The easily accessible C-nucleoside 2-amino-5-(2‘-deoxy-β-d-ribofuranosyl)pyridine (P) and its 3-methyl ( Me P) and 2‘-O-methyl (P OMe ) derivatives were synthesized and incorporated as protonated cytidine equivalents in homopyrimidine oligodeoxynucleotides. T m measurements indicate that oligonucleotides containing P or Me P have a higher affinity to double-stranded DNA over the pH range of 6−8 than 5-methylcytidine (MeC) containing oligonucleotides. This increase in stability is most pronounced above pH 7.0. The average increase in T m/modification for the dissociation of oligonucleotide d(TTTTTMePTMePTMePTMePTMePT) from a 21-mer target duplex at pH 7.5 is 2.3 °C relative to oligonucleotide d(TTTTTMeCTMeCTMeCTMeCTMeCT). The pH dependence and sequence composition effects are much less pronounced for Me P (and also P) containing oligonucleotides than for MeC containing ones. While oligonucleotide d(TTTMeCMeCMeCMeCTTTTMeCTTT) shows no longer any affinity to the target duplex above pH 6.5, oligonucleotide d(TTTMePMePMePMePTTTTMePTTT) displays preserved binding with a T m of 32.5 °C at pH 7.0 and even binds with a T m of 23.3 °C at pH 8.0. Oligonucleotides containing P OMe show distinctly less stable triple helices. The average decrease in T m/modification for oligonucleotide d(TTTTTPOMeTPOMeTPOMeTPOMeTPOMeT) at pH 6.5 is 6.7 °C relative to the MeC containing oligonucleotide. DNase I footprint titration experiments indicate that d(TTTTTMePTMePTMePTMePTMePT) binds not only five times stronger to a 229 base pair DNA fragment than d(TTTTTMeCTMeCTMeCTMeCTMeCT) but also with higher selectivity. UV-melting experiments show that duplexes of d(TTTTTCTXTCTCTCT) (where X = P, Me P, or P OMe ) with their antiparallel Watson−Crick complement are dramatically less stable (ΔT m < −12 °C) at pH 8.0 than the corresponding natural duplex. Thus the new bases P and Me P show Hoogsteen specific pairing behavior.

Fluorometric imaging plate reader (FLIPR) membrane potential dyes (FMP-Red-Dye and FMP-Blue-Dye) were evaluated for the detection of compounds acting either as positive allosteric modulators or agonists on the GABA A receptor (GABA A R). A stable HEK293 cell line with constitutive expression of the rat GABA A R α1, β2, and γ2 genes was used to establish a functional high-throughput screening (HTS) assay based on measurement of the membrane potential change in living cells. The assay was validated with the FLIPR technology for identification of agonists and positive allosteric modulators using GABA and diazepam as model compounds. The FMP-Red-Dye showed better performance than the FMP-Blue-Dye, and the effects induced by GABA and diazepam were comparable to electrophysiology data. Subsequently, the assay was also validated with an ultra-HTS approach known as microarrayed compound screening (μARCS). The LOPAC library was used in a test screen for an initial assessment of the technology. Finally, the FLIPR and μARCS technologies were tested with a larger screening campaign. A focused library of 3520 putative positive modulators was tested with the FLIPR assay, and a diverse subset of 84,480 compounds was selected for screening with the μARCS technology. All hits were subjected to verification using the FLIPR technology, and confirmed hits were subsequently evaluated by EC50 determination. Finally, selected hits were further confirmed with electrophysiology testing. (Journal of Biomolecular Screening 2008:218-228)

Ligand-based virtual screening and computational hit expansion methods undoubtedly facilitate the finding of novel active chemical entities, utilizing already existing knowledge of active compounds. It has been demonstrated that the parallel execution of complementary similarity search methods enhances the performance of such virtual screening campaigns. In this article, we examine the use of virtualized template (query, seed) structures as an extension to common search methods, such as fingerprint and pharmacophore graph-based similarity searches. We demonstrate that template virtualization by bioisosteric enumeration and other rule-based methods, in combination with standard similarity search techniques, represents a powerful approach for hit expansion following high-throughput screening campaigns. The reliability of the methods is demonstrated by four different test data sets representing different target classes and two hit finding case studies on the epigenetic targets G9a and LSD1.

Malaria is responsible for approximately 1 million deaths annually; thus, continued efforts to discover new antimalarials are required. A HTS screen was established to identify novel inhibitors of the parasite's mitochondrial enzyme NADH:quinone oxidoreductase (PfNDH2). On the basis of only one known inhibitor of this enzyme, the challenge was to discover novel inhibitors of PfNDH2 with diverse chemical scaffolds. To this end, using a range of ligand-based chemoinformatics methods, ∼17000 compounds were selected from a commercial library of ∼750000 compounds. Forty-eight compounds were identified with PfNDH2 enzyme inhibition IC50 values ranging from 100 nM to 40 μM and also displayed exciting whole cell antimalarial activity. These novel inhibitors were identified through sampling 16% of the available chemical space, while only screening 2% of the library. This study confirms the added value of using multiple ligand-based chemoinformatic approaches and has successfully identified novel distinct chemotypes primed for development as new agents against malaria.

The iron release mechanism from protonated ferric enterobactin [Fe(III)(enterobactinH(3))] via the salicylate binding mode was probed. For this purpose, a tripodal dodecadentate ligand incorporating three salicylamide (OO) and three bipyridine (NN) binding sites was synthesized as well as iron complexes thereof. It was shown that a ferric ion coordinates selectively to the hard salicylamides and a ferrous ion binds to the softer bipyridines. Upon reduction or oxidation, the iron translocates reversibly and intramolecularly from one site to the other, thus displaying switchlike properties. Both states were characterized by cyclic voltammetry and visible and Mössbauer spectroscopy. The Mössbauer spectrum for the ferric complex is fully consistent with that obtained by Pecoraro et al. upon lowering the pH of [Fe(III)(enterobactin)](3)(-) solutions (Pecoraro, V. L., et al. J. Am. Chem. Soc. 1983, 105, 4617), thus supporting the alternative iron release mechanism from enterobactin via the salicylate binding mode.