http://www.mdpi.com/section/molecular_diversity http://www.mdpi.com/1420-3049/15/3/1690/ West Nile virus (WNV) is a positive sense, single-stranded RNA virus that can cause illness in humans when transmitted via mosquito vectors. Unfortunately, no antivirals or vaccines are currently available, and therefore efficient and safe antivirals are urgently needed. We developed a high throughput screen to discover small molecule probes that inhibit virus infection of Vero E6 cells. A primary screen of a 13,001 compound library at a 10 µM final concentration was conducted using the 384-well format. Z′ values ranged from 0.54–0.83 with a median of 0.74. Average S/B was 17 and S/N for each plate ranged from 10.8 to 23.9. Twenty-six compounds showed a dose response in the HT screen and were further evaluated in a time of addition assay and in a titer reduction assay. Seven compounds showed potential as small molecule probes directed at WNV. The hit rate from the primary screen was 0.185% (24 compounds out of 13,001 compounds) and from the secondary screens was 0.053% (7 out of 13,001 compounds) respectively. http://www.mdpi.com/1420-3049/15/3/1690/ Fri, 12 Mar 2010 00:00:00 CET Molecules 2010-03-12 15 3 Article 1690 1704 1420-3049 HTS-Driven Discovery of New Chemotypes with West Nile Virus Inhibitory Activity 2010-03-12 doi: 10.3390/molecules15031690 Chung Jonsson Maddox McKellip Moore Heil White Ananthan Li Feng Rasmussen http://www.mdpi.com/1420-3049/15/3/1645/ We instituted a quasi-quality assurance program for demonstrating coniferyl alcohol’s fluorescence and fluorescence diminishment following enzymatic oxidation. The magnitude of diminishment was a measure of catalysis. High throughput screening was performed in pseudo-kinetic and endpoint modes by measuring the fluorescence at 416 nm following excitation at 290, 310 or 340 nm. Dose-response tracings were linear between two and three orders of magnitude with average limits of detection and quantitation of 1.8 and 6.9 mM coniferyl alcohol, respectively. Oxidation was evident with 0.025 mg/mL laccase or 0.003 mg/mL peroxidase or inside 5 min using 0.5 mg/mL laccase or 5 mM substrate. Sodium chloride inhibited (IC50, 25 mM) laccase oxidation of coniferyl alcohol. Fluorescence from 10 concentrations (1 to 1000 mM) of coniferyl alcohol was stable for 24 hours over 14 excitation/emission cycles at 3 different combinations of excitation and emission wavelengths. In conclusion, coniferyl alcohol absorption and fluorescence assays should facilitate biomass lignin analyses and improve delignification. http://www.mdpi.com/1420-3049/15/3/1645/ Thu, 11 Mar 2010 00:00:00 CET Molecules 2010-03-11 15 3 Article 1645 1667 1420-3049 Hitherto Unrecognized Fluorescence Properties of Coniferyl Alcohol 2010-03-11 doi: 10.3390/molecules15031645 Komandoor Elayavalli Achyuthan Paul David Adams Supratim Datta Blake Alexander Simmons Anup Kumar Singh http://www.mdpi.com/1420-3049/15/3/1590/ Combinatorial chemistry is a powerful tool used to rapidly generate a large number of potentially biologically active compounds. In our goal to develop bisubstrate inhibitors of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) that interact with both the substrate (estrone or estradiol) and the cofactor (NAD(P)H) binding sites, we used parallel solid-phase synthesis to prepare three libraries of 16β-estradiol derivatives with two or three levels of molecular diversity. From estrone, we first synthesized a sulfamate precursor that we loaded on trityl chloride resin using the efficient multidetachable sulfamate linker strategy recently developed in our laboratory. We then introduced molecular diversity [one or two amino acid(s) followed by a carboxylic acid] on steroid nucleus by Fmoc peptide chemistry. Finally, after a nucleophilic cleavage, libraries of 30, 63 and 25 estradiol derivatives were provided. A library of 30 sulfamoylated estradiol derivatives was also generated by acidic cleavage and its members were screened for inhibition of steroid sulfatase. Biological evaluation on homogenated HEK-293 cells overexpressing 17β-HSD1 of the estradiol derivatives carrying different oligoamide-type chains at C-16 first revealed that three levels of molecular diversity (a spacer of two amino acids) were necessary to interact with the adenosine part of the cofactor binding site. Second, the best inhibition was obtained when hydrophobic residues (phenylalanine) were used as building blocks. http://www.mdpi.com/1420-3049/15/3/1590/ Wed, 10 Mar 2010 00:00:00 CET Molecules 2010-03-10 15 3 Article 1590 1631 1420-3049 Preparation of 16β-Estradiol Derivative Libraries as Bisubstrate Inhibitors of 17β-Hydroxysteroid Dehydrogenase Type 1 Using the Multidetachable Sulfamate Linker 2010-03-10 doi: 10.3390/molecules15031590 Marie Bérubé Florian Delagoutte Donald Poirier http://www.mdpi.com/1420-3049/15/1/215/ To obtain an aptamer with a high affinity for vascular endothelial growth factor (VEGF), we focused on the receptor-binding domain (RBD) of VEGF as a target epitope. Three rounds of screening gave Vap7, which bound to the VEGF isoforms VEGF121 and VEGF165 with KD values of 1.0 nM and 20 nM, respectively. Moreover, Vap7 showed specificity within the VEGF family. Secondary structure predictions and circular dicrhoism suggested that Vap7 folds into a G-quadruplex structure. We obtained a mutant aptamer that contains only this region of the aptamer sequence. This truncated mutant (V7t1) bound to both VEGF121 and VEGF165 with KD values of 1.1 nM and 1.4 nM, respectively. Its sequence was 5'-TGTGGGGGTGGACGGGCCGGGTAGA-3', and it appeared to form a G-quadruplex structure. We also produced an aptamer heterodimer consisting of our previously derived aptamer (del5-1), which binds to the heparin-binding domain of VEGF, linked to V7t1. The resulting heterodimer bound strongly to VEGF165 with a KD value of 4.7 × 102 pM. http://www.mdpi.com/1420-3049/15/1/215/ Thu, 07 Jan 2010 00:00:00 CET Molecules 2010-01-07 15 1 Article 215 225 1420-3049 Screening and Improvement of an Anti-VEGF DNA Aptamer 2010-01-07 doi: 10.3390/molecules15010215 Yoshihiko Nonaka Koji Sode Kazunori Ikebukuro http://www.mdpi.com/1420-3049/15/1/1/ The generation of single-stranded DNA (ssDNA) molecules plays a key role in the SELEX (Systematic Evolution of Ligands by EXponential enrichment) combinatorial chemistry process and numerous molecular biology techniques and applications, such as DNA sequencing, single-nucleotide polymorphism (SNP) analysis, DNA chips, DNA single-strand conformation polymorphism (SSCP) analysis and many other techniques. The purity and yield of ssDNA can affect the success of each application. This study compares the two ssDNA production methods, the strand separation by streptavidin-coated magnetic beads and alkaline denaturation and the lambda exonuclease digestion, in regard to the purity of generated ssDNA and the efficiency. Here, we demonstrate the considerable benefits of ssDNA production by lambda exonuclease digestion for in vitro selection of DNA aptamers. We believe that the generation of ssDNA aptamers using this method will greatly improve the success rate of SELEX experiments concerning the recovery of target-specific aptamers. http://www.mdpi.com/1420-3049/15/1/1/ Thu, 24 Dec 2009 00:00:00 CET Molecules 2009-12-24 15 1 Article 1 11 1420-3049 Upgrading SELEX Technology by Using Lambda Exonuclease Digestion for Single-Stranded DNA Generation 2009-12-24 doi: 10.3390/molecules15010001 Meltem Avci-Adali Angel Paul Nadj Wilhelm Gerhard Ziemer Hans Peter Wendel http://www.mdpi.com/1420-3049/14/12/5223/ Representative benzimidazopyrimidinones were previously reported to be intercalating antitumor agents. In this work, we used 2-substituted 4,10-dihydrobenzo [4,5]imidazo[1,2-α]pyriminin-4-ones for their diversification by regioselective alkylation. Under the conditions established, the alkylation gave 10-alkyl derivatives which permitted the parallel generation of a 500-member library of the title compounds. http://www.mdpi.com/1420-3049/14/12/5223/ Tue, 15 Dec 2009 00:00:00 CET Molecules 2009-12-15 14 12 Article 5223 5234 1420-3049 Generation of 500-Member Library of 10-Alkyl-2-R1,3-R2-4,10-Dihydrobenzo[4,5]imidazo[1,2-α]pyrimidin-4-ones 2009-12-15 doi: 10.3390/molecules14125223 Svetlana M. Sirko Nikolay Yu. Gorobets Vladimir I. Musatov Sergey M. Desenko http://www.mdpi.com/1420-3049/14/12/5179/ The pore forming properties of synthetic cecropin-melittin hybrid peptide (Acetyl-KWKLFKKIGAVLKVL-CONH2; CM15) were investigated by using photoreceptor rod outer segments (OS) isolated from frog retinae obtained by using the whole-cell configuration of the patch-clamp technique. CM15 was applied (and removed) to (from) the OS in ~50 ms with a computer-controlled microperfusion system. Once the main OS endogenous conductance was blocked with light, the OS membrane resistance was ≥1 GΩ, allowing high resolution, low-noise recordings. Different to alamethicines, CM15 produced voltage-independent membrane permeabilisation, repetitive peptide application caused a progressive permeabilisation increase, and no single-channel events were detected at low peptide concentrations. Collectively, these results indicate a toroidal mechanism of pore formation by CM15. http://www.mdpi.com/1420-3049/14/12/5179/ Fri, 11 Dec 2009 00:00:00 CET Molecules 2009-12-11 14 12 Article 5179 5188 1420-3049 Pore Forming Properties of Cecropin-Melittin Hybrid Peptide in a Natural Membrane 2009-12-11 doi: 10.3390/molecules14125179 Alberto Milani Mascia Benedusi Marco Aquila Giorgio Rispoli http://www.mdpi.com/1420-3049/14/11/4758/ Lignin composition (monolignol types of coniferyl, sinapyl or p-coumaryl alcohol) is causally related to biomass recalcitrance. We describe multiwavelength (220, 228, 240, 250, 260, 290, 295, 300, 310 or 320 nm) absorption spectroscopy of coniferyl alcohol and its laccase- or peroxidase-catalyzed products during real time kinetic, pseudokinetic and endpoint analyses, in optical turn on or turn off modes, under acidic or basic conditions. Reactions in microwell plates and 100 μL volumes demonstrated assay miniaturization and high throughput screening capabilities. Bathochromic and hypsochromic shifts along with hyperchromicity or hypochromicity accompanied enzymatic oxidations by laccase or peroxidase. The limits of detection and quantitation of coniferyl alcohol averaged 2.4 and 7.1 μM respectively, with linear trend lines over 3 to 4 orders of magnitude. Coniferyl alcohol oxidation was evident within 10 minutes or with 0.01 μg/mL laccase and 2 minutes or 0.001 μg/mL peroxidase. Detection limit improved to 1.0 μM coniferyl alcohol with Km of 978.7 ± 150.7 μM when examined at 260 nm following 30 minutes oxidation with 1.0 μg/mL laccase. Our assays utilized the intrinsic spectroscopic properties of coniferyl alcohol or its oxidation products for enabling detection, without requiring chemical synthesis or modification of the substrate or product(s). These studies facilitate lignin compositional analyses and augment pretreatment strategies for reducing biomass recalcitrance. http://www.mdpi.com/1420-3049/14/11/4758/ Mon, 23 Nov 2009 00:00:00 CET Molecules 2009-11-23 14 11 Article 4758 4778 1420-3049 Spectroscopic Analyses of the Biofuels-Critical Phytochemical Coniferyl Alcohol and Its Enzyme-Catalyzed Oxidation Products 2009-11-23 doi: 10.3390/molecules14114758 Komandoor Elayavalli Achyuthan Paul David Adams Blake Alexander Simmons Anup Kumar Singh http://www.mdpi.com/1420-3049/14/11/4707/ In the framework of the correlation between chemotaxonomy and chemical analysis studies, the chemical composition of the essential oils of four varieties of Thuja species cultivated in Poland − T. occidentalis ‘globosa’, T. occidentalis ‘aurea’, T. plicata and T. plicata ‘gracialis’ − were investigated by GC and GC-MS. Thirty-one compounds were identified from T. occidentalis ‘globosa’, representing 96.92% of the total oil; twenty-seven from T. occidentalis ‘aurea’ (94.34%); thirty-one from T. plicata (94.75%); and thirty compounds from T. plicata ‘gracialis’ (96.36%). The main constituents in all samples were the monoterpene ketones α- and β-thujone, fenchone and sabinene, as well as the diterpenes beyerene and rimuene.The chemosystematic value of the total ketone content of all samples (which varied from 54.30–69.18%) has been discussed and investigated. The constituents, beyerene and the mixture of α- and β-thujone, were isolated from the oils and tested against six Gram-positive and -negative bacteria and three pathogenic fungi. The oils of the two T. plicata species exhibited significant antimicrobial activity, while the mixture of α- and β-thujone showed very strong activity as well. http://www.mdpi.com/1420-3049/14/11/4707/ Thu, 19 Nov 2009 00:00:00 CET Molecules 2009-11-19 14 11 Article 4707 4715 1420-3049 Chemosystematic Value of the Essential Oil Composition of Thuja species Cultivated in Poland—Antimicrobial Activity 2009-11-19 doi: 10.3390/molecules14114707 Dimitroula Tsiri Konstantia Graikou Loretta Pobłocka-Olech Miroslawa Krauze-Baranowska Caroline Spyropoulos Ioanna Chinou http://www.mdpi.com/1420-3049/14/11/4546/ Evaluation of the genetic diversity among populations is an essential prerequisite for the preservation of endangered species. Thousands of new accessions are introduced into germplasm institutes each year, thereby necessitating assessment of their molecular diversity before elimination of the redundant genotypes. Of the protocols that facilitate the assessment of molecular diversity, SSRPs (simple sequence repeat polymorphisms) or microsatellite variation is the preferred system since it detects a large number of DNA polymorphisms with relatively simple technical complexity. The paucity of information on DNA sequences has limited their widespread utilization in the assessment of genetic diversity of minor or neglected crop species. However, recent advancements in DNA sequencing and PCR technologies in conjunction with sophisticated computer software have facilitated the development of SSRP markers in minor crops. This review examines the development and molecular nature of SSR markers, and their utilization in many aspects of plant genetics and ecology. http://www.mdpi.com/1420-3049/14/11/4546/ Tue, 10 Nov 2009 00:00:00 CET Molecules 2009-11-10 14 11 Review 4546 4569 1420-3049 Simple Sequence Repeat Polymorphisms (SSRPs) for Evaluation of Molecular Diversity and Germplasm Classification of Minor Crops 2009-11-10 doi: 10.3390/molecules14114546 Yong-Jin Park Ju Kyong Lee Nam-Soo Kim http://www.mdpi.com/1420-3049/14/9/3436/ MoStBioDat is a uniform data storage and extraction system with an extensive array of tools for structural similarity measures and pattern matching which is essential to facilitate the drug discovery process. Structure-based database screening has recently become a common and efficient technique in early stages of the drug development, shifting the emphasis from rational drug design into the probability domain of more or less random discovery. The virtual ligand screening (VLS), an approach based on high-throughput flexible docking, samples a virtually infinite molecular diversity of chemical libraries increasing the concentration of molecules with high binding affinity. The rapid process of subsequent examination of a large number of molecules in order to optimize the molecular diversity is an attractive alternative to the traditional methods of lead discovery. This paper presents the application of the MoStBioDat package not only as a data management platform but mainly in substructure searching. In particular, examples of the applications of MoStBioDat are discussed and analyzed. http://www.mdpi.com/1420-3049/14/9/3436/ Tue, 08 Sep 2009 00:00:00 CEST Molecules 2009-09-08 14 9 Article 3436 3445 1420-3049 The Use of MoStBioDat for Rapid Screening of Molecular Diversity 2009-09-08 doi: 10.3390/molecules14093436 Andrzej Bak Jaroslaw Polanski Agata Kurczyk http://www.mdpi.com/1420-3049/14/8/2717/ Volatile organic compounds of Amorpha fruticosa honey samples were isolated by headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE), followed by gas chromatography and mass spectrometry analyses (GC, GC-MS), in order to obtain complementary data for overall characterization of the honey aroma. The headspace of the honey was dominated by 2-phenylethanol (38.3–58.4%), while other major compounds were trans- and cis-linalool oxides, benzaldehyde and benzyl alcohol. 2-Phenylethanol (10.5–16.8%) and methyl syringate (5.8–8.2%) were the major compounds of ultrasonic solvent extracts, with an array of small percentages of linalool, benzene and benzoic acid derivatives, aliphatic hydrocarbons and alcohols, furan derivatives and others. The scavenging ability of the series of concentrations of the honey ultrasonic solvent extracts and the corresponding honey samples was tested by a DPPH (1,1-diphenyl-2-picrylhydrazyl) assay. Approximately 25 times lower concentration ranges (up to 2 g/L) of the extracts exhibited significantly higher free radical scavenging potential with respect to the honey samples. http://www.mdpi.com/1420-3049/14/8/2717/ Mon, 27 Jul 2009 00:00:00 CEST Molecules 2009-07-27 14 8 Article 2717 2728 1420-3049 Headspace, Volatile and Semi-Volatile Organic Compounds Diversity and Radical Scavenging Activity of Ultrasonic Solvent Extracts from Amorpha fruticosa Honey Samples 2009-07-27 doi: 10.3390/molecules14082717 Igor Jerković Zvonimir Marijanović Janja Kezić Mirko Gugić http://www.mdpi.com/1420-3049/14/5/1660/ Validation is a crucial aspect of quantitative structure–activity relationship (QSAR) modeling. The present paper shows that traditionally used validation parameters (leave-one-out Q2 for internal validation and predictive R2 for external validation) may be supplemented with two novel parameters rm2 and Rp2 for a stricter test of validation. The parameter rm2(overall) penalizes a model for large differences between observed and predicted values of the compounds of the whole set (considering both training and test sets) while the parameter Rp2 penalizes model R2 for large differences between determination coefficient of nonrandom model and square of mean correlation coefficient of random models in case of a randomization test. Two other variants of rm2 parameter, rm2(LOO) and rm2(test), penalize a model more strictly than Q2 and R2pred respectively. Three different data sets of moderate to large size have been used to develop multiple models in order to indicate the suitability of the novel parameters in QSAR studies. The results show that in many cases the developed models could satisfy the requirements of conventional parameters (Q2 and R2pred) but fail to achieve the required values for the novel parameters rm2 and Rp2. Moreover, these parameters also help in identifying the best models from among a set of comparable models. Thus, a test for these two parameters is suggested to be a more stringent requirement than the traditional validation parameters to decide acceptability of a predictive QSAR model, especially when a regulatory decision is involved. http://www.mdpi.com/1420-3049/14/5/1660/ Wed, 29 Apr 2009 00:00:00 CEST Molecules 2009-04-29 14 5 Article 1660 1701 1420-3049 On Two Novel Parameters for Validation of Predictive QSAR Models 2009-04-29 doi: 10.3390/molecules14051660 Partha Pratim Roy Somnath Paul Indrani Mitra Kunal Roy