Exploring the structure-activity relationship of benzylidene-2,3-dihydro-1H-inden-1-one compared to benzofuran-3(2H)-one derivatives as inhibitors of tau amyloid fibers.

Tauopathies, such as Alzheimer's disease, have been the subject of several hypotheses regarding the way to treat them. Hyperphosphorylation of tau protein leading to its aggregation is widely recognized as a key step in the development of these diseases resulting in neuronal dysfunction. The AcPHF6 model of tau that includes the shorter critical fragment involved in the protein aggregation was used in vitro to identify new potential inhibitors. Following a previous study on aurone derivatives, we herein compare this polyphenol family to a very close one, the benzylidene-2,3-dihydro-1H-inden-1-one (also named indanone). The structure activity relationship studies bring to light the importance of the hydroxylation pattern in both series: the more hydroxylated, the more active. In addition, the three-dimensional shape of the molecules is involved in their interaction mode with their target, thus defining their role either as inhibitors of fiber elongation or as fiber-binding molecules. Indanone 13a was identified as a promising inhibitor: its activity was confirmed by circular dichroism and atomic force microscopy studies.

Targeting different binding sites in the CFTR structures allows to synergistically potentiate channel activity.

Recent evidence shows that combination of correctors and potentiators, such as the drug ivacaftor (VX-770), can significantly restore the functional expression of mutated Cystic Fibrosis Transmembrane conductance Regulator (CFTR), an anion channel which is mutated in cystic fibrosis (CF). The success of these combinatorial therapies highlights the necessity of identifying a broad panel of specific binding mode modulators, occupying several distinct binding sites at structural level. Here, we identified two small molecules, SBC040 and SBC219, which are two efficient cAMP-independent potentiators, acting at low concentration of forskolin with EC50 close to 1 µM and in a synergic way with the drug VX-770 on several CFTR mutants of classes II and III. Molecular dynamics simulations suggested potential SBC binding sites at the vicinity of ATP-binding sites, distinct from those currently proposed for VX-770, outlining SBC molecules as members of a new family of potentiators.

Chimeric Protein-Protein Interface Inhibitors Allow Efficient Inhibition of Type III Secretion Machinery and Pseudomonas aeruginosa Virulence.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen naturally resistant to many common antibiotics and acquires new resistance traits at an alarming pace. Targeting the bacterial virulence factors by an antivirulence strategy, therefore, represents a promising alternative approach besides antibiotic therapy. The Type III secretion system (T3SS) of P. aeruginosa is one of its main virulence factors. It consists of more than 20 proteins building a complex syringe-like machinery enabling the injection of toxin into host cells. Previous works showed that disrupting interactions between components of this machinery efficiently lowers the bacterial virulence. Using automated target-based screening of commercial and in-house libraries of small molecules, we identified compounds inhibiting the protein-protein interaction between PscE and PscG, the two cognate chaperones of the needle subunit PscF of P. aeruginosa T3SS. Two hits were selected and assembled using Split/Mix/Click chemistry to build larger hybrid analogues. Their efficacy and toxicity were evaluated using phenotypic analysis including automated microscopy and image analysis. Two nontoxic hybrid leads specifically inhibited the T3SS and reduced the ex vivo cytotoxicity of bacteria and their virulence in Galleria mellonella.

New pseudodimeric aurones as palm pocket inhibitors of Hepatitis C virus RNA-dependent RNA polymerase.

The NS5B RNA-dependent RNA polymerase (RdRp) is a key enzyme for Hepatitis C Virus (HCV) replication. In addition to the catalytic site, this enzyme is characterized by the presence of at least four allosteric pockets making it an interesting target for development of inhibitors as potential anti-HCV drugs. Based on a previous study showing the potential of the naturally occurring aurones as inhibitors of NS5B, we pursued our efforts to focus on pseudodimeric aurones that have never been investigated so far. Hence, 14 original compounds characterized by the presence of a spacer between the benzofuranone moieties were synthesized and investigated as HCV RdRp inhibitors by means of an in vitro assay. The most active inhibitor, pseudodimeric aurone 4, induced high inhibition activity (IC50 = 1.3 µM). Mutagenic and molecular modeling studies reveal that the binding site for the most active derivatives probably is the palm pocket I instead of the thumb pocket I as for the monomeric derivatives.

A new 9-alkyladenine-cyclic methylglyoxal diadduct activates wt- and F508del-cystic fibrosis transmembrane conductance regulator (CFTR) in vitro and in vivo.

Cystic fibrosis transmembrane conductance regulator (CFTR) is the main chloride channel present in the apical membrane of epithelial cells and the F508 deletion (F508del-CFTR) in the CF gene is the most common cystic fibrosis-causing mutation. In the search for a pharmacotherapy of cystic fibrosis caused by the F508del-CFTR, a bi-therapy could be developed associating a corrector of F508del-CFTR trafficking and an activator of the channel activity of CFTR. Here, we report on the synthesis of 9-alkyladenine derivatives analogues of our previously discovered activator of wt-CFTR and F508del-CFTR, GPact-11a, and the identification of a new activator of these channels, GPact-26a, through various flux assays on human airway epithelial CF and non-CF cell lines and in vivo measurement of rat salivary secretion. This study reveals that the possible modifications of the side chain introduced at the N9 position of the main pharmacophore are highly limited since only an allyl group can replace the propyl side chain present in GPact-11a to lead to a strong activation of wt-CFTR in CHO cells. Docking simulations of the synthesised compounds and of four described modulators performed using a 3D model of the wt-type CFTR protein suggest five possible binding sites located at the interface of the nucleotide binding domains NBD1/NBD2. However, the docking study did not allow the differentiation between active and non-active compounds.

New 7-methylguanine derivatives targeting the influenza polymerase PB2 cap-binding domain.

The heterotrimeric influenza virus polymerase performs replication and transcription of viral RNA in the nucleus of infected cells. Transcription by "cap-snatching" requires that host-cell pre-mRNAs are bound via their 5' cap to the PB2 subunit. Thus, the PB2 cap-binding site is potentially a good target for new antiviral drugs that will directly inhibit viral replication. Docking studies using the structure of the PB2 cap-binding domain suggested that 7-alkylguanine derivatives substituted at position N-9 and N-2 could be good candidates. Four series of 7,9-di- and 2,7,9-trialkyl guanine derivatives were synthesized and evaluated by an AlphaScreen assay in competition with a biotinylated cap analogue. Three synthesized compounds display potent in vitro activity with IC50 values lower than 10 µM. High-resolution X-ray structures of three inhibitors in complex with the H5N1 PB2 cap-binding domain confirmed the binding mode and provide detailed information for further compound optimization.

Discovery of naturally occurring aurones that are potent allosteric inhibitors of hepatitis C virus RNA-dependent RNA polymerase.

We have identified naturally occurring 2-benzylidenebenzofuran-3-ones (aurones) as new templates for non-nucleoside hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) inhibitors. The aurone target site, identified by site-directed mutagenesis, is located in thumb pocket I of HCV RdRp. The RdRp inhibitory activity of 42 aurones was rationally explored in an enzyme assay. Molecular docking studies were used to determine how aurones bind to HCV RdRp and to predict their range of inhibitory activity. Seven aurone derivatives were found to have potent inhibitory effects on HCV RdRp, with IC(50) below 5 µM and excellent selectivity index (inhibition activity versus cellular cytotoxicity). The most active aurone analogue was (Z)-2-((1-butyl-1H-indol-3-yl)methylene)-4,6-dihydroxybenzofuran-3(2H)-one (compound 51), with an IC(50) of 2.2 µM. Their potent RdRp inhibitory activity and their low toxicity make these molecules attractive candidates as direct-acting anti-HCV agents.

2-Arylidenedihydroindole-3-ones: design, synthesis, and biological activity on bladder carcinoma cell lines.

2-Arylidenedihydroindole-3-ones were assayed for their antiproliferative and apoptotic abilities as potential drug candidates to treat bladder tumor. These compounds were tested on cell lines obtained from bladder tumors of various stages [superficial (pTa and pT1) vs. invasive (pT2)]. The most active compound (3c) inhibited the proliferation, induced apoptosis, and decreased the expression of p-Stat5 and p-Pyk2 in DAG-1 and RT112 lines in which the FGFR3 is either mutated or overexpressed. Knowing that FGFR3 is involved in cell proliferation, differentiation, and migration through cell signaling pathways including p-Stat5 way via p-Pyk2, let us assume that compound 3c may probably act through FGFR3 pathway.

Toward a better understanding of the basis of the molecular mimicry of polysaccharide antigens by peptides: the example of Shigella flexneri 5a.

Protein conjugates of oligosaccharides or peptides that mimic complex bacterial polysaccharide antigens represent alternatives to the classical polysaccharide-based conjugate vaccines developed so far. Hence, a better understanding of the molecular basis ensuring appropriate mimicry is required in order to design efficient carbohydrate mimic-based vaccines. This study focuses on the following two unrelated sets of mimics of the Shigella flexneri 5a O-specific polysaccharide (O-SP): (i) a synthetic branched pentasaccharide known to mimic the average solution conformation of S. flexneri 5a O-SP, and (ii) three nonapeptides selected upon screening of phage-displayed peptide libraries with two protective murine monoclonal antibodies (mAbs) of the A isotype specific for S. flexneri 5a O-SP. By inducing anti-O-SP antibodies upon immunization in mice when appropriately presented to the immune system, the pentasaccharide and peptides p100c and p115, but not peptide p22, were qualified as mimotopes of the native antigen. NMR studies based on transferred NOE (trNOE) experiments revealed that both kinds of mimotopes had an average conformation when bound to the mAbs that was close to that of their free form. Most interestingly, saturation transfer difference (STD) experiments showed that the characteristic turn conformations adopted by the major conformers of p100c and p115, as well as of p22, are clearly involved in mAb binding. These latter experiments also showed that the branched glucose residue of the pentasaccharide was a key part of the determinant recognized by the protective mAbs. Finally, by using NMR-derived pentasaccharide and peptide conformations coupled to STD information, models of antigen-antibody interaction were obtained. Most interestingly, only one model was found compatible with experimental data when large O-SP fragments were docked into one of the mIgA-binding sites. This newly made available system provides a new contribution to the understanding of the molecular mimicry of complex polysaccharides by peptides and short oligosaccharides.