RESUMO
The continuing emergence of antibiotic-resistant microbes highlights the need for the identification of new chemotypes with antimicrobial activity. One of the most prolific sources of antimicrobial molecules has been the systematic screening of natural product samples. The National Institute of Allergy and Infectious Diseases and the National Cancer Institute here report a large screen of 326,656 partially purified natural product fractions against a panel of four microbial pathogens, resulting in the identification of >3000 fractions with antifungal and/or antibacterial activity. A small sample of these active fractions was further purified and the chemical structures responsible for the antimicrobial activity were elucidated. The proof-of-concept study identified many different chemotypes, several of which have not previously been reported to have antimicrobial activity. The results show that there remain many unidentified antibiotic compounds from nature.
Assuntos
Anti-Infecciosos , Produtos Biológicos , Estados Unidos , Produtos Biológicos/farmacologia , Produtos Biológicos/química , National Cancer Institute (U.S.) , Anti-Infecciosos/farmacologia , Antibacterianos/farmacologia , Extratos VegetaisRESUMO
An automated, high-capacity, and high-throughput procedure for the rapid isolation and identification of biologically active natural products from a prefractionated library is presented. The semipreparative HPLC method uses 1 mg of the primary hit fraction and produces 22 subfractions in an assay-ready format. Following screening, all active fractions are analyzed by NMR, LCMS, and FTIR, and the active principle structural classes are elucidated. In the proof-of-concept study, we show the processes involved in generating the subfractions, the throughput of the structural elucidation work, as well as the ability to rapidly isolate and identify new and biologically active natural products. Overall, the rapid second-stage purification conserves extract mass, requires much less chemist time, and introduces knowledge of structure early in the isolation workflow.
Assuntos
Antineoplásicos/análise , Produtos Biológicos/análise , Ensaios de Triagem em Larga Escala/métodos , Bibliotecas de Moléculas Pequenas/análise , Animais , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Descoberta de Drogas , Gastrópodes/química , Haliclona/química , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , National Cancer Institute (U.S.) , Estudo de Prova de Conceito , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Bibliotecas de Moléculas Pequenas/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier , Estados UnidosRESUMO
Our initial discovery of 8-hydroxyquinoline inhibitors of BoNT/A and separation/testing of enantiomers of one of the more active leads indicated considerable flexibility in the binding site. We designed a limited study to investigate this flexibility and probe structure-activity relationships; utilizing the Betti reaction, a 36 compound matrix of quinolinol BoNT/A LC inhibitors was developed using three 8-hydroxyquinolines, three heteroaromatic amines, and four substituted benzaldehydes. This study has revealed some of the most effective quinolinol-based BoNT/A inhibitors to date, with 7 compounds displaying IC50 values ⩽1µM and 11 effective at ⩽2µM in an ex vivo assay.