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1.
J Am Chem Soc ; 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38918178

RESUMO

Metals are important cofactors in the metabolic processes of cyanobacteria, including photosynthesis, cellular respiration, DNA replication, and the biosynthesis of primary and secondary metabolites. In adaptation to the marine environment, cyanobacteria use metallophores to acquire trace metals when necessary as well as to reduce potential toxicity from excessive metal concentrations. Leptochelins A-C were identified as structurally novel metallophores from three geographically dispersed cyanobacteria of the genus Leptothoe. Determination of the complex structures of these metabolites presented numerous challenges, but they were ultimately solved using integrated data from NMR, mass spectrometry and deductions from the biosynthetic gene cluster. The leptochelins are comprised of halogenated linear NRPS-PKS hybrid products with multiple heterocycles that have potential for hexadentate and tetradentate coordination with metal ions. The genomes of the three leptochelin producers were sequenced, and retrobiosynthetic analysis revealed one candidate biosynthetic gene cluster (BGC) consistent with the structure of leptochelin. The putative BGC is highly homologous in all three Leptothoe strains, and all possess genetic signatures associated with metallophores. Postcolumn infusion of metals using an LC-MS metabolomics workflow performed with leptochelins A and B revealed promiscuous binding of iron, copper, cobalt, and zinc, with greatest preference for copper. Iron depletion and copper toxicity experiments support the hypothesis that leptochelin metallophores may play key ecological roles in iron acquisition and in copper detoxification. In addition, the leptochelins possess significant cytotoxicity against several cancer cell lines.

2.
ACS Infect Dis ; 9(6): 1245-1256, 2023 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-37163243

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 Vegetais
3.
ACS Omega ; 8(10): 9250-9256, 2023 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-36936303

RESUMO

National Cancer Institute (NCI) Program for Natural Product Discovery is a new initiative aimed at creating new technologies for natural product-based drug discovery. Here, we present the development of a neural network-based bioinformatics platform for visualization and analysis of natural product high-throughput screening data using the NCI's 60 human tumor cell anticancer drug screen. We demonstrate how the tool enables visualization of similar patterns of response that can be parsed both chemically and taxonomically, grouping NCI-60 biological profiles in one easy-to-use bioinformatics interface.

4.
ACS Chem Biol ; 15(8): 2125-2136, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32608972

RESUMO

Coibamide A (CbA) is a marine natural product with potent antiproliferative activity against human cancer cells and a unique selectivity profile. Despite promising antitumor activity, the mechanism of cytotoxicity and specific cellular target of CbA remain unknown. Here, we develop an optimized synthetic CbA photoaffinity probe (photo-CbA) and use it to demonstrate that CbA directly targets the Sec61α subunit of the Sec61 protein translocon. CbA binding to Sec61 results in broad substrate-nonselective inhibition of ER protein import and potent cytotoxicity against specific cancer cell lines. CbA targets a lumenal cavity of Sec61 that is partially shared with known Sec61 inhibitors, yet profiling against resistance conferring Sec61α mutations identified from human HCT116 cells suggests a distinct binding mode for CbA. Specifically, despite conferring strong resistance to all previously known Sec61 inhibitors, the Sec61α mutant R66I remains sensitive to CbA. A further unbiased screen for Sec61α resistance mutations identified the CbA-resistant mutation S71P, which confirms nonidentical binding sites for CbA and apratoxin A and supports the susceptibility of the Sec61 plug region for channel inhibition. Remarkably, CbA, apratoxin A, and ipomoeassin F do not display comparable patterns of potency and selectivity in the NCI60 panel of human cancer cell lines. Our work connecting CbA activity with selective prevention of secretory and membrane protein biogenesis by inhibition of Sec61 opens up possibilities for developing new Sec61 inhibitors with improved drug-like properties that are based on the coibamide pharmacophore.


Assuntos
Depsipeptídeos/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Canais de Translocação SEC/efeitos dos fármacos , Sítios de Ligação , Células Cultivadas , Depsipeptídeos/metabolismo , Humanos , Proteínas de Membrana/biossíntese , Marcadores de Fotoafinidade/química , Canais de Translocação SEC/metabolismo
5.
ACS Chem Biol ; 15(4): 1104-1114, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32223208

RESUMO

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 Unidos
6.
Nat Prod Rep ; 37(7): 893-918, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32186299

RESUMO

Covering: up to 2020The National Cancer Institute of the United States (NCI) has initiated a Cancer Moonshot program entitled the NCI Program for Natural Product Discovery. As part of this effort, the NCI is producing a library of 1 000 000 partially purified natural product fractions which are being plated into 384-well plates and provided to the research community free of charge. As the first 326 000 of these fractions have now been made available, this review seeks to describe the general methods used to collect organisms, extract those organisms, and create a prefractionated library. Importantly, this review also details both cell-based and cell-free bioassay methods and the adaptations necessary to those methods to productively screen natural product libraries. Finally, this review briefly describes post-screen dereplication and compound purification and scale up procedures which can efficiently identify active compounds and produce sufficient quantities of natural products for further pre-clinical development.


Assuntos
Produtos Biológicos/química , Bibliotecas de Moléculas Pequenas/química , Bioensaio/métodos , Produtos Biológicos/farmacologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Humanos
7.
Fitoterapia ; 137: 104285, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31386897

RESUMO

Botanical-based natural products are an important resource for medicinal drug discovery and continue to provide diverse pharmacophores with therapeutic potential against cancer and other human diseases. A prototype Traditional Chinese Medicine (TCM) plant extract library has been established at the US National Cancer Institute, which contains both the organic and aqueous extracts of 132 authenticated medicinal plant species that collectively represent the potential therapeutic contents of most commonly used TCM herbal prescriptions. This library is publicly available in 96- and 384- well plates for high throughput screening across a broad array of biological targets, as well as in larger quantities for isolation of active chemical ingredients. Herein, we present the methodology used to generate the library and the preliminary assessment of the anti-proliferative activity of this crude extract library in NCI-60 human cancer cell lines screen. Particularly, we report the chemical profiling and metabolome comparison analysis of four commonly used TCM plants, namely Brucea javanica, Dioscorea nipponica, Cynanchum atratum, and Salvia miltiorrhiza. Bioassay-guided isolation resulted in the identification of the active compounds, and different extraction methods were compared for their abilities to extract cytotoxic compounds and to concentrate biologically active natural products.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Plantas Medicinais/química , Antineoplásicos Fitogênicos/isolamento & purificação , Brucea/química , Linhagem Celular Tumoral , China , Cynanchum/química , Dioscorea/química , Descoberta de Drogas , Humanos , Medicina Tradicional Chinesa , National Cancer Institute (U.S.) , Compostos Fitoquímicos/isolamento & purificação , Salvia miltiorrhiza/química , Estados Unidos
8.
ACS Chem Biol ; 13(9): 2484-2497, 2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-29812901

RESUMO

The US National Cancer Institute's (NCI) Natural Product Repository is one of the world's largest, most diverse collections of natural products containing over 230,000 unique extracts derived from plant, marine, and microbial organisms that have been collected from biodiverse regions throughout the world. Importantly, this national resource is available to the research community for the screening of extracts and the isolation of bioactive natural products. However, despite the success of natural products in drug discovery, compatibility issues that make extracts challenging for liquid handling systems, extended timelines that complicate natural product-based drug discovery efforts and the presence of pan-assay interfering compounds have reduced enthusiasm for the high-throughput screening (HTS) of crude natural product extract libraries in targeted assay systems. To address these limitations, the NCI Program for Natural Product Discovery (NPNPD), a newly launched, national program to advance natural product discovery technologies and facilitate the discovery of structurally defined, validated lead molecules ready for translation will create a prefractionated library from over 125,000 natural product extracts with the aim of producing a publicly-accessible, HTS-amenable library of >1,000,000 fractions. This library, representing perhaps the largest accumulation of natural-product based fractions in the world, will be made available free of charge in 384-well plates for screening against all disease states in an effort to reinvigorate natural product-based drug discovery.


Assuntos
Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Descoberta de Drogas/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Triagem em Larga Escala/métodos , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/química , Produtos Biológicos/química , Humanos , National Cancer Institute (U.S.) , Estados Unidos , Fluxo de Trabalho
9.
J Nat Prod ; 76(9): 1781-8, 2013 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-24016099

RESUMO

Cultivation of the marine cyanobacterium Moorea producens, collected from the Nabq Mangroves in the Gulf of Aqaba (Red Sea), led to the isolation of new apratoxin analogues apratoxin H (1) and apratoxin A sulfoxide (2), together with the known apratoxins A-C, lyngbyabellin B, and hectochlorin. The absolute configuration of these new potent cytotoxins was determined by chemical degradation, MS, NMR, and CD spectroscopy. Apratoxin H (1) contains pipecolic acid in place of the proline residue present in apratoxin A, expanding the known suite of naturally occurring analogues that display amino acid substitutions within the final module of the apratoxin biosynthetic pathway. The oxidation site of apratoxin A sulfoxide (2) was deduced from MS fragmentation patterns and IR data, and 2 could not be generated experimentally by oxidation of apratoxin A. The cytotoxicity of 1 and 2 to human NCI-H460 lung cancer cells (IC50 = 3.4 and 89.9 nM, respectively) provides further insight into the structure-activity relationships in the apratoxin series. Phylogenetic analysis of the apratoxin-producing cyanobacterial strains belonging to the genus Moorea, coupled with the recently annotated apratoxin biosynthetic pathway, supports the notion that apratoxin production and structural diversity may be specific to their geographical niche.


Assuntos
Cianobactérias/química , Citotoxinas/isolamento & purificação , Citotoxinas/farmacologia , Depsipeptídeos/isolamento & purificação , Depsipeptídeos/farmacologia , Toxinas de Lyngbya/isolamento & purificação , Toxinas de Lyngbya/farmacologia , Safrol/análogos & derivados , Tiazóis/isolamento & purificação , Tiazóis/farmacologia , Citotoxinas/química , Depsipeptídeos/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Oceano Índico , Concentração Inibidora 50 , Lactonas/química , Lactonas/isolamento & purificação , Lactonas/farmacologia , Toxinas de Lyngbya/química , Biologia Marinha , Estrutura Molecular , Oxirredução , Ácidos Pipecólicos/química , Safrol/química , Safrol/isolamento & purificação , Safrol/farmacologia , Tiazóis/química
10.
J Nat Prod ; 74(8): 1677-85, 2011 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-21806012

RESUMO

Two new grassypeptolides and a lyngbyastatin analogue, together with the known dolastatin 12, have been isolated from field collections and laboratory cultures of the marine cyanobacterium Leptolyngbya sp. collected from the SS Thistlegorm shipwreck in the Red Sea. The overall stereostructures of grassypeptolides D (1) and E (2) and Ibu-epidemethoxylyngbyastatin 3 (3) were determined by a combination of 1D and 2D NMR experiments, MS analysis, Marfey's methodology, and HPLC-MS. Compounds 1 and 2 contain 2-methyl-3-aminobutyric acid and 2-aminobutyric acid, while biosynthetically distinct 3 contains 3-amino-2-methylhexanoic acid and the ß-keto amino acid 4-amino-2,2-dimethyl-3-oxopentanoic acid (Ibu). Grassypeptolides D (1) and E (2) showed significant cytotoxicity to HeLa (IC50 = 335 and 192 nM, respectively) and mouse neuro-2a blastoma cells (IC50 = 599 and 407 nM, respectively), in contrast to Ibu-epidemethoxylyngbyastatin 3 (neuro-2a cells, IC50 > 10 µM) and dolastatin 12 (neuro-2a cells, IC50 > 1 µM).


Assuntos
Antineoplásicos/isolamento & purificação , Cianobactérias/química , Depsipeptídeos/isolamento & purificação , Peptídeos Cíclicos/isolamento & purificação , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Oceano Índico , Concentração Inibidora 50 , Camundongos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia
11.
J Nat Prod ; 73(3): 489-99, 2010 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-20099811

RESUMO

Deep-sea hydrothermal vents are among the most extreme and dynamic environments on Earth. However, islands of highly dense and biologically diverse communities exist in the immediate vicinity of hydrothermal vent flows, in stark contrast to the surrounding bare seafloor. These communities comprise organisms with distinct metabolisms based on chemosynthesis and growth rates comparable to those from shallow water tropical environments, which have been rich sources of biologically active natural products. The geological setting and geochemical nature of deep-sea vents that impact the biogeography of vent organisms, chemosynthesis, and the known biological and metabolic diversity of Eukarya, Bacteria, and Archaea, including the handful of natural products isolated to date from deep-sea vent organisms, are considered here in an assessment of deep-sea hydrothermal vents as potential hot spots for natural products investigations. Of critical importance too are the logistics of collecting deep vent organisms, opportunities for re-collection considering the stability and longevity of vent sites, and the ability to culture natural product-producing deep vent organisms in the laboratory. New cost-effective technologies in deep-sea research and more advanced molecular techniques aimed at screening a more inclusive genetic assembly are poised to accelerate natural product discoveries from these microbial diversity hot spots.


Assuntos
Produtos Biológicos , Descoberta de Drogas , Temperatura Alta , Oceanos e Mares
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