-type-dependent anti-influenza properties of BBR in vitro which suggests that the compound acts on the cell and not the virus. Importantly, BBR hampers influenza replication in primary human airway epithelium 3D cultures that mimic the natural replication site of the virus. Studies show that the influenza A virus upregulates the mitogen-activated protein kinase/extracellular signal-related kinase (MAPK/ERK) pathway and hijacks this pathway for nucleolar export of the viral ribonucleoprotein. Our results suggest that BBR interferes with this process and hampers influenza A replication.

Synthetic sulfonated derivatives of poly(allylamine hydrochloride) as inhibitors of human metapneumovirus.

Human metapneumovirus (hMPV) is a widely distributed pathogen responsible for acute upper and lower respiratory infections of varying severity. Previously, we reported that N-sulfonated derivatives of poly(allylamine hydrochloride) (NSPAHs) efficiently inhibit replication of the influenza virus in vitro and ex vivo. Here, we show a dose dependent inhibition of hMPV infection by NSPAHs in LLC-MK2 cells. The results showed strong antiviral properties of NSPAHs. While the activity of NSPAHs is comparable to those of carrageenans, they show better physicochemical properties and may be delivered at high concentrations. The functional assays showed that tested polymers block hMPV release from infected cells and, consequently, constrain virus spread. Moreover, further studies on viruses utilizing different egress mechanisms suggest that observed antiviral effect depend on selective inhibition of viruses budding from the cell surface.