RESUMO
Pyrrole-containing natural products form a large group of structurally diverse compounds that occur in both terrestrial and marine organisms. In the present study the formation of trideuteromethylated artifacts of pyrrole-containing natural products was investigated, focusing on the discorhabdins. Three deuterated discorhabdins, 1, 3, and 5, were identified to be isolation procedure artifacts caused by the presence of DMSO-d6 during NMR sample preparation and handling. Three additional semisynthetic derivatives, 7-9, were made during the investigation of the mechanism of formation, which was shown to be driven by trideuteromethyl radicals in the presence of water, methanol, TFA, and traces of iron in the deuterated solvent. Generation of trideuteromethylated artifacts was also confirmed for other classes of pyrrole-containing metabolites, namely, makaluvamines, tambjamines, and dibromotryptamines, which had also been dissolved in DMSO-d6 during the structure elucidation process. Semisynthetic discorhabdins were assessed for antiproliferative activity against a panel of human tumor cell lines, and 14-trideuteromethyldiscorhabdin L (3) averaged low micromolar potency.
Assuntos
Produtos Biológicos , Dimetil Sulfóxido , Humanos , Dimetil Sulfóxido/química , Pirróis/química , Produtos Biológicos/farmacologia , Artefatos , Solventes/químicaRESUMO
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
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.
RESUMO
Modifications at the glycolate moiety of englerin A were made to explore variations at the most sensitive site on the molecule for activity in the NCI 60 screen, wherein englerin A is highly potent and selective for renal cancer cells. Replacement of the glycolate by other functionalities as well as esterification of the glycolate hydroxyl yielded compounds which displayed excellent selectivity and potency compared with the natural product. TRPC4/5 ion channel experiments with five compounds showed delayed or reduced agonism with TRPC5, at much higher concentrations than englerin A. With TRPC4, these compounds all had no effect at 10 µM. The same compounds were not detectable in mouse serum after a single oral dose of 12.5 mg/kg. At 100 mg/kg p.o., no toxicity was observed, and blood levels were barely detectable. Intravenous administration led to toxicity but at substantially lower doses than for englerin A.
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 UnidosRESUMO
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 UnidosRESUMO
Two new cassaine-type diterpenoids, namely erythrofordins D (1) and E (2), sourced from a Cameroon collection of Erythrophleum suaveolens were isolated and assessed for anti-tumor activity. In the NCI-60 cancer cell assay, erythrofordins D (1) and E (2) were found to be cytotoxic in the low micro molar ranges with a mean GI50 value of 2.45 and 0.71⯵M, mean TGI value of 9.77 and 2.29⯵M, and a mean LC50 of 26.92 and 11.48⯵M for 1 and 2 respectively. Using the COMPARE algorithm, the new compounds were found to have similar NCI-60 response profiles to the known cardiac glycosides hyrcanoside and strophanthin. In addition, in an assay examining the viability and contractile function in human cardiomyocytes derived from induced pluripotent stem-cells, erythrofordins showed cardiotoxicity effects at concentrations as low as 0.03⯵g/mL.
Assuntos
Caesalpinia/química , Diterpenos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Diterpenos/química , Diterpenos/isolamento & purificação , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
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 TrabalhoRESUMO
Several basic leucine zipper (B-ZIP) transcription factors have been implicated in cancer, substance abuse, and other pathological conditions. We previously identified arylstibonic acids that bind to B-ZIP proteins and inhibit their interaction with DNA. In this study, we used electrophoretic mobility shift assay to analyze 46 arylstibonic acids for their activity to disrupt the DNA binding of three B-ZIP [CCAAT/enhancer-binding protein α, cyclic AMP-response element-binding protein (CREB), and vitellogenin gene-binding protein (VBP)] and two basic helix-loop-helix leucine zipper (B-HLH-ZIP) [USF (upstream stimulating factor) and Mitf] proteins. Twenty-five arylstibonic acids showed activity at micromolar concentrations. The most active compound, P6981 [2-(3-stibonophenyl)malonic acid], had half-maximal inhibition at ~5 nM for CREB. Circular dichroism thermal denaturation studies indicated that P6981 binds both the B-ZIP domain and the leucine zipper. The crystal structure of an arylstibonic acid, NSC13778, bound to the VBP leucine zipper identified electrostatic interactions between both the stibonic and carboxylic acid groups of NSC13778 [(E)-3-(3-stibonophenyl)acrylic acid] and arginine side chains of VBP, which is also involved in interhelical salt bridges in the leucine zipper. P6981 induced GFP-B-ZIP chimeric proteins to partially localize to the cytoplasm, demonstrating that it is active in cells. P6981 inhibited the growth of a patient-derived clear cell sarcoma cell line whose oncogenic potential is driven by a chimeric protein EWS-ATF1 (Ewing's sarcoma protein-activating transcription factor 1), which contains the DNA binding domain of ATF1, a B-ZIP protein. NSC13778 inhibited the growth of xenografted clear cell sarcoma, and no toxicity was observed. These experiments suggest that antimony containing arylstibonic acids are promising leads for suppression of DNA binding activities of B-ZIP and B-HLH-ZIP transcription factors.
Assuntos
Ácidos não Carboxílicos/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , DNA/metabolismo , Compostos Organometálicos/farmacologia , Ácidos não Carboxílicos/química , Animais , Antimônio/química , Fatores de Transcrição de Zíper de Leucina Básica/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/antagonistas & inibidores , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cinamatos/química , Dicroísmo Circular , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Zíper de Leucina , Camundongos , Camundongos SCID , Modelos Moleculares , Estrutura Molecular , Compostos Organometálicos/química , Desnaturação Proteica , Transplante Heterólogo , Vitelogeninas/genéticaRESUMO
The chlorinated englerins (3-9) were isolated from Phyllanthus engleri and shown to selectively inhibit the growth of renal cancer cells. The compounds were shown to be extraction artifacts produced by exposure to chloroform decomposition products during their isolation. The most active compound, 3, was synthesized from englerin A (1).
Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Hidrocarbonetos Clorados/isolamento & purificação , Hidrocarbonetos Clorados/farmacologia , Phyllanthus/química , Sesquiterpenos de Guaiano/isolamento & purificação , Sesquiterpenos de Guaiano/farmacologia , Antineoplásicos Fitogênicos/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Hidrocarbonetos Clorados/química , Rim , Neoplasias Renais/tratamento farmacológico , Estrutura Molecular , National Cancer Institute (U.S.) , Sesquiterpenos de Guaiano/química , Estereoisomerismo , Tanzânia , Estados UnidosRESUMO
Human topoisomerase IB (hTopo) forms a covalent phosphotyrosyl linkage with the DNA backbone, and controls genomic DNA topology by relaxing DNA supercoils during the processes of DNA replication, transcription, chromosome condensation and decondensation. The essential role of hTopo in these processes has made it a preeminent anticancer drug target. We have screened a small library of arylstibonic acids for their effects on plasmid supercoil relaxation catalyzed by hTopo. Despite the similar structures of the library compounds, some compounds were found to be effective competitive inhibitors, and others, nonessential activators. Some arylstibonic acids show selectivity in their action against hTopo and the related enzyme from poxvirus (vTopo). Structure-activity relationships and structural modeling suggest that competitive inhibition may result from positioning of the negatively charged stibonic acid and carboxylate groups of the inhibitors into DNA phosphate binding pockets on hTopo. The hTopo activators act by a surprising allosteric mechanism without interfering with DNA binding or binding of the widely used hTopo poison camptothecin. Arylstibonic acid competitive inhibitors may become useful small molecules for elucidating the cellular functions of hTopo.
Assuntos
Ácidos/química , Ácidos/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores da Topoisomerase I , Camptotecina/química , Camptotecina/farmacologia , DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Estrutura Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Human apurinic/apyrimidinic endonuclease (Ape1) plays an important role by processing the >10,000 highly toxic abasic sites generated in the genome of each cell every day. Ape1 has recently emerged as a target for inhibition, in that its overexpression in tumors has been linked with poor response to both radiation and chemotherapy and lower overall patient survival. Inhibition of Ape1 using siRNA or the expression of a dominant-negative form of the protein has been shown to sensitize cells to DNA-damaging agents, including various chemotherapeutic agents. However, potent small-molecule inhibitors of Ape1 remain to be found. To this end, we screened Ape1 against the NCI Diversity Set of small molecules and discovered aromatic nitroso, carboxylate, sulfonamide, and arylstibonic acid compounds with micromolar affinities for the protein. A further screen of a 37-compound arylstibonic acid sublibrary identified ligands with IC(50) values in the range of 4 to 300 nM. The negatively charged stibonic acids act by a partial-mixed mode and probably serve as DNA phosphate mimics. These compounds provide a useful scaffold for development of chemotherapeutic agents against Ape1.
Assuntos
Antimônio/farmacologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/antagonistas & inibidores , Endonucleases/antagonistas & inibidores , Antimônio/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , DNA/análise , DNA/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/isolamento & purificação , Humanos , Concentração Inibidora 50 , Cinética , Ligantes , Modelos Químicos , Osteossarcoma/tratamento farmacológico , Osteossarcoma/enzimologia , Osteossarcoma/genética , Osteossarcoma/patologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/isolamento & purificação , Eletricidade Estática , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The crucial functions of HIV-1 nucleocapsid-p7 protein (NC-p7) at different stages of HIV replication are dependent on its nucleic acid binding properties. In this study, a search has been made to identify antagonists of the interaction between NC-p7 and d(TG)(4). A chemical library of approximately 2000 small molecules (the NCI Diversity Set) was screened, of the 26 active inhibitors that were identified, five contained a xanthenyl ring structure. Further analysis of 63 structurally related compounds led to the identification of 2,3,4,5-tetrachloro-6-(4('),5('),6(')-trihydroxy-3(')-oxo-3H-xanthen-9(')-yl)benzoic acid, which binds to NC-p7 stoichiometrically. This compound exerted a significant anti-HIV activity in vitro with an IC(50) of 16.6+/-4.3 microM (means+/-SD). Synthetic variants lacking the two hydroxyls at positions 4(') and 5(') in the xanthenyl ring system failed to bind NC-p7 and showed significantly less protection against HIV infection. Molecular modeling predicts that these hydroxyl groups would bind to the amide nitrogen of Gly(35) with other contacts at the carbonyl oxygens of Gly(40) and Lys(33).