RESUMO
Codification of DNA Encoded Libraries (DELs) is critical for successful ligand identification of molecules that bind a protein of interest (POI). There are different encoding strategies that permit, for instance, the customization of a DEL for testing single or dual pharmacophores (single strand DNA) or for producing and screening large diversity libraries of small molecules (double strand DNA). Both approaches challenges, either from the synthetic and encoding point of view, or from the selection methodology to be utilized for the screening. The Head-Piece contains the DNA sequence that is attached to a chemical compound, allowing the encoding of each molecule with a unique DNA tag. Designing the Head-Piece for a DNA-encoded library involves careful consideration of several key aspects including DNA barcode identity, sequence length and attachment chemistry. Here we describe a double stranded DNA versatile Head-Piece that can be used for the generation of single or dual pharmacophore libraries, but also shows other advanced DEL functionalities, stability and enlarged encoding capacity.
Assuntos
Descoberta de Drogas , Bibliotecas de Moléculas Pequenas , Descoberta de Drogas/métodos , Bibliotecas de Moléculas Pequenas/química , DNA/química , Biblioteca Gênica , DNA de Cadeia SimplesRESUMO
The output of an affinity selection screening results in a huge amount of valuable data that, after conducting the appropriate analysis, lead to the correct identification of the compounds enriched in the target of interest. The approach chosen to perform these analyses has become a key step in the development of a successful DNA Encoded Library platform. In this paper, we describe the combination of High Performance Liquid Chromatography purification during the library production with the Next Generation Sequencing analysis of the libraries to assess the yield of the chemical reactions prior to the affinity selection. This process allows us, apart from achieving higher quality libraries, to enable a normalization analysis of the affinity selection output, thus minimizing the bias induced by the chemical yield of each reaction as a misleading factor within the analysis and subsequent compound short-listing for off-DNA synthesis.
Assuntos
DNA/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Bibliotecas de Moléculas Pequenas/farmacologia , Cromatografia Líquida de Alta Pressão , DNA/síntese química , DNA/química , Relação Dose-Resposta a Droga , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
Conformational analysis in solution of beta-secretase inhibitors 1 and 2 by NMR spectroscopy reveals that the hydroxyethylene isostere, an apparently flexible fragment widely used as a scissile bond replacement in aspartic protease inhibitors, exists in one predominant conformation in solution. This preferred conformation is similar to that adopted by the hydroxyethylene core of 1 in complex with beta-secretase and that adopted by hydroxyethylene cores of related compounds when bound to aspartic proteases, indicating that this structural unit is preorganized in solution.
Assuntos
Dipeptídeos/química , Endopeptidases/química , Inibidores de Proteases/química , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Cristalografia por Raios X , Humanos , Leucina/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Fenilalanina/química , Soluções , Valina/químicaRESUMO
In the course of our screening efforts to discover small molecules as selective inhibitors of vacuolar-type H+-ATPase of Saccharomyces cerevisiae, we have identified eight active destruxins, 1-8, from the fungus Metarhizium anisopliae. The structures were elucidated by extensive 1D- and 2D-NMR spectroscopy, and MS spectrometry. One of these compounds, 8, a regioisomer of chlorohydrin destruxin E (7), is a new destruxin.
Assuntos
Depsipeptídeos/metabolismo , Depsipeptídeos/farmacologia , Saccharomyces cerevisiae/enzimologia , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Animais , Galinhas , Relação Dose-Resposta a Droga , Fermentação , Concentração Inibidora 50 , Metarhizium/metabolismo , Estrutura Molecular , Osteoclastos , Fatores de Tempo , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
In the course of a search for small-molecule inhibitors of 5-hydroxytryptamine receptors we have identified a novel ergoline derivative (1) from the fungal culture of Dicyma sp. This compound has a pK(i) of 10.2 versus the 5-hydroxytryptamine(1A) receptor subtype. The structure was elucidated by extensive NMR spectroscopy and mass spectrometry.
Assuntos
Ascomicetos/química , Ergolinas/isolamento & purificação , Alcaloides de Claviceps/isolamento & purificação , Receptor 5-HT1A de Serotonina/efeitos dos fármacos , Antagonistas da Serotonina/isolamento & purificação , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ergolinas/química , Ergolinas/farmacologia , Alcaloides de Claviceps/química , Alcaloides de Claviceps/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Ensaio Radioligante , Receptor 5-HT1A de Serotonina/metabolismo , Antagonistas da Serotonina/química , Antagonistas da Serotonina/farmacologiaRESUMO
Ammonium hydrogencarbonate buffer has been found to be especially useful for high-pH HPLC analysis of samples from both combinatorial and medicinal chemistry sources. Satisfactory results were obtained by the standard diode array, evaporative light-scattering, and MS detection by using this buffer at a concentration of 10 mM. From a practical standpoint, ammonium hydrogencarbonate is an ideal buffer for chromatographers since it provides excellent chromatographic behaviour and reproducible separation. In addition to this, its volatility makes it an essential tool for rapid LC-MS product identification. Ammonium hydrogencarbonate was tested for a number of drug-like compounds analysed as mixtures, and data obtained were compared to those from the classical and MS-friendly buffers widely used by chromatographers: trifluoroacetic and formic acids. The results of this study revealed the suitability of this buffer for routine HPLC application in research laboratories.
Assuntos
Soluções Tampão , Carbonatos/química , Cromatografia Líquida de Alta Pressão/métodos , Concentração de Íons de Hidrogênio , Espectrometria de Massas/métodosRESUMO
Novel normal-phase gradient systems have been employed for fast high-throughput chiral analyses of Discovery compounds in our research laboratories in Eli Lilly and Company. In this report, we describe an automated screening approach based on gradient elution, in order to achieve accurate enantiomeric excess determinations, and chiral separations when needed, in the shortest possible timeframe. Baseline resolution of enantiomers has been obtained for over 85% of the samples so tested. For the remaining cases, complete enantioseparation by isocratic optimisation is generally achieved in a single shot. This technique has been proven to be robust and is now standard operating procedure at our analytical research laboratories.
Assuntos
Cromatografia Líquida/métodos , Química Farmacêutica , EstereoisomerismoRESUMO
The combination of HPLC and MS has become the most valuable analytical tool to determine the identity and purity of a drug substance in the drug discovery arena over the past decade. This article describes different LC/MS configurations and their broad applicability to meet the fundamental analytical requirements involved in discovering new drugs. In addition, the value of chemiluminescence nitrogen detection for absolute purity determination and the convenience of CE as an orthogonal separation technique to HPLC are also discussed. In summary, LC/MS-based methodologies that implicate automation, various levels of throughput and open access systems have proved to be an integral part of the drug discovery process. As a result, the paradigm of high-quality/-quantity information is fulfilled in a timely fashion.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Preparações Farmacêuticas/química , Preparações Farmacêuticas/isolamento & purificação , Cafeína/química , Cafeína/isolamento & purificação , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/instrumentação , História do Século XX , História do Século XXI , Espectrometria de Massas/instrumentação , Preparações Farmacêuticas/históriaRESUMO
The combination of 1D and 2D high-resolution magic angle spinning NMR experiments led to the assignment of the proton and carbon resonances for several disubstituted benzoates bound to a polystyrene resin through a Wang linker. It is shown that the signal corresponding to the methylene protons of the linker can be utilized to monitor the solid-phase reactions and determine the loading of the compounds on the resin.
RESUMO
This work describes the discovery and characterization of a novel series of tricyclic natural product-derived metallo-beta-lactamase inhibitors. Natural product screening of the Bacillus cereus II enzyme identified an extract from a strain of Chaetomium funicola with inhibitory activity against metallo-beta-lactamases. SB236050, SB238569, and SB236049 were successfully extracted and purified from this extract. The most active of these compounds was SB238569, which possessed K(i) values of 79, 17, and 3.4 microM for the Bacillus cereus II, Pseudomonas aeruginosa IMP-1, and Bacteroides fragilis CfiA metallo-beta-lactamases, respectively, yet none of the compounds exhibited any inhibitory activity against the Stenotrophomonas maltophilia L-1 metallo-beta-lactamase (50% inhibitory concentration > 1,000 microM). The lack of activity against angiotensin-converting enzyme and serine beta-lactamases demonstrated the selective nature of these compounds. The crystal structure of SB236050 complexed in the active site of CfiA has been obtained to a resolution of 2.5 A. SB236050 exhibits key polar interactions with Lys184, Asn193, and His162 and a stacking interaction with the indole ring of Trp49 in the flap, which is in the closed conformation over the active site groove. SB236050 and SB238569 also demonstrate good antibacterial synergy with meropenem. Eight micrograms of SB236050 per ml gave rise to an eightfold drop in the MIC of meropenem for two clinical isolates of B. fragilis producing CfiA, making these strains sensitive to meropenem (MIC < or = 4 microg/ml). Consequently, this series of metallo-beta-lactamase inhibitors exhibit the most promising antibacterial synergy activity so far observed against organisms producing metallo-beta-lactamases.