Lead Optimization of Influenza Virus RNA Polymerase Inhibitors Targeting PA-PB1 Interaction.

Influenza viruses are responsible for contagious respiratory illnesses in humans and cause seasonal epidemics and occasional pandemics worldwide. Previously, we identified a quinolinone derivative PA-49, which inhibited the influenza virus RNA-dependent RNA polymerase (RdRp) by targeting PA-PB1 interaction. This paper reports the structure optimization of PA-49, which resulted in the identification of 3-((dibenzylamino)methyl)quinolinone derivatives with more potent anti-influenza virus activity. During the optimization, the hit compound 89, which was more active than PA-49, was identified. Further optimization and scaffold hopping of 89 led to the most potent compounds 100 and a 1,8-naphthyridinone derivative 118, respectively. We conclusively determined that compounds 100 and 118 suppressed the replication of influenza virus and exhibited anti-influenza virus activity against both influenza virus types A and B in the range of 50% effective concentration (EC50) = 0.061-0.226 µM with low toxicity (50% cytotoxic concentration (CC50) >10 µM).

Novel and potent antimicrobial effects of caspofungin on drug-resistant Candida and bacteria.

Echinocandins, including caspofungin, micafungin, and anidulafungin, are first-line antifungal agents for the treatment of invasive candidiasis. They exhibit fungicidal activity by inhibiting the synthesis of ß-1,3-D-glucan, an essential component of the fungal cell wall. However, they are active only against proliferating fungal cells and unable to completely eradicate fungal cells even after a 24 h drug exposure in standard time-kill assays. Surprisingly, we found that caspofungin, when dissolved in low ionic solutions, had rapid and potent antimicrobial activities against multidrug-resistant (MDR) Candida and bacteria cells even in non-growth conditions. This effect was not observed in 0.9% NaCl or other ion-containing solutions and was not exerted by other echinocandins. Furthermore, caspofungin dissolved in low ionic solutions drastically reduced mature biofilm cells of MDR Candida auris in only 5 min, as well as Candida-bacterial polymicrobial biofilms in a catheter-lock therapy model. Caspofungin displayed ion concentration-dependent conformational changes and intracellular accumulation with increased reactive oxygen species production, indicating a novel mechanism of action in low ionic conditions. Importantly, caspofungin dissolved in 5% glucose water did not exhibit increased toxicity to human cells. This study facilitates the development of new therapeutic strategies in the management of catheter-related biofilm infections.

Design and Development of an HBT-Based Ratiometric Fluorescent Probe to Monitor Stress-Induced Premature Senescence.

Stress-induced premature senescence (SIPS) can be induced in tumor cells by reactive oxygen species (ROS) or oncogenes. The antineoplastic drugs cause apoptosis and senescence by damaging the DNA. Although the detection of cellular senescence is important to monitor drug response during anticancer therapy, only a few probes have been studied for imaging SIPS. In this study, we developed 2-(2'-hydroxyphenyl)benzothiazole (HBT)-based fluorescent probes to determine SIPS by monitoring the oxidative stress and ß-galactosidase activity. HBT is a commonly used fluorophore because of its luminescence mechanism via excited-state intramolecular proton transfer, and it has attractive properties, such as a four-level photochemical process and large Stokes shift (151 nm). A novel fluorescent probe, (2-(benzo[d]thiazol-2-yl)phenyl)boronic acid, was prepared for the detection of ROS, including H2O2, via the oxidation reaction of arylboronic acids to form the fluorescent phenol, HBT. In addition, to determine the enzymatic activity of ß-galactosidase, a 2-(4'-chloro-2'-hydroxyphenyl)benzothiazole (CBT)-based enzymatic turn-on probe (CBT-ß-Gal) was designed and synthesized. ß-Galactosidase catalyzed the hydrolysis of ß-galactopyranoside from CBT-ß-Gal to release the fluorescent CBT. These probes were capable of ratiometric imaging the accumulation of H2O2 and the degree of ß-galatosidase activity in contrast to H2O2-untreated and H2O2-treated HeLa cells. Furthermore, these probes were successfully employed for imaging the increased levels of ROS and ß-galactosidase activity in the doxorubicin-treated HeLa cells.

A Quinolinone Compound Inhibiting the Oligomerization of Nucleoprotein of Influenza A Virus Prevents the Selection of Escape Mutants.

The emergence of resistance to currently available anti-influenza drugs has heightened the need for antivirals with novel mechanisms of action. The influenza A virus (IAV) nucleoprotein (NP) is highly conserved and essential for the formation of viral ribonucleoprotein (vRNP), which serves as the template for replication and transcription. Recently, using in silico screening, we identified an antiviral compound designated NUD-1 (a 4-hydroxyquinolinone derivative) as a potential inhibitor of NP. In this study, we further analyzed the interaction between NUD-1 and NP and found that the compound interferes with the oligomerization of NP, which is required for vRNP formation, leading to the suppression of viral transcription, protein synthesis, and nuclear export of NP. We further assessed the selection of resistant variants by serially passaging a clinical isolate of the 2009 H1N1 pandemic influenza virus in the presence of NUD-1 or oseltamivir. NUD-1 did not select for resistant variants after nine passages, whereas oseltamivir selected for resistant variants after five passages. Our data demonstrate that NUD-1 interferes with the oligomerization of NP and less likely induces drug-resistant variants than oseltamivir; hence, it is a potential lead compound for the development of novel anti-influenza drugs.

Synthesis of Trifluoromethyl-α,ß-unsaturated Lactones and Pyrazolinones and Discovery of Influenza Virus Polymerase Inhibitors.

To explore the potential biological activities of trifluoromethyl heterocycles, we recently developed a synthetic approach to access a series of α-trifluoromethyl-α,ß-unsaturated lactones and trifluoromethyl pyrazolinones. The compounds were tested for their antimicrobial activity, and we found that some compounds had anti-influenza viral activity. The ß-aryl-α-trifluoromethyl α,ß-unsaturated lactone derivatives 5 g (5-(4-chlorophenyl)-5-methyl-4-phenyl-3-(trifluoromethyl)furan-2-one), 7 b (4-(4-methoxyphenyl)-3-(trifluoromethyl)spiro[furan-5,1'-indane]-2-one), and the trifluoromethyl pyrazolinone 12 c (1-(6-methoxy-2-naphthyl)-2-(trifluoromethyl)-5,6,7,8-tetrahydropyrazolo[1,2-a]pyridazin-3-one) were found to possess promising inhibitory activity against influenza virus type A, strain A/WSN/33 (H1N1). These three hit compounds were successfully optimized, and we identified that the most potent compound 5 h (5-(4-chlorophenyl)-4-(6-methoxy-2-naphthyl)-5-methyl-3-(trifluoromethyl)furan-2-one) showed inhibitory activity against various types of influenza A and B viruses in the low-micromolar range without showing cytotoxicity. Moreover, 5 h was more effective against the clinical isolate A/California/7/2009 (H1N1pdm) strain than the influenza viral polymerase inhibitor, favipiravir (T-705). We also delineated the structure-activity relationship and obtained mechanistic insight into inhibition of the viral polymerase.

Antiviral Activity of Peanut (Arachis hypogaea L.) Skin Extract Against Human Influenza Viruses.

The high propensity of influenza viruses to develop resistance to antiviral drugs necessitates the continuing search for new therapeutics. Peanut skins, which are low-value byproducts of the peanut industry, are known to contain high levels of polyphenols. In this study, we investigated the antiviral activity of ethanol extracts of peanut skins against various influenza viruses using cell-based assays. Extracts with a higher polyphenol content exhibited higher antiviral activities, suggesting that the active components are the polyphenols. An extract prepared from roasted peanut skins effectively inhibited the replication of influenza virus A/WSN/33 with a half maximal inhibitory concentration of 1.3 µg/mL. Plaque assay results suggested that the extract inhibits the early replication stages of the influenza virus. It demonstrated activity against both influenza type A and type B viruses. Notably, the extract exhibited a potent activity against a clinical isolate of the 2009 H1N1 pandemic, which had reduced sensitivity to oseltamivir. Moreover, a combination of peanut skin extract with the anti-influenza drugs, oseltamivir and amantadine, synergistically increased their antiviral activity. These data demonstrate the potential application of peanut skin extract in the development of new therapeutic options for influenza management.

Identification of small molecule inhibitors for influenza a virus using in silico and in vitro approaches.

Influenza viruses have acquired resistance to approved neuraminidase-targeting drugs, increasing the need for new drug targets for the development of novel anti-influenza drugs. Nucleoprotein (NP) is an attractive target since it has an indispensable role in virus replication and its amino acid sequence is well conserved. In this study, we aimed to identify new inhibitors of the NP using a structure-based drug discovery algorithm, named Nagasaki University Docking Engine (NUDE), which has been established especially for the Destination for GPU Intensive Machine (DEGIMA) supercomputer. The hit compounds that showed high binding scores during in silico screening were subsequently evaluated for anti-influenza virus effects using a cell-based assay. A 4-hydroxyquinolinone compound, designated as NUD-1, was found to inhibit the replication of influenza virus in cultured cells. Analysis of binding between NUD-1 and NP using surface plasmon resonance assay and fragment molecular orbital calculations confirmed that NUD-1 binds to NP and could interfere with NP-NP interactions essential for virus replication. Time-of-addition experiments showed that the compound inhibited the mid-stage of infection, corresponding to assembly of the NP and other viral proteins. Moreover, NUD-1 was also effective against various types of influenza A viruses including a clinical isolate of A(H1N1)pdm09 influenza with a 50% inhibitory concentration range of 1.8-2.1 µM. Our data demonstrate that the combined use of NUDE system followed by the cell-based assay is useful to obtain lead compounds for the development of novel anti-influenza drugs.