Discovery of 2-phenylquinazolines as potent Mycobacterium avium efflux pump inhibitors able to synergize with clarithromycin against clinical isolate.

Nontuberculous mycobacteria (NTM), which include the Mycobacterium avium complex, are classified as difficult-to-treat pathogens due to their ability to quickly develop drug resistance against the most common antibiotics used to treat NTM infections. The overexpression of efflux pumps (EPs) was demonstrated to be a key mechanism of clarithromycin (CLA) resistance in NTM. Therefore, in this work, 24 compounds from an in-house library, characterized by chemical diversity, were tested as potential NTM EP inhibitors (EPIs) against Mycobacterium smegmatis mc2 155 and M. avium clinical isolates. Based on the acquired results, 12 novel analogs of the best derivatives 1b and 7b were designed and synthesized to improve the NTM EP inhibition activity. Among the second set of compounds, 13b emerged as the most potent NTM EPI. At a concentration of 4 µg/mL, it reduced the CLA minimum inhibitory concentration by 16-fold against the clinical isolate M. avium 2373 overexpressing EPs as primary mechanism of CLA resistance.

Fighting Antimicrobial Resistance: Insights on How the Staphylococcus aureus NorA Efflux Pump Recognizes 2-Phenylquinoline Inhibitors by Supervised Molecular Dynamics (SuMD) and Molecular Docking Simulations.

The superbug Staphylococcus aureus (S. aureus) exhibits several resistance mechanisms, including efflux pumps, that strongly contribute to antimicrobial resistance. In particular, the NorA efflux pump activity is associated with S. aureus resistance to fluoroquinolone antibiotics (e.g., ciprofloxacin) by promoting their active extrusion from cells. Thus, since efflux pump inhibitors (EPIs) are able to increase antibiotic concentrations in bacteria as well as restore their susceptibility to these agents, they represent a promising strategy to counteract bacterial resistance. Additionally, the very recent release of two NorA efflux pump cryo-electron microscopy (cryo-EM) structures in complex with synthetic antigen-binding fragments (Fabs) represents a real breakthrough in the study of S. aureus antibiotic resistance. In this scenario, supervised molecular dynamics (SuMD) and molecular docking experiments were combined to investigate for the first time the molecular mechanisms driving the interaction between NorA and efflux pump inhibitors (EPIs), with the ultimate goal of elucidating how the NorA efflux pump recognizes its inhibitors. The findings provide insights into the dynamic NorA-EPI intermolecular interactions and lay the groundwork for future drug discovery efforts aimed at the identification of novel molecules to fight antimicrobial resistance.

Identification of Anti-Influenza A Compounds Inhibiting the Viral Non-Structural Protein 1 (NS1) Using a Type I Interferon-Driven Screening Strategy.

There is an urgent need to identify efficient antiviral compounds to combat existing and emerging RNA virus infections, particularly those related to seasonal and pandemic influenza outbreaks. While inhibitors of the influenza viral integral membrane proton channel protein (M2), neuraminidase (NA), and cap-dependent endonuclease are available, circulating influenza viruses acquire resistance over time. Thus, the need for the development of additional anti-influenza drugs with novel mechanisms of action exists. In the present study, a cell-based screening assay and a small molecule library were used to screen for activities that antagonized influenza A non-structural protein 1 (NS1), a highly conserved, multifunctional accessory protein that inhibits the type I interferon response against influenza. Two potential anti-influenza agents, compounds 157 and 164, were identified with anti-NS1 activity, resulting in the reduction of A/PR/8/34(H1N1) influenza A virus replication and the restoration of IFN-ß expression in human lung epithelial A549 cells. A 3D pharmacophore modeling study of the active compounds provided a glimpse of the structural motifs that may contribute to anti-influenza virus activity. This screening approach is amenable to a broader analysis of small molecule compounds to inhibit other viral targets.

Drug efflux transporters in Staphylococcus pseudintermedius: in silico prediction and characterization of resistance.

OBJECTIVES: To perform an in silico prediction of drug efflux pumps (EPs) in Staphylococcus pseudintermedius and investigate their role in conferring resistance to antibiotic and biocidal agents and biofilm formation. METHODS: A S. pseudintermedius efflux mutant was obtained by stimulating an isogenic line (ATCC 49444) with increasing concentrations of an efflux system substrate. Changes in antimicrobial susceptibility and biofilm-forming capability were evaluated in the presence/absence of the EP inhibitors (EPIs) thioridazine and reserpine and the efflux activity was assayed by fluorometry. Homologues of EPs of Staphylococcus aureus and Staphylococcus epidermidis were searched by exploratory GenBank investigations. Gene expression analyses and sequencing were then conducted on selected genes. RESULTS: Susceptibility to chlorhexidine, gentamicin and ciprofloxacin, but not enrofloxacin, was affected by the increased efflux and it was variably restored by the EPIs. The efflux mutant showed much greater biofilm formation that the original strain, which was significantly inhibited by thioridazine and reserpine at MIC/2. A high expression of norA, which was mgrA-independent, was found in the S. pseudintermedius efflux mutant, apparently regulated by an 11 bp deletion in its promoter region, whilst lmrB was transitorily overexpressed. icaA, which encodes the polysaccharide intercellular adhesin forming the extracellular matrix of staphylococcal biofilm, was also up-regulated. CONCLUSIONS: EPs, particularly NorA, are supposed to have complex involvement in multiple stages of resistance development. Overexpression of EPs appears to be correlated with a remarkable increase of S. pseudintermedius biofilm production; however, the regulatory mechanisms remain to be explored.

Q-raKtion: A Semiautomated KNIME Workflow for Bioactivity Data Points Curation.

The recent increase of bioactivity data freely available to the scientific community and stored as activity data points in chemogenomic repositories provides a huge amount of ready-to-use information to support the development of predictive models. However, the benefits provided by the availability of such a vast amount of accessible information are strongly counteracted by the lack of uniformity and consistency of data from multiple sources, requiring a process of integration and harmonization. While different automated pipelines for processing and assessing chemical data have emerged in the last years, the curation of bioactivity data points is a less investigated topic, with useful concepts provided but no tangible tools available. In this context, the present work represents a first step toward the filling of this gap, by providing a tool to meet the needs of end-user in building proprietary high-quality data sets for further studies. Specifically, we herein describe Q-raKtion, a systematic, semiautomated, flexible, and, above all, customizable KNIME workflow that effectively aggregates information on biological activities of compounds retrieved by two of the most comprehensive and widely used repositories, PubChem and ChEMBL.

Unconventional Extraction of Total Non-Polar Carotenoids from Pumpkin Pulp and Their Nanoencapsulation.

Pumpkin is considered a functional food with beneficial effects on human health due to the presence of interesting bioactives. In this research, the impact of unconventional ultrasound-assisted extraction (UAE) and microwave-assisted extraction techniques on the recovery of total non-polar carotenoids from Cucurbita moschata pulp was investigated. A binary (hexane:isopropanol, 60:40 v/v) and a ternary (hexane:acetone:ethanol, 50:25:25 v/v/v) mixture were tested. The extracts were characterized for their antioxidant properties by in vitro assays, while the carotenoid profiling was determined by high-performance liquid chromatography coupled with a diode array detector. UAE with the binary mixture (30 min, 45 °C) was the most successful extracting technique, taking into consideration all analytical data and their correlations. In parallel, solid lipid nanoparticles (SLN) were optimized for the encapsulation of the extract, using ß-carotene as a reference compound. SLN, loaded with up to 1% ß-carotene, had dimensions (~350 nm) compatible with increased intestinal absorption. Additionally, the ABTS ((2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay showed that the technological process did not change the antioxidant capacity of ß-carotene. These SLN will be used to load an even higher percentage of the extract without affecting their dimensions due to its liquid nature and higher miscibility with the lipid with respect to the solid ß-carotene.

Ethidium bromide exposure unmasks an antibiotic efflux system in Rhodococcus equi.

BACKGROUND: This study introduces a newly created strain (Rhodococcus equiEtBr25) by exposing R. equi ATCC 33701 to ethidium bromide (EtBr), a substrate for MDR transporters. Such an approach allowed us to investigate the resulting phenotype and genetic mechanisms underlying the efflux-mediated resistance in R. equi. METHODS: R. equi ATCC 33701 was stimulated with increasing concentrations of EtBr. The antimicrobial susceptibility of the parental strain and R. equiEtBr25 was investigated in the presence/absence of efflux pump inhibitors (EPIs). EtBr efflux was evaluated by EtBr-agar method and flow cytometry. The presence of efflux pump genes was determined by conventional PCR before to quantify the expression of 30 genes coding for membrane transporters by qPCR. The presence of erm(46) and mutations in 23S rRNA, and gyrA/gyrB was assessed by PCR and DNA sequencing to exclude the occurrence of resistance mechanisms other than efflux. RESULTS: R. equi EtBr25 showed an increased EtBr efflux. Against this strain, the activity of EtBr, azithromycin and ciprofloxacin was more affected than that of rifampicin and azithromycin/rifampicin combinations. Resistances were reversed by combining the antimicrobials with EPIs. Gene expression analysis detected a marked up-regulation of REQ_RS13460 encoding for a Major Facilitator Superfamily (MFS) transporter. G→A transition occurred in the transcriptional repressor tetR/acrR adjacent to REQ_RS13460. CONCLUSIONS: Exposure of R. equi to EtBr unmasked an efflux-mediated defence against azithromycin and ciprofloxacin, which seemingly correlates with the overexpression of a specific MFS transporter. This genotype may mirror an insidious low-level resistance of clinically important isolates that could be countered by EPI-based therapies.

Microbial Efflux Pump Inhibitors: A Journey around Quinoline and Indole Derivatives.

Antimicrobial resistance (AMR) is a complex threat to human health and, to date, it represents a hot topic in drug discovery. The use of non-antibiotic molecules to block resistance mechanisms is a powerful alternative to the identification of new antibiotics. Bacterial efflux pumps exert the early step of AMR development, allowing the bacteria to grow in presence of sub-inhibitory drug concentration and develop more specific resistance mechanisms. Thus, efflux pump inhibitors (EPIs) offer a great opportunity to fight AMR, potentially restoring antibiotic activity. Based on our experience in designing and synthesizing novel EPIs, herein, we retrieved information around quinoline and indole derivatives reported in literature on this topic. Thus, our aim was to collect all data around these promising classes of EPIs in order to delineate a comprehensive structure-activity relationship (SAR) around each core for different microbes. With this review article, we aim to help future research in the field in the discovery of new microbial EPIs with improved activity and a better safety profile.

Alternate NF-κB-Independent Signaling Reactivation of Latent HIV-1 Provirus.

Current combination antiretroviral therapies (cART) are unable to eradicate HIV-1 from infected individuals because of the establishment of proviral latency in long-lived cellular reservoirs. The shock-and-kill approach aims to reactivate viral replication from the latent state (shock) using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells (kill) by specific therapeutics. The NF-κB RelA/p50 heterodimer has been characterized as an essential component of reactivation of the latent HIV-1 long terminal repeat (LTR). Nevertheless, prolonged NF-κB activation contributes to the development of various autoimmune, inflammatory, and malignant disorders. In the present study, we established a cellular model of HIV-1 latency in J-Lat CD4+ T cells that stably expressed the NF-κB superrepressor IκB-α 2NΔ4 and demonstrate that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivated HIV-1 from latency, even under conditions where NF-κB activation was repressed. Using specific calcineurin phosphatase, p38, and MEK1/MEK2 kinase inhibitors or specific short hairpin RNAs, c-Jun was identified to be an essential factor binding to the LTR enhancer κB sites and mediating the combined synergistic reactivation effect. Furthermore, acetylsalicylic acid (ASA), a potent inhibitor of the NF-κB activator kinase IκB kinase ß (IKK-ß), did not significantly diminish reactivation in a primary CD4+ T central memory (TCM) cell latency model. The present work demonstrates that the shock phase of the shock-and-kill approach to reverse HIV-1 latency may be achieved in the absence of NF-κB, with the potential to avoid unwanted autoimmune- and or inflammation-related side effects associated with latency-reversing strategies.IMPORTANCE The shock-and-kill approach consists of the reactivation of HIV-1 replication from latency using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells. The cellular transcription factor NF-κB is considered a master mediator of HIV-1 escape from latency induced by LRAs. Nevertheless, a systemic activation of NF-κB in HIV-1-infected patients resulting from the combined administration of different LRAs could represent a potential risk, especially in the case of a prolonged treatment. We demonstrate here that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivate HIV-1 from latency, even under conditions where NF-κB activation is repressed. Our study provides a molecular proof of concept for the use of anti-inflammatory drugs, like aspirin, capable of inhibiting NF-κB in patients under combination antiretroviral therapy during the shock-and-kill approach, to avoid potential autoimmune and inflammatory disorders that can be elicited by combinations of LRAs.

C-2 phenyl replacements to obtain potent quinoline-based Staphylococcus aureus NorA inhibitors.

NorA is the most studied efflux pump of Staphylococcus aureus and is responsible for high level resistance towards fluoroquinolone drugs. Although along the years many NorA efflux pump inhibitors (EPIs) have been reported, poor information is available about structure-activity relationship (SAR) around their nuclei and reliability of data supported by robust assays proving NorA inhibition. In this regard, we focussed efforts on the 2-phenylquinoline as a promising chemotype to develop potent NorA EPIs. Herein, we report SAR studies about the introduction of different aryl moieties on the quinoline C-2 position. The new derivative 37a showed an improved EPI activity (16-fold) with respect to the starting hit 1. Moreover, compound 37a exhibited a high potential in time-kill curves when combined with ciprofloxacin against SA-1199B (norA+). Also, 37a exhibited poor non-specific effect on bacterial membrane polarisation and showed an improvement in terms of "selectivity index" in comparison to 1.

Structural Modifications of the Quinolin-4-yloxy Core to Obtain New Staphylococcus aureus NorA Inhibitors.

Tackling antimicrobial resistance (AMR) represents a social responsibility aimed at renewing the antimicrobial armamentarium and identifying novel therapeutical approaches. Among the possible strategies, efflux pumps inhibition offers the advantage to contrast the resistance against all drugs which can be extruded. Efflux pump inhibitors (EPIs) are molecules devoid of any antimicrobial activity, but synergizing with pumps-substrate antibiotics. Herein, we performed an in silico scaffold hopping approach starting from quinolin-4-yloxy-based Staphylococcus aureus NorA EPIs by using previously built pharmacophore models for NorA inhibition activity. Four scaffolds were identified, synthesized, and modified with appropriate substituents to obtain new compounds, that were evaluated for their ability to inhibit NorA and synergize with the fluoroquinolone ciprofloxacin against resistant S. aureus strains. The two quinoline-4-carboxamide derivatives 3a and 3b showed the best results being synergic (4-fold MIC reduction) with ciprofloxacin at concentrations as low as 3.13 and 1.56 µg/mL, respectively, which were nontoxic for human THP-1 and A549 cells. The NorA inhibition was confirmed by SA-1199B ethidium bromide efflux and checkerboard assays against the isogenic pair SA-K2378 (norA++)/SA-K1902 (norA-). These in vitro results indicate the two compounds as valuable structures for designing novel S. aureus NorA inhibitors to be used in association with fluoroquinolones.

1,2,4-Triazolo[1,5-a]pyrimidines as a Novel Class of Inhibitors of the HIV-1 Reverse Transcriptase-Associated Ribonuclease H Activity.

Despite great efforts have been made in the prevention and therapy of human immunodeficiency virus (HIV-1) infection, however the difficulty to eradicate latent viral reservoirs together with the emergence of multi-drug-resistant strains require the search for innovative agents, possibly exploiting novel mechanisms of action. In this context, the HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H), which is one of the few HIV-1 encoded enzymatic function still not targeted by any current drug, can be considered as an appealing target. In this work, we repurposed in-house anti-influenza derivatives based on the 1,2,4-triazolo[1,5-a]-pyrimidine (TZP) scaffold for their ability to inhibit HIV-1 RNase H function. Based on the results, a successive multi-step structural exploration around the TZP core was performed leading to identify catechol derivatives that inhibited RNase H in the low micromolar range without showing RT-associated polymerase inhibitory activity. The antiviral evaluation of the compounds in the MT4 cells showed any activity against HIV-1 (IIIB strain). Molecular modelling and mutagenesis analysis suggested key interactions with an unexplored allosteric site providing insights for the future optimization of this class of RNase H inhibitors.

From cycloheptathiophene-3-carboxamide to oxazinone-based derivatives as allosteric HIV-1 ribonuclease H inhibitors.

The paper focussed on a step-by-step structural modification of a cycloheptathiophene-3-carboxamide derivative recently identified by us as reverse transcriptase (RT)-associated ribonuclease H (RNase H) inhibitor. In particular, its conversion to a 2-aryl-cycloheptathienoozaxinone derivative and the successive thorough exploration of both 2-aromatic and cycloheptathieno moieties led to identify oxazinone-based compounds as new anti-RNase H chemotypes. The presence of the catechol moiety at the C-2 position of the scaffold emerged as critical to achieve potent anti-RNase H activity, which also encompassed anti-RNA dependent DNA polymerase (RDDP) activity for the tricyclic derivatives. Benzothienooxazinone derivative 22 resulted the most potent dual inhibitor exhibiting IC50s of 0.53 and 2.90 µM against the RNase H and RDDP functions. Mutagenesis and docking studies suggested that compound 22 binds two allosteric pockets within the RT, one located between the RNase H active site and the primer grip region and the other close to the DNA polymerase catalytic centre.

Deciphering the Molecular Recognition Mechanism of Multidrug Resistance Staphylococcus aureus NorA Efflux Pump Using a Supervised Molecular Dynamics Approach.

The use and misuse of antibiotics has resulted in critical conditions for drug-resistant bacteria emergency, accelerating the development of antimicrobial resistance (AMR). In this context, the co-administration of an antibiotic with a compound able to restore sufficient antibacterial activity may be a successful strategy. In particular, the identification of efflux pump inhibitors (EPIs) holds promise for new antibiotic resistance breakers (ARBs). Indeed, bacterial efflux pumps have a key role in AMR development; for instance, NorA efflux pump contributes to Staphylococcus aureus (S. aureus) resistance against fluoroquinolone antibiotics (e.g., ciprofloxacin) by promoting their active extrusion from the cells. Even though NorA efflux pump is known to be a potential target for EPIs development, the absence of structural information about this protein and the little knowledge available on its mechanism of action have strongly hampered rational drug discovery efforts in this area. In the present work, we investigated at the molecular level the substrate recognition pathway of NorA through a Supervised Molecular Dynamics (SuMD) approach, using a NorA homology model. Specific amino acids were identified as playing a key role in the efflux pump-mediated extrusion of its substrate, paving the way for a deeper understanding of both the mechanisms of action and the inhibition of such efflux pumps.

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.

p38α MAPK and Type I Inhibitors: Binding Site Analysis and Use of Target Ensembles in Virtual Screening.

Mitogen-activated protein kinase p38α plays an essential role in the regulation of pro-inflammatory signaling, and selective blockade of this kinase could be efficacious in many pathological processes. Despite considerable research efforts focused on the discovery and development of p38α MAPK inhibitors, no drug targeting this protein has been approved for clinical use so far. We herein analyze the available crystal structures of p38α MAPK in complex with ATP competitive type I inhibitors, getting insights into ATP binding site conformation and its influence on automated molecular docking results. The use of target ensembles, rather than single conformations, resulted in a performance improvement in both the ability to reproduce experimental bound conformations and the capability of mining active molecules from compound libraries. The information gathered from this study can be exploited in structure-based drug discovery programs having as the ultimate aim the identification of novel p38α MAPK type I inhibitors.

Accounting for target flexibility and water molecules by docking to ensembles of target structures: the HCV NS5B palm site I inhibitors case study.

The introduction of new anti-HCV drugs in therapy is an imperative need and is necessary with a view to develop an interferon-free therapy. Thus, the discovery and development of novel small molecule inhibitors of the viral NS5B polymerase represent an exciting area of research for many pharmaceutical companies and academic groups. This study represents a contribution to this field and relies on the identification of the best NS5B model(s) to be used in structure-based computational approaches aimed at identifying novel non-nucleoside inhibitors of one of the protein allosteric sites, namely, palm site I. First, the NS5B inhibitors at palm site I were classified as water-mediated or nonwater-mediated ligands depending on their ability to interact with or displace a specific water molecule. Then, we took advantage of the available X-ray structures of the NS5B/ligand complexes to build different models of protein/water combinations, which were used to investigate the influence on docking studies of solvent sites as well as of the influence of the protein conformations. As the overall trend, we observed improved performance in the docking results of the water-mediated inhibitors by inclusion of explicit water molecules, with an opposite behavior generally happening for the nonwater-mediated inhibitors. The best performing target structures for the two ligand sets were then used for virtual screening simulations of a library containing the known NS5B inhibitors along with related decoys to assess the best performing targets ensembles on the basis of their ability to discriminate active and inactive compounds as well as to generate the correct binding modes. The parallel use of different protein structures/water sets outperformed the use of a single target structure, with the two-protein 3H98/2W-2FVC/7W and 3HKY/NoW-3SKE/NoW models resulting in the best performing ensembles for water-mediated inhibitors and nonwater-mediated inhibitors, respectively. The information gathered from this work confirms the primary role of water molecules and protein flexibility in docking-based studies and can be exploited to aid NS5B-directed HCV drug discovery efforts.

Inhibition of Staphylococcus pseudintermedius Efflux Pumps by Using Staphylococcus aureus NorA Efflux Pump Inhibitors.

One promising approach in treating antibiotic-resistant bacteria is to "break" resistances connected with antibacterial efflux by co-administering efflux pump inhibitors (EPIs) with antibiotics. Here, ten compounds, previously optimized to restore the susceptibility to ciprofloxacin (CIP) of norA-overexpressing Staphylococcus aureus, were evaluated for their ability to inhibit norA-mediated efflux in Staphylococcus pseudintermedius and synergize with CIP, ethidium bromide (EtBr), gentamycin (GEN), and chlorhexidine digluconate (CHX). We focused efforts on S. pseudintermedius as a pathogenic bacterium of concern within veterinary and human medicine. By combining data from checkerboard assays and EtBr efflux inhibition experiments, the hits 2-arylquinoline 1, dihydropyridine 6, and 2-phenyl-4-carboxy-quinoline 8 were considered the best EPIs for S. pseudintermedius. Overall, most of the compounds, except for 2-arylquinoline compound 2, were able to fully restore the susceptibility of S. pseudintermedius to CIP and synergize with GEN as well, while the synergistic effect with CHX was less significant and often did not show a dose-dependent effect. These are valuable data for medicinal chemistry optimization of EPIs for S. pseudintermedius and lay the foundation for further studies on successful EPIs to treat staphylococcal infections.

Functionalized sulfonyl anthranilic acid derivatives inhibit replication of all the four dengue serotypes.

Dengue virus (DENV), a mosquito-borne flavivirus, continues to be a major public health threat in many countries and no approved antiviral therapeutics are available yet. In this work, we designed and synthesized a series of sulfonyl anthranilic acid (SAA) derivatives using a ligand-based scaffold morphing approach of the 2,1-benzothiazine 2,2-dioxide core, previously used by us to develop DENV polymerase inhibitors resulting devoid of any cell-based antiviral activity. Several derivatives based on the new SAA chemotype exhibited potent inhibition against DENV infection in the cell-based assay but did not inhibit DENV NS5 polymerase activity in the in vitro de novo initiation and elongation assays. Notably, best compounds 26 and 39 showed EC50 values in the range of 0.54-1.36 µM against cells infected with the four dengue serotypes (DENV-1-4). Time-of-drug-addition assay revealed that analogue 26 is a post-entry replication inhibitor that appears to be specific for cells of primate origin, implicating a host target with a high barrier to resistance. In conclusion, SAA derivatives offer a valuable starting point for developing effective Dengue antiviral therapeutics.

Pyridobenzothiazole derivatives as new chemotype targeting the HCV NS5B polymerase.

Hepatitis C virus (HCV) infection has been recognized as the major cause of liver failure that can lead to hepatocellular carcinoma. Among all the HCV proteins, NS5B polymerase represents a leading target for drug discovery strategies. Herein, we describe our initial research efforts towards the identification of new chemotypes as allosteric NS5B inhibitors. In particular, the design, synthesis, in vitro anti-NS5B and in cellulo anti-HCV evaluation of a series of 1-oxo-1H-pyrido[2,1-b][1,3]benzothiazole-4-carboxylate derivatives are reported. Some of the newly synthesized compounds showed an IC(50) ranging from 11 to 23 µM, and molecular modeling and biochemical studies suggested that the thumb domain could be the target site for this new class of NS5B inhibitors.