Novel broad-spectrum virucidal antivirals

The current COVID-19 pandemic has showed the threats that viruses can cause to the whole world. Over 200 million infections and over 4.5 million deaths and enormous socio-economic impact were the output of the emergence of a single new pathogen: SARS-CoV-2. The development of vac-cines is the paramount solution to overcome the pandemic. However, even with extraordinary and unprecedented worldwide efforts, it took almost one year to have a first validated vaccine and it will take longer to have a global vaccination. In addition, vaccines are preventive drugs, that need to be administered before the infection to stimulate the immune-system of the patient. In the meanwhile, in case of an infection, we were harmless in the fight against the virus.Antivirals are curative drugs, that inhibit one step of the viral replication. Many approaches have been investigated: antibodies, peptides, peptomimics, small molecules. Most of them are specif-ic to a single family of virus, making the development slow and expensive. Entry inhibitors are a class of antivirals that binds to the virus, preventing the attachment to the cell membrane. Such antivirals usually have a broad-spectrum of action, since they mimic cell receptors, such as heparan sulfate pro-teoglycans (HSPGs), exploited by different viruses. Many compounds have been developed throughout the years; however, the reversible nature of the virus inhibition mechanism has prevented their trans-lation to the clinic. Our group has recently shown that the simple addition of a long hydrophobic linker makes the inhibition irreversible, proving this hypothesis with two compounds: gold-nanoparticles and beta-cyclodextrins. The goal of this thesis is to develop novel antivirals able to inhibit a broad-spectrum of viruses with an irreversible mechanism of action and to investigate the key features responsible of such properties. In this thesis, we further expand the broad-spectrum activity of gold-nanoparticles and beta-cyclodextrins showing that they are capable of inhibiting viruses beyond HSPG-dependent ones. In addition, we show their ability of reversibly inactivating SARS-CoV-2. Moreover, we have investigated the role of the hydrophobic linker's length in the virucidal inhibition in case of cyclodextrins, proving the need of such moiety to have an irreversible viral inactivation. Envisioning a translation of such drug, we have performed a preliminar pharmacokinetic study performed via intranasal administration.A novel class of compounds has been also proposed: sulfated/sulfonated dendritic polyglyc-erol. A broad investigation of the molecular design rules needed to achieve nanomolar irreversible viral inhibition is discussed. We also show that the most active compound proves to be active against HSV-2 and RSV, thus with a broad-spectrum activity. In addition, we demonstrate that the virucidal activity is leading to the release of viral DNA upon interaction between the virus and our dendritic polyglycerol. A different modification of dendritic polyglycerol, bearing carboxylic acid, showed activi-ty against SARS-CoV-2; such inhibition results to be irreversible. This compound has been further test-ed in a hamster model, where initial studies confirm its feasibility as antiviral. In addition to these studies on development of virucidal antivirals, different technological so-lutions to prevent viral spreading are proposed. In particular, we focused on an antiviral filter and antiviral surfaces.

Interferon Lambda Delays the Emergence of Influenza Virus Resistance to Oseltamivir

Paulo Henrique Jacob Silva, Chiara Medaglia, Francesco Stellacci

Influenza viruses are a leading cause of morbidity and mortality worldwide. These air-borne pathogens are able to cross the species barrier, leading to regular seasonal epidemics and sporadic pandemics. Influenza viruses also possess a high genetic variability, which allows for the acquisition of resistance mutations to antivirals. Combination therapies with two or more drugs targeting different mechanisms of viral replication have been considered an advantageous option to not only enhance the effectiveness of the individual treatments, but also reduce the likelihood of resistance emergence. Using an in vitro infection model, we assessed the barrier to viral resistance of a combination therapy with the neuraminidase inhibitor oseltamivir and human interferon lambda against the pandemic H1N1 A/Netherlands/602/2009 (H1N1pdm09) virus. We serially passaged the virus in a cell line derived from human bronchial epithelial cells in the presence or absence of increasing concentrations of oseltamivir alone or oseltamivir plus interferon lambda. While the treatment with oseltamivir alone quickly induced the emergence of antiviral resistance through a single mutation in the neuraminidase gene, the co-administration of interferon lambda delayed the emergence of drug-resistant influenza virus variants. Our results suggest a possible clinical application of interferon lambda in combination with oseltamivir to treat influenza.

2021

Polyanionic Amphiphilic Dendritic Polyglycerols as Broad-Spectrum Viral Inhibitors with a Virucidal Mechanism

Valeria Cagno, Matteo Gasbarri, Francesco Stellacci

Heparin has been known to be a broad-spectrum inhibitor of viral infection for almost 70 years, and it has been used as a medication for almost 90 years due to its anticoagulant effect. This nontoxic biocompatible polymer efficiently binds to many types of viruses and prevents their attachment to cell membranes. However, the anticoagulant properties are limiting their use as an antiviral drug. Many heparin-like compounds have been developed throughout the years; however, the reversible nature of the virus inhibition mechanism has prevented their translation to the clinics. In vivo, such a mechanism requires the unrealistic maintenance of the concentration above the binding constant. Recently, we have shown that the addition of long hydrophobic linkers to heparin-like compounds renders the interaction irreversible while maintaining the low-toxicity and broad-spectrum activity. To date, such hydrophobic linkers have been used to create heparin-like gold nanopartides and beta-cydodextrins. The former achieves a nanomolar inhibition concentration on a non-biodegradable scaffold. The latter, on a fully biodegradable scaffold, shows only a micromolar inhibition concentration. Here, we report that the addition of hydrophobic linkers to a new type of multifunctional scaffold (dendritic polyglycerol, dPG) creates biocompatible compounds endowed with nanomolar activity. Furthermore, we present an in-depth analysis of the molecular design rules needed to achieve irreversible virus inhibition. The most active compound (dPG-5) showed nanomolar activity against herpes simplex virus 2 (HSV-2) and respiratory syncytial virus (RSV), giving a proof-of-principle for broad-spectrum while keeping low-toxicity. In addition, we demonstrate that the virucidal activity leads to the release of viral DNA upon the interaction between the virus and our polyanionic dendritic polymers. We believe that this paper will be a stepping stone toward the design of a new dass of irreversible nontoxic broad-spectrum antivirals.

AMER CHEMICAL SOC2022

Adeno-Associated Virus Activates an Innate Immune Response in Normal Human Cells but Not in Osteosarcoma Cells

Peter Martin Beard

Adeno-associated virus (AAV) is a small, DNA-containing dependovirus with promising potential as a gene delivery vehicle. Given the variety of applications of AAV-based vectors in the treatment of genetic disorders, numerous studies have focused on the immunogenicity of recombinant AAV. In general, AAV vectors appear not to induce strong inflammatory responses. We have found that AAV2, when it infects the osteosarcoma cells U2OS, can initiate part of its replicative cycle in the absence of helper virus. This does not occur in untransformed cells. We set out to test whether the cellular innate antiviral defenses control this susceptibility and found that, in nonimmune normal human fibroblasts, AAV2 induces type I interferon production and release and the accumulation of nuclear promyelocytic leukemia bodies. AAV fails to mobilize this defense pathway in the U2OS cells. This permissiveness is in large part due to impairment of the viral sensing machinery in these cells. Our investigations point to Toll-like receptor 9 as a potential intracellular sensor that detects AAV2 and triggers the antiviral state in AAV-infected untransformed cells. Efficient sensing of the AAV genome and the ensuing activation of an innate antiviral response are thus crucial cellular events dictating the parvovirus infectivity in host cells.

2011

Polyanionic Amphiphilic Dendritic Polyglycerols as Broad-Spectrum Viral Inhibitors with a Virucidal Mechanism

Valeria Cagno, Matteo Gasbarri, Francesco Stellacci

AMER CHEMICAL SOC2022