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1.
Mol Cancer Ther ; 16(7): 1236-1245, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28468779

RESUMO

Cancer cells are highly reliant on NAD+-dependent processes, including glucose metabolism, calcium signaling, DNA repair, and regulation of gene expression. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ salvage from nicotinamide, has been investigated as a target for anticancer therapy. Known NAMPT inhibitors with potent cell activity are composed of a nitrogen-containing aromatic group, which is phosphoribosylated by the enzyme. Here, we identified two novel types of NAM-competitive NAMPT inhibitors, only one of which contains a modifiable, aromatic nitrogen that could be a phosphoribosyl acceptor. Both types of compound effectively deplete cellular NAD+, and subsequently ATP, and produce cell death when NAMPT is inhibited in cultured cells for more than 48 hours. Careful characterization of the kinetics of NAMPT inhibition in vivo allowed us to optimize dosing to produce sufficient NAD+ depletion over time that resulted in efficacy in an HCT116 xenograft model. Our data demonstrate that direct phosphoribosylation of competitive inhibitors by the NAMPT enzyme is not required for potent in vitro cellular activity or in vivo antitumor efficacy. Mol Cancer Ther; 16(7); 1236-45. ©2017 AACR.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Citocinas/antagonistas & inibidores , Inibidores Enzimáticos/administração & dosagem , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Sinalização do Cálcio/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Citocinas/genética , Reparo do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Camundongos , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Ensaios Antitumorais Modelo de Xenoenxerto
2.
J Biomol Screen ; 17(8): 1005-17, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22706350

RESUMO

Efficient elucidation of the biological mechanism of action of novel compounds remains a major bottleneck in the drug discovery process. To address this need in the area of oncology, we report the development of a multiparametric high-content screening assay panel at the level of single cells to dramatically accelerate understanding the mechanism of action of cell growth-inhibiting compounds on a large scale. Our approach is based on measuring 10 established end points associated with mitochondrial apoptosis, cell cycle disruption, DNA damage, and cellular morphological changes in the same experiment, across three multiparametric assays. The data from all of the measurements taken together are expected to help increase our current understanding of target protein functions, constrain the list of possible targets for compounds identified using phenotypic screens, and identify off-target effects. We have also developed novel data visualization and phenotypic classification approaches for detailed interpretation of individual compound effects and navigation of large collections of multiparametric cellular responses. We expect this general approach to be valuable for drug discovery across multiple therapeutic areas.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromos c/metabolismo , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Mitocôndrias/efeitos dos fármacos
3.
J Biomol Screen ; 13(6): 527-37, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18566484

RESUMO

Prioritization of compounds based on human hepatotoxicity potential is currently a key unmet need in drug discovery, as it can become a major problem for several lead compounds in later stages of the drug discovery pipeline. The authors report the validation and implementation of a high-content multiparametric cytotoxicity assay based on simultaneous measurement of 8 key cell health indicators associated with nuclear morphology, plasma membrane integrity, mitochondrial function, and cell proliferation. Compounds are prioritized by (a) computing an in vitro safety margin using the minimum cytotoxic concentration (IC(20)) across all 8 indicators and cell-based efficacy data and (b) using the minimal cytotoxic concentration alone to take into account concentration of drug in tissues. Feasibility data using selected compounds, including quinolone antibiotics, thiazolidinediones, and statins, suggest the viability of this approach. To increase overall throughput of compound prioritization, the authors have identified the higher throughput, plate reader-based CyQUANT assay that is similar to the high-content screening (HCS) assay in sensitivity of measuring inhibition of cell proliferation. It is expected that the phenotypic output from the multiparametric HCS assay in combination with other highly sensitive approaches, such as microarray-based expression analysis of toxic signatures, will contribute to a better understanding and predictivity of human hepatotoxicity potential.


Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Hepatócitos/efeitos dos fármacos , Testes de Toxicidade/métodos , Xenobióticos/toxicidade , Bioensaio , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/classificação , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Concentração Inibidora 50 , Neoplasias Hepáticas/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Xenobióticos/classificação
4.
J Biomol Screen ; 12(7): 938-45, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17942786

RESUMO

A 384-well soft agar assay was developed to identify potential novel anticancer compounds. Normally used to detect cell transformation, the assay is used here to quantitate cell proliferation in a 3-dimensional (3-D) anchorage-independent format. HCC827 cells, which are highly sensitive to epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors, were used to develop the method and a set of 9600 compounds used to validate the assay. Results were compared to a monolayer assay using the same compound set. The assay provides a robust method to discover compounds that could be missed using traditional monolayer formats.


Assuntos
Antineoplásicos/química , Inibidores Enzimáticos/química , Ágar , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Humanos
5.
Antimicrob Agents Chemother ; 47(12): 3831-9, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14638491

RESUMO

We report the discovery and characterization of a novel ribosome inhibitor (NRI) class that exhibits selective and broad-spectrum antibacterial activity. Compounds in this class inhibit growth of many gram-positive and gram-negative bacteria, including the common respiratory pathogens Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Moraxella catarrhalis, and are nontoxic to human cell lines. The first NRI was discovered in a high-throughput screen designed to identify inhibitors of cell-free translation in extracts from S. pneumoniae. The chemical structure of the NRI class is related to antibacterial quinolones, but, interestingly, the differences in structure are sufficient to completely alter the biochemical and intracellular mechanisms of action. Expression array studies and analysis of NRI-resistant mutants confirm this difference in intracellular mechanism and provide evidence that the NRIs inhibit bacterial protein synthesis by inhibiting ribosomes. Furthermore, compounds in the NRI series appear to inhibit bacterial ribosomes by a new mechanism, because NRI-resistant strains are not cross-resistant to other ribosome inhibitors, such as macrolides, chloramphenicol, tetracycline, aminoglycosides, or oxazolidinones. The NRIs are a promising new antibacterial class with activity against all major drug-resistant respiratory pathogens.


Assuntos
Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Aminoacil-tRNA Sintetases/genética , Animais , Bacillus subtilis/efeitos dos fármacos , DNA Girase/genética , DNA Girase/metabolismo , Desenho de Fármacos , Farmacorresistência Bacteriana , Escherichia coli/enzimologia , Escherichia coli/genética , Células Eucarióticas/metabolismo , Genes Reporter/genética , Indicadores e Reagentes , Luciferases/genética , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Coelhos , Proteínas Ribossômicas/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Fatores de Transcrição/genética , Transcrição Gênica , beta-Galactosidase/genética
6.
J Biomol Screen ; 7(5): 429-32, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14599358

RESUMO

A strategy is described for the development of high-throughput screening assays against targets of unknown function that involves the use of nuclear magnetic resonance (NMR) spectroscopy. Using this approach, molecules that bind to the protein target are identified from an NMR-based screen of a library of substrates, cofactors, and other compounds that are known to bind to many proteins and enzymes. Once a ligand has been discovered, a fluorescent or radiolabeled analog of the ligand is synthesized that can be used in a high-throughput screen. The approach is illustrated in the development of a high-throughput screening assay against HI-0033, a conserved protein from Haemophilus influenzae whose function is currently unknown. Adenosine was found to bind to HI-0033 by NMR, and fluorescent analogs were rapidly identified that bound to HI-0033 in the submicromolar range. Using these fluorescent compounds, a fluorescence polarization assay was developed that is suitable for high-throughput screening and obtaining detailed structure-activity relationships for lead optimization.


Assuntos
Proteínas de Bactérias/análise , Bioensaio/métodos , Técnicas de Química Combinatória/métodos , Espectroscopia de Ressonância Magnética/métodos , Proteínas de Bactérias/metabolismo , Haemophilus influenzae/química , Haemophilus influenzae/metabolismo , Ligantes , Espectrometria de Fluorescência/métodos
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