Ebola virus disease: In vivo protection provided by the PAMP restricted TLR3 agonist rintatolimod and its mechanism of action

Ebola virus (EBOV) is a highly infectious and lethal pathogen responsible for sporadic self-limiting clusters of Ebola virus disease (EVD) in Central Africa capable of reaching epidemic status. 100% protection from lethal EBOV-Zaire in Balb/c mice was achieved by rintatolimod (Ampligen) at the well tolerated human clinical dose of 6 mg/kg. The data indicate that the mechanism of action is rintatolimod's dual ability to act as both a competitive decoy for the IID domain of VP35 blocking viral dsRNA sequestration and as a pathogen-associated molecular pattern (PAMP) restricted agonist for direct TLR3 activation but lacking RIG-1-like cytosolic helicase agonist properties. These data show promise for rintatolimod as a prophylactic therapy against human Ebola outbreaks

Broad-spectrum coronavirus 3C-like protease peptidomimetic inhibitors effectively block SARS-CoV-2 replication in cells: Design, synthesis, biological evaluation, and X-ray structure determination

Despite the approval of vaccines, monoclonal antibodies and restrictions during the pandemic, the demand for new efficacious and safe antivirals is compelling to boost the therapeutic arsenal against the COVID-19. The viral 3-chymotrypsin-like protease (3CLpro) is an essential enzyme for replication with high homology in the active site across CoVs and variants showing an almost unique specificity for Leu-Gln as P2–P1 residues, allowing the development of broad-spectrum inhibitors. The design, synthesis, biological activity, and cocrystal structural information of newly conceived peptidomimetic covalent reversible inhibitors are herein described. The inhibitors display an aldehyde warhead, a Gln mimetic at P1 and modified P2–P3 residues. Particularly, functionalized proline residues were inserted at P2 to stabilize the β-turn like bioactive conformation, modulating the affinity. The most potent compounds displayed low/sub-nM potency against the 3CLpro of SARS-CoV-2 and MERS-CoV and inhibited viral replication of three human CoVs, i.e. SARS-CoV-2, MERS-CoV, and HCoV 229 in different cell lines. Particularly, derivative 12 exhibited nM-low μM antiviral activity depending on the virus, and the highest selectivity index. Some compounds were co-crystallized with SARS-CoV-2 3CLpro validating our design. Altogether, these results foster future work toward broad-spectrum 3CLpro inhibitors to challenge CoVs related pandemics

New Thiazolidine-4-One Derivatives as SARS-CoV-2 Main Protease Inhibitors

It has been more than four years since the first report of SARS-CoV-2, and humankind has experienced a pandemic with an unprecedented impact. Moreover, the new variants have made the situation even worse. Among viral enzymes, the SARS-CoV-2 main protease (Mpro) has been deemed a promising drug target vs. COVID-19. Indeed, Mpro is a pivotal enzyme for viral replication, and it is highly conserved within coronaviruses. It showed a high extent of conservation of the protease residues essential to the enzymatic activity, emphasizing its potential as a drug target to develop wide-spectrum antiviral agents effective not only vs. SARS-CoV-2 variants but also against other coronaviruses. Even though the FDA-approved drug nirmatrelvir, a Mpro inhibitor, has boosted the antiviral therapy for the treatment of COVID-19, the drug shows several drawbacks that hinder its clinical application. Herein, we report the synthesis of new thiazolidine-4-one derivatives endowed with inhibitory potencies in the micromolar range against SARS-CoV-2 Mpro. In silico studies shed light on the key structural requirements responsible for binding to highly conserved enzymatic residues, showing that the thiazolidinone core acts as a mimetic of the Gln amino acid of the natural substrate and the central role of the nitro-substituted aromatic portion in establishing π-π stacking interactions with the catalytic His-41 residue

Exploring antiviral strategies: From innate immunity modulation to viral proteins targeting

The search for new targets and antiviral agents is continuously growing, especially for agents with broad-spectrum activity. In fact, even though vaccines are the elective way to limit diseases and possibly eradicate pathogens, they are not always effective, particularly in people with compromised immune defenses, and they can also lose activity against rapidly evolving pathogens. Hence the identification of novel antiviral agents is a priority for the health systems, and the number of approved antiviral drugs is increasing yearly: until today over one hundred antivirals have been approved. The most common strategies to develop antiviral drugs are based on the identification of molecules targeting viral proteins and blocking viral replication as direct acting agents. Although successful, this strategy must consider that most viruses are easily capable to select drug resistant strains. A novel approach for the identification of potential broad-spectrum antivirals is to target cellular proteins inducing innate immune response, so avoiding the high mutagenesis rate occurring for viral proteins. An important help in fighting viral spread can arrive from the Traditional Chinese Medicine (TCM), widely used in China to treat infectious diseases both alone and in cooperation with western medicines. This thesis will investigate three possible paths for the identification of potential antivirals i) the identification of cellular targets to develop broad-spectrum antivirals, ii) target viral proteins in order to impair viral replication selectively, iii) study the effect of immunomodulators with unknown targets in order to identify potential broad-spectrum antiviral. Among the cellular proteins possibly used as drug target, a recently discovered one is STING: a downstream actor in the detection of non-self cytosolic nucleic acids related to viral infections and tumor conditions. The Stimulator of Interferon Genes (STING) plays a pivotal role in counteracting viral infections, independently from whether the viral genome is DNA or RNA, mounting a strong innate immune response driven principally by type I Interferon (IFN-I). On the one side, when cytosolic DNA is detected cGAS produces cyclic GMP-AMP (2’3’cGAMP) that directly binds STING determining TBK1 phosphorylation (pTBK1) and hence transcription of IFN-I. On the other side, when the RIG-I pathway is activated in response to viral RNA detection, STING interacts with activated Mitochondrial Antiviral Signaling protein (MAVS) determining pTBK1 mediated IFN-I production. The SARS-CoV-2 pandemic have highlighted the need of specific as well as potent antivirals; among SARS-CoV-2 proteins the Papain Like protease (PL-pro) is an important protein involved in viral life cycle and in the innate immune evasion, hence it will be investigated as potential viral target for the development of selective antiviral drugs. Finally, we will investigate the mechanism of action of a series of compounds derived from one molecule isolated from herbal extracts used as immune stimulator in the TCM, in order to verify their potency in inducing the innate immune response

Unlocking STING as a Therapeutic Antiviral Strategy

Invading pathogens have developed weapons that subvert physiological conditions to weaken the host and permit the spread of infection. Cells, on their side, have thus developed countermeasures to maintain cellular physiology and counteract pathogenesis. The cyclic GMP-AMP (cGAMP) synthase (cGAS) is a pattern recognition receptor that recognizes viral DNA present in the cytosol, activating the stimulator of interferon genes (STING) protein and leading to the production of type I interferons (IFN-I). Given its role in innate immunity activation, STING is considered an interesting and innovative target for the development of broad-spectrum antivirals. In this review, we discuss the function of STING; its modulation by the cellular stimuli; the molecular mechanisms developed by viruses, through which they escape this defense system; and the therapeutical strategies that have been developed to date to inhibit viral replication restoring STING functionality