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

Boosting Effect of 2‑Phenylquinoline Efflux Inhibitors in Combination with Macrolides against Mycobacterium smegmatis and Mycobacterium avium

The identification of efflux inhibitors to be used as adjuvants alongside existing drug regimens could have a tremendous value in the treatment of any mycobacterial infection. Here, we investigated the ability of four 2-(4′-propoxyphenyl)­quinoline Staphylococcus aureus NorA efflux inhibitors (<b>1</b>–<b>4</b>) to reduce the efflux activity in Mycobacterium smegmatis and Mycobacterium avium strains. All four compounds were able to inhibit efflux pumps in both mycobacterial species; in particular, <i>O</i>-ethylpiperazinyl derivative <b>2</b> showed an efflux inhibitory activity comparable to that of verapamil, the most potent mycobacterial efflux inhibitor reported to date, and was able to significantly reduce the MIC values of macrolides against different <i>M. avium</i> strains. The contribution of the <i>M. avium</i> efflux pumps MAV_1406 and MAV_1695 to clarithromycin resistance was proved because they were found to be overexpressed in two <i>M. avium</i> 104 isogenic strains showing high-level clarithromycin resistance. These results indicated a correlation between increased expression of efflux pumps, increased efflux, macrolide resistance, and reduction of resistance by efflux pump inhibitors such as compound <b>2</b>. Additionally, compound <b>2</b> showed synergistic activity with clarithromycin, at a concentration below the cytotoxicity threshold, in an ex vivo experiment against <i>M. avium</i> 104-infected macrophages. In summary, the 2-(4′-propoxyphenyl)­quinoline scaffold is suitable to obtain compounds endowed with good efflux pump inhibitory activity against both <i>S. aureus</i> and nontuberculous mycobacteria

Pyrazolo[4,3-<i>c</i>][1,2]benzothiazines 5,5-Dioxide: A Promising New Class of Staphylococcus aureus NorA Efflux Pump Inhibitors

The increasing resistance to antibacterials commonly employed in the clinic and the growth of multidrug resistant strains suggest that the development of new therapeutic approaches should be of primary concern. In this context, EPIs may restore life to old drugs. In the present work, the EPI activity of the COX-2 inhibitor celecoxib was confirmed and a new class of pyrazolo­[4,3-<i>c</i>]­[1,2]­benzothiazine 5,5-dioxide analogues acting as inhibitors of the Staphylococcus aureus NorA multidrug efflux pump was identified

Re-evolution of the 2‑Phenylquinolines: Ligand-Based Design, Synthesis, and Biological Evaluation of a Potent New Class of Staphylococcus aureus NorA Efflux Pump Inhibitors to Combat Antimicrobial Resistance

Overexpression of efflux pumps is an important mechanism by which bacteria evade the effects of antimicrobial agents that are substrates. NorA is a Staphylococcus aureus efflux pump that confers reduced susceptibility to many structurally unrelated agents, including fluoroquinolones, biocides, and dyes, resulting in a multidrug resistant (MDR) phenotype. In this work, a series of 2-phenylquinoline derivatives was designed by means of ligand-based pharmacophore modeling in an attempt to identify improved S. aureus NorA efflux pump inhibitors (EPIs). Most of the 2-phenylquinoline derivatives displayed potent EPI activity against the <i>norA</i> overexpressing strain SA-1199B. The antibacterial activity of ciprofloxacin, when used in combination with some of the synthesized compounds, was completely restored in SA-1199B and SA-K2378, a strain overexpressing <i>norA</i> from a multicopy plasmid. Compounds <b>3m</b> and <b>3q</b> also showed potent synergistic activity with the ethidium bromide dye in a strain overexpressing the MepA MDR efflux pump

Ethyl 1,8-Naphthyridone-3-carboxylates Downregulate Human Papillomavirus-16 E6 and E7 Oncogene Expression

Strong epidemiological and molecular data associate cervical cancer (CC) with high-risk human papillomavirus (HPV) infections. The carcinogenic mechanism depends mainly on the expression of E6 and E7 oncoproteins encoded by the viral genome. Using a cell-based high-throughput assay, an in-house library of compounds was screened identifying the 1,8-naphthyridone <b>1</b> that efficiently inhibited the transcription driven by the long control region of the HPV genome. A series of analogues were then synthesized, obtaining more potent derivatives able to downregulate E6 and E7 transcripts in HPV-16-positive CC CaSki cells. An unusual structural insight emerged for the C-3 position of the 1,8-naphthyridone core, where the ethyl carboxylate esters, but not the carboxylic acids, are responsible for the activity. In vitro uptake studies showed that the 3-ethyl carboxylates do not act as prodrugs. The 1,8-naphthyridones emerged as valid starting points for the development of innovative agents potentially useful for the treatment of HPV-induced CC

Pharmacophore-Based Repositioning of Approved Drugs as Novel <i>Staphylococcus aureus</i> NorA Efflux Pump Inhibitors

An intriguing opportunity to address antimicrobial resistance is represented by the inhibition of efflux pumps. Focusing on NorA, the most important efflux pump of <i>Staphylococcus aureus</i>, an efflux pump inhibitors (EPIs) library was used for ligand-based pharmacophore modeling studies. By exploitation of the obtained models, an in silico drug repositioning approach allowed for the identification of novel and potent NorA EPIs

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

none13nomixedMassari, Serena; Nannetti, Giulio; Desantis, Jenny; Muratore, Giulia; Sabatini, Stefano; Manfroni, Giuseppe; Mercorelli, Beatrice; Cecchetti, Violetta; Palù, Giorgio; Cruciani, Gabriele; Loregian, Arianna; Goracci, Laura; Tabarrini, OrianaMassari, Serena; Nannetti, Giulio; Desantis, Jenny; Muratore, Giulia; Sabatini, Stefano; Manfroni, Giuseppe; Mercorelli, Beatrice; Cecchetti, Violetta; Palu', Giorgio; Cruciani, Gabriele; Loregian, Arianna; Goracci, Laura; Tabarrini, Orian

Structure-Based Discovery of Pyrazolobenzothiazine Derivatives As Inhibitors of Hepatitis C Virus Replication

The NS5B RNA-dependent RNA polymerase is an attractive target for the development of novel and selective inhibitors of hepatitis C virus replication. To identify novel structural hits as anti-HCV agents, we performed structure-based virtual screening of our in-house library followed by rational drug design, organic synthesis, and biological testing. These studies led to the identification of pyrazolobenzothiazine scaffold as a suitable template for obtaining novel anti-HCV agents targeting the NS5B polymerase. The best compound of this series was the <i>meta</i>-fluoro-<i>N</i>-1-phenyl pyrazolobenzothiazine derivative <b>4a</b>, which exhibited an EC₅₀ = 3.6 μM, EC₉₀ = 25.6 μM, and CC₅₀ > 180 μM in the Huh 9–13 replicon system, thus providing a good starting point for further hit evolution