Structural Investigations on Novel Non-Nucleoside Inhibitors of Human Norovirus Polymerase.

Human norovirus is the first cause of foodborne disease worldwide, leading to extensive outbreaks of acute gastroenteritis, and causing around 200,000 children to die annually in developing countries. No specific vaccines or antiviral agents are currently available, with therapeutic options limited to supportive care to prevent dehydration. The infection can become severe and lead to life-threatening complications in young children, the elderly and immunocompromised individuals, leading to a clear need for antiviral agents, to be used as treatments and as prophylactic measures in case of outbreaks. Due to the key role played by the viral RNA-dependent RNA polymerase (RdRp) in the virus life cycle, this enzyme is a promising target for antiviral drug discovery. In previous studies, following in silico investigations, we identified different small-molecule inhibitors of this enzyme. In this study, we rationally modified five identified scaffolds, to further explore structure-activity relationships, and to enhance binding to the RdRp. The newly designed compounds were synthesized according to multiple-step synthetic routes and evaluated for their inhibition of the enzyme in vitro. New inhibitors with low micromolar inhibitory activity of the RdRp were identified, which provide a promising basis for further hit-to-lead optimization.

Targeting the Complement Serine Protease MASP-2 as a Therapeutic Strategy for Coronavirus Infections.

MASP-2, mannose-binding protein-associated serine protease 2, is a key enzyme in the lectin pathway of complement activation. Hyperactivation of this protein by human coronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2 has been found to contribute to aberrant complement activation in patients, leading to aggravated lung injury with potentially fatal consequences. This hyperactivation is triggered in the lungs through a conserved, direct interaction between MASP-2 and coronavirus nucleocapsid (N) proteins. Blocking this interaction with monoclonal antibodies and interfering directly with the catalytic activity of MASP-2, have been found to alleviate coronavirus-induced lung injury both in vitro and in vivo. In this study, a virtual library of 8736 licensed drugs and clinical agents has been screened in silico according to two parallel strategies. The first strategy aims at identifying direct inhibitors of MASP-2 catalytic activity, while the second strategy focusses on finding protein-protein interaction inhibitors (PPIs) of MASP-2 and coronaviral N proteins. Such agents could represent promising support treatment options to prevent lung injury and reduce mortality rates of infections caused by both present and future-emerging coronaviruses. Forty-six drug repurposing candidates were purchased and, for the ones selected as potential direct inhibitors of MASP-2, a preliminary in vitro assay was conducted to assess their interference with the lectin pathway of complement activation. Some of the tested agents displayed a dose-response inhibitory activity of the lectin pathway, potentially providing the basis for a viable support strategy to prevent the severe complications of coronavirus infections.

Synthesis and characterization of 1,2,4-triazolo[1,5-a]pyrimidine-2-carboxamide-based compounds targeting the PA-PB1 interface of influenza A virus polymerase.

Influenza viruses (Flu) are responsible for seasonal epidemics causing high rates of morbidity, which can dramatically increase during severe pandemic outbreaks. Antiviral drugs are an indispensable weapon to treat infected people and reduce the impact on human health, nevertheless anti-Flu armamentarium still remains inadequate. In search for new anti-Flu drugs, our group has focused on viral RNA-dependent RNA polymerase (RdRP) developing disruptors of PA-PB1 subunits interface with the best compounds characterized by cycloheptathiophene-3-carboxamide and 1,2,4-triazolo[1,5-a]pyrimidine-2-carboxamide scaffolds. By merging these moieties, two very interesting hybrid compounds were recently identified, starting from which, in this paper, a series of analogues were designed and synthesized. In particular, a thorough exploration of the cycloheptathiophene-3-carboxamide moiety led to acquire important SAR insight and identify new active compounds showing both the ability to inhibit PA-PB1 interaction and viral replication in the micromolar range and at non-toxic concentrations. For few compounds, the ability to efficiently inhibit PA-PB1 subunits interaction did not translate into anti-Flu activity. Chemical/physical properties were investigated for a couple of compounds suggesting that the low solubility of compound 14, due to a strong crystal lattice, may have impaired its antiviral activity. Finally, computational studies performed on compound 23, in which the phenyl ring suitably replaced the cycloheptathiophene, suggested that, in addition to hydrophobic interactions, H-bonds enhanced its binding within the PAC cavity.

Potent and broad-spectrum cycloheptathiophene-3-carboxamide compounds that target the PA-PB1 interaction of influenza virus RNA polymerase and possess a high barrier to drug resistance.

Influenza viruses are major respiratory pathogens responsible for both seasonal epidemics and occasional pandemics worldwide. The current available treatment options have limited efficacy and thus the development of new antivirals is highly needed. We previously reported the identification of a series of cycloheptathiophene-3-carboxamide compounds as influenza A virus inhibitors that act by targeting the protein-protein interactions between the PA-PB1 subunits of the viral polymerase. In this study, we characterized the antiviral properties of the most promising compounds as well as investigated their propensity to induce drug resistance. Our results show that some of the selected compounds possess potent, broad-spectrum anti-influenza activity as they efficiently inhibited the replication of several strains of influenza A and B viruses, including an oseltamivir-resistant clinical isolate, with nanomolar or low-micromolar potency. The most promising compounds specifically inhibited the PA-PB1 binding in vitro and interfered with the influenza A virus polymerase activity in a cellular context, without showing cytotoxicity. The most active PA-PB1 inhibitors showed to possess a drug resistance barrier higher than that of oseltamivir. Indeed, no viral variants with reduced susceptibility to the selected compounds emerged after serial passages of influenza A virus under drug selective pressure. Overall, our studies identified potent PA-PB1 inhibitors as promising candidates for the development of new anti-influenza drugs.

Synthesis and biological evaluation of a library of hybrid derivatives as inhibitors of influenza virus PA-PB1 interaction.

The limited treatment options against influenza virus along with the growing public health concerns regarding the continuous emergence of drug-resistant viruses make essential the development of new anti-flu agents with novel mechanisms of action. One of the most attractive targets is the interaction between two subunits of the RNA-dependent RNA polymerase, PA and PB1. Herein we report the rational design of hybrid compounds starting from a 3-cyano-4,6-diphenylpyridine scaffold recently identified as disruptor of PA-PB1 interactions. Guided by the previously reported SAR data, a library of amino acid derivatives was synthesized. The biological evaluation led to the identification of new PA-PB1 inhibitors, that do not show appreciable toxicity. Molecular modeling shed further lights on the inhibition mechanism of these compounds.

A sensitive and validated HPLC-UV method for the quantitative determination of the new antifungal drug isavuconazole in human plasma.

Isavuconazole is a broad-spectrum triazole antifungal drug recently approved for the therapy of both invasive aspergillosis and mucormycosis. To support a widespread therapeutic drug monitoring of isavuconazole, a simple, sensitive, and precise high-performance liquid chromatography method with UV detection was developed and fully validated for the quantification of this drug in human plasma. The method involved a combined protein precipitation-solid-phase extraction and a chromatographic separation on a Waters XTerra RP18 (150 × 4.6 mm, 3.5 µm) column using an isocratic mobile phase of ammonium acetate buffer (pH 8.0, 10 mm) and acetonitrile (45:55, v/v). The UV detection was performed at 285 nm. This method was linear (correlation coefficients ≥0.998), specific (no interference with plasma components or various potentially co-administrated drugs), sensitive (lower limit of quantification of 0.025 µg/mL), reproducible (coefficients of variation were ≤7.9%) and accurate (deviations ranged from -5.0 to 8.0%) over the range of 0.025-10 µg/mL. The method fulfilled all of the US Food and Drug Administration guidelines validation criteria and performed well in an international proficiency testing program. The assay was also successfully applied to routine therapeutic drug monitoring of patients and to drug stability investigations under various conditions.

A quantitative LumiFluo assay to test inhibitory compounds blocking p53 degradation induced by human papillomavirus oncoprotein E6 in living cells.

High-risk human papillomaviruses (HR-HPVs) are the causative agents for the onset of several epithelial cancers in humans. The deregulated expression of the viral oncoproteins E6 and E7 is the driving force sustaining the progression of malignant transformation in pre-neoplastic lesions. Targeting the viral E6 oncoprotein through inhibitory compounds can counteract the survival of cancer cells due to the reactivation of p53-mediated pathways and represents an intriguing strategy to treat HPV-associated neoplasias. Here, we describe the development of a quantitative and easy-to-perform assay to monitor the E6-mediated degradation of p53 in living cells to be used for small-molecule testing. This assay allows to unbiasedly determine whether a compound can protect p53 from the E6-mediated degradation in cells, through a simple 3-step protocol. We validated the assay by testing two small molecules, SAHA and RITA, reported to impair the E6-mediated p53 degradation. Interestingly, we observed that only SAHA efficiently rescued p53, while RITA could not provide the same degree of protection. The possibility to specifically and quantitatively monitor the ability of a selected compound to rescue p53 in a cellular context through our LumiFluo assay could represent an important step towards the successful development of anti-HPV drugs.

Efficient and regioselective one-step synthesis of 7-aryl-5-methyl- and 5-aryl-7-methyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidine derivatives.

Two facile and efficient one-step procedures for the regioselective synthesis of 7-aryl-5-methyl- and 5-aryl-7-methyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidines have been developed, via reactions of 3,5-diamino-1,2,4-triazole with variously substituted 1-aryl-1,3-butanediones and 1-aryl-2-buten-1-ones, respectively. The excellent yield and/or regioselectivity shown by the reactions decreased when ethyl 5-amino-1,2,4-triazole-3-carboxylate was used. [1,2,4]Triazolo[1,5-a]pyrimidine being a privileged scaffold, the procedures herein reported may be useful for the preparation of biologically active compounds. In this study, the preparation of a set of compounds based on the [1,2,4]triazolo[1,5-a]pyrimidine scaffold led to the identification of compound 20 endowed with a very promising ability to inhibit influenza virus RNA polymerase PA-PB1 subunit heterodimerization.

Exploring the cycloheptathiophene-3-carboxamide scaffold to disrupt the interactions of the influenza polymerase subunits and obtain potent anti-influenza activity.

With the aim to identify small molecules able to disrupt PA-PB1 subunits interaction of influenza virus (flu) RNA-dependent RNA polymerase, and based on previous structural and computational information, in this paper we have designed and synthesized a new series of cycloheptathiophene-3-carboxamide (cHTC) derivatives. Their biological evaluation led to highlight important structural insights along with new interesting compounds, such as the 2-hydroxybenzamido derivatives 29, 31, and 32, and the 4-aminophenyl derivative 54, which inhibited viral growth in the low micromolar range (EC50 = 0.18-1.2 µM) at no toxic concentrations (CC50 > 250 µM). This study permitted to obtain among the most potent anti-flu compounds within the PA-PB1 interaction inhibitors, confirming the cHTC scaffold as particularly suitable to achieve innovative anti-flu agents.

Development and validation of a simple and robust HPLC method with UV detection for quantification of the hepatitis C virus inhibitor daclatasvir in human plasma.

Daclatasvir is an inhibitor of hepatitis C virus NS5A protein that is used for the therapy of chronic hepatitis. So far, published methods for analysis of daclatasvir in plasma are exclusively based on mass spectrometry, which is not always available in standard clinical laboratories. Thus, we wished to develop and validate a simple, but still reliable and sensitive high-performance liquid chromatography (HPLC) assay with UV detection for the quantification of daclatasvir, feasible for a wide-spread clinical routine use. The method consisted of solid-phase extraction of daclatasvir using Waters Oasis HLB 1cc cartridges, reversed-phase liquid chromatography with a Waters XTerra RP18 (150mm×4.6mm, 3.5µm) column and a mobile phase of ammonium acetate buffer (pH 5.0, 10mM) and acetonitrile (56:44, v/v), and UV detection at 318nm. This assay proved to be sensitive (lower limit of quantification of 0.05µg/mL), linear (correlation coefficients ≥0.997), specific (no interference with various potentially co-administrated drugs), reproducible (both intra-day and inter-day coefficients of variation ≤8.9%), and accurate (deviations ranged from -2.2 to 8.0% and from -6.5 to 9.2% for intra-day and inter-day assays, respectively). The method was applied to therapeutic monitoring of patients undergoing daclatasvir therapy for hepatitis C and showed to be reliable and robust. Thus, this method provides a simple, sensitive, precise, and reproducible assay for dosing daclatasvir that can be readily adaptable to routine use by clinical laboratories with standard equipment. In addition, the stability of daclatasvir in plasma was evaluated under various conditions, including after the heating procedure required for inactivation of infectious viruses and in different light exposure conditions. These studies evidenced photo-instability of the compound under sunlight exposure over time. Thus, blood sampling and the whole handling procedure have to be performed quickly and with minimal light exposure.

Daclatasvir plasma level and resistance selection in HIV patients with hepatitis C virus cirrhosis treated with daclatasvir, sofosbuvir, and ribavirin.

OBJECTIVES: Effective treatment with direct-acting antiviral drugs against hepatitis C virus (HCV) is a medical need in cirrhotic HIV-HCV co-infected patients. METHODS: This study investigated the plasma levels of daclatasvir (DCV) and ribavirin (RBV) in HIV-HCV co-infected subjects treated with DCV, sofosbuvir, and RBV. Drug concentrations were quantified using validated high-performance liquid chromatography methods with ultraviolet detection. The HCV non-structural protein 5A and non-structural protein 5B coding regions were analyzed by population-based sequencing. RESULTS: DCV was dosed at week 4 and at week 8 of treatment, and RBV at week 8. One patient had the lowest DCV level, corresponding to 32.7% of the overall median value of the other patients at week 4 and about 40% at week 8. The Y93H variant was detected in this subject at weeks 8, 16, and 20 of treatment, but not before treatment or at day 2, and the patient experienced virological failure. Another subject with the Y93H variant at baseline and appropriate DCV levels had HCV RNA <12 IU/ml at week 12 and undetectable at week 16. CONCLUSIONS: Sub-optimal DCV drug levels allow the selection of resistance-associated variants and fail to contribute to antiviral activity. No definite reason for the low DCV level was found. Quantifying the drug is suggested in difficult-to-treat patients.

4,6-Diphenylpyridines as Promising Novel Anti-Influenza Agents Targeting the PA-PB1 Protein-Protein Interaction: Structure-Activity Relationships Exploration with the Aid of Molecular Modeling.

Influenza is an infectious disease that represents an important public health burden, with high impact on the global morbidity, mortality, and economy. The poor protection and the need of annual updating of the anti-influenza vaccine, added to the rapid emergence of viral strains resistant to current therapy make the need for antiviral drugs with novel mechanisms of action compelling. In this regard, the viral RNA polymerase is an attractive target that allows the design of selective compounds with reduced risk of resistance. In previous studies we showed that the inhibition of the polymerase acidic protein-basic protein 1 (PA-PB1) interaction is a promising strategy for the development of anti-influenza agents. Starting from the previously identified 3-cyano-4,6-diphenyl-pyridines, we chemically modified this scaffold and explored its structure-activity relationships. Noncytotoxic compounds with both the ability of disrupting the PA-PB1 interaction and antiviral activity were identified, and their mechanism of target binding was clarified with molecular modeling simulations.

Drug Repurposing Approach Identifies Inhibitors of the Prototypic Viral Transcription Factor IE2 that Block Human Cytomegalovirus Replication.

New targets for antiviral strategies are needed against human cytomegalovirus (HCMV), a major human pathogen. A cell-based screen aimed at finding inhibitors of the viral transcription factor Immediate-Early 2 (IE2) was performed in HCMV-infected cells expressing EGFP under the control of an IE2-inducible viral promoter. Screening of a library of bioactive small molecules led to the identification of several compounds able to inhibit EGFP expression and also HCMV replication with potency in the low-micromolar range. Follow-up studies with four selected hits indicated that they all block viral DNA synthesis as well as viral Early and Late gene expression. Furthermore, mechanistic studies confirmed that the compounds specifically act via inhibition of IE2 transactivating activity, thus blocking viral Early gene expression and the progression of virus replication. These results provide proof of concept for identifying small molecules that modulate the activity of a microbial transcription factor to control pathogen replication.

Development of a simple HPLC-UV method for the determination of the hepatitis C virus inhibitor simeprevir in human plasma.

A simple high-performance liquid chromatography method for the determination of the hepatitis C virus protease inhibitor simeprevir in human plasma was developed and validated. The method involved a rapid and simple solid-phase extraction of simeprevir using Oasis HLB 1cc cartridges, isocratic reversed-phase liquid chromatography on an XTerra RP18 (150 mm×4.6 mm, 3.5 µm) column, and ultraviolet detection at 225 nm. The mobile phase consisted of phosphate buffer (pH 6, 52.5 mM) and acetonitrile (30:70, v/v). This assay proved to be sensitive (lower limit of quantification of 0.05 µg/mL), linear (correlation coefficients ≥0.99), specific (no interference with various potentially co-administrated drugs), reproducible (both intra-day and inter-day coefficients of variation ≤8.3%), and accurate (deviations ranged from -8.0 to 1.2% and from -3.3 to 6.0% for intra-day and inter-day analysis, respectively). The method was applied to therapeutic monitoring of patients undergoing simeprevir treatment for hepatitis C and proved to be robust and reliable. Thus, this method provides a simple, sensitive, precise and reproducible assay for dosing simeprevir that can be readily adaptable to routine use by clinical laboratories with standard equipment.

A Broad Anti-influenza Hybrid Small Molecule That Potently Disrupts the Interaction of Polymerase Acidic Protein-Basic Protein 1 (PA-PB1) Subunits.

In continuing our efforts to identify small molecules able to disrupt the interaction of the polymerase acidic protein-basic protein 1 (PA-PB1) subunits of influenza virus (Flu) RNA-dependent RNA polymerase, this paper is devoted to the optimization of a dihydrotriazolopyrimidine derivative, previously identified through structure-based drug discovery. The structure modifications performed around the bicyclic core led to the identification of compounds endowed with both the ability to disrupt PA-PB1 subunits interaction and anti-Flu activity with no cytotoxicity. Very interesting results were obtained with the hybrid molecules 36 and 37, designed by merging some peculiar structural features known to impart PA-PB1 interaction inhibition, with compound 36 that emerged as the most potent PA-PB1 interaction inhibitor (IC50 = 1.1 µM) among all the small molecules reported so far. Calculations showed a very favored H-bonding between the 2-amidic carbonyl of 36 and Q408, which seems to justify its potent ability to interfere with the interaction of the polymerase subunits.

Optimization of small-molecule inhibitors of influenza virus polymerase: from thiophene-3-carboxamide to polyamido scaffolds.

Influenza virus infections represent a serious concern to public health, being characterized by high morbidity and significant mortality. To date, compounds targeting the viral ion-channel M2 or the viral neuraminidase are the drugs available for treatment of influenza, but the emergence of drug-resistant viral mutants renders the search for novel targets and their possible inhibitors a major priority. Recently, we demonstrated that the viral RNA-dependent RNA polymerase (RdRP) complex can be an optimal target of protein-protein disruption by small molecules, with thiophene-3-carboxamide derivatives emerging as promising candidates for the development of new anti-influenza drugs with broad-spectrum activity. Here, we report a further dissection of the thiophene-3-carboxamide structure. By using a GRID molecular interaction field (MIF)-based scaffold-hopping approach, more potent and nontoxic polyamido derivatives were identified, highlighting a new space in the chemical variability of RdRP inhibitors. Finally, a possible pharmacophoric model highlighting the key features required for RdRP inhibition is proposed.

Antiviral strategies against influenza virus: towards new therapeutic approaches.

Influenza viruses are major human pathogens responsible for respiratory diseases affecting millions of people worldwide and characterized by high morbidity and significant mortality. Influenza infections can be controlled by vaccination and antiviral drugs. However, vaccines need annual updating and give limited protection. Only two classes of drugs are currently approved for the treatment of influenza: M2 ion channel blockers and neuraminidase inhibitors. However, they are often associated with limited efficacy and adverse side effects. In addition, the currently available drugs suffer from rapid and extensive emergence of drug resistance. All this highlights the urgent need for developing new antiviral strategies with novel mechanisms of action and with reduced drug resistance potential. Several new classes of antiviral agents targeting viral replication mechanisms or cellular proteins/processes are under development. This review gives an overview of novel strategies targeting the virus and/or the host cell for counteracting influenza virus infection.

Structural investigation of cycloheptathiophene-3-carboxamide derivatives targeting influenza virus polymerase assembly.

The limited number of drug classes licensed for treatment of influenza virus (Flu), together with the continuous emergence of viral variants and drug resistant mutants, highlights the urgent need to find antivirals with novel mechanisms of action. In this context, the viral RNA-dependent RNA polymerase (RdRP) subunits assembly has emerged as an attractive target. Starting from a cycloheptathiophene-3-carboxamide derivative recently identified by us for its ability to disrupt the interaction between the PA and PB1 subunits of RdRP, we have designed and synthesized a series of analogues. Their biological evaluation led to the identification of more potent protein-protein interaction inhibitors, endowed with antiviral activity that also encompassed a number of clinical isolates of FluA, including an oseltamivir-resistant strain, and FluB, without showing appreciable toxicity. From this study, the cycloheptathiophene-3-carboxamide scaffold emerged as being particularly suitable to impart anti-Flu activity.

Near full-length sequence analysis of HIV type 1 BF recombinants from Italy.

Recombination between HIV-1 subtypes B and F has generated several circulating and unique recombinant forms, particularly in Latin American areas. In Italy, subtype B is highly prevalent while subtype F is the most common pure non-B subtype. To investigate the recombination pattern in Italian BF recombinant viruses, we characterized full-length sequences derived from 15 adult patients, mostly Italian and infected by the heterosexual route. One of the BF mosaics was a CRF29, three sequences clustered with low bootstrap values with CRF39, CRF40, and CRF42. With the exception of the CRF29-like sequence, the other recombination patterns were unique, but two possible clusters were identified. Analysis of the gp120 V3 domain suggested a possible link with subtype F from Eastern Europe rather than from Latin America, favoring the hypothesis of local recombination between clade B and F viruses over that of import of BF recombinants from Latin America. HIV-1 subtypes B and F appear prone to generation of unique recombinants in Italy, warranting epidemiological surveillance and investigation of a possible clinical significance.