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1.
Assay Drug Dev Technol ; 11(8): 478-88, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24127661

RESUMO

The 90 kDa heat-shock protein (Hsp90) and other cochaperones allow for proper folding of nascent or misfolded polypeptides. Cancer cells exploit these chaperones by maintaining the stability of mutated and misfolded oncoproteins and allowing them to evade proteosomal degradation. Inhibiting Hsp90 is an attractive strategy for cancer therapy, as the concomitant degradation of multiple oncoproteins may lead to effective anti-neoplastic agents. Unfortunately, early clinical trials have been disappointing with N-terminal Hsp90 inhibitors, as it is unclear whether the problems that plague current Hsp90 inhibitors in clinical trials are related to on-target or off-target activity. One approach to overcome these pitfalls is to identify structurally diverse scaffolds that improve Hsp90 inhibitory activity in the cancer cell milieu. Utilizing a panel of cancer cell lines that express luciferase, we have designed an in-cell Hsp90-dependent luciferase refolding assay. The assay was optimized using previously identified Hsp90 inhibitors and experimental novobiocin analogues against prostate, colon, and lung cancer cell lines. This assay exhibits good interplate precision (% CV), a signal-to-noise ratio (S/N) of ≥7, and an approximate Z-factor ranging from 0.5 to 0.7. Novobiocin analogues that revealed activity in this assay were examined via western blot experiments for client protein degradation, a hallmark of Hsp90 inhibition. Subsequently, a pilot screen was conducted using the Prestwick library, and two compounds, biperiden and ethoxyquin, revealed significant activity. Here, we report the development of an in-cell Hsp90-dependent luciferase refolding assay that is amenable across cancer cell lines for the screening of inhibitors in their specific milieu.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Luciferases/química , Redobramento de Proteína/efeitos dos fármacos , Animais , Bioensaio , Western Blotting , Linhagem Celular Tumoral , Corantes , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Luciferases/antagonistas & inibidores , Novobiocina/análogos & derivados , Novobiocina/farmacologia , Desnaturação Proteica , Coelhos , Reticulócitos/efeitos dos fármacos , Reticulócitos/metabolismo , Rodaminas , Bibliotecas de Moléculas Pequenas
2.
J Med Chem ; 54(11): 3839-53, 2011 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-21553822

RESUMO

Development of the DNA gyrase inhibitor, novobiocin, into a selective Hsp90 inhibitor was accomplished through structural modifications to the amide side chain, coumarin ring, and sugar moiety. These species exhibit ∼700-fold improved anti-proliferative activity versus the natural product as evaluated by cellular efficacies against breast, colon, prostate, lung, and other cancer cell lines. Utilization of structure-activity relationships established for three novobiocin synthons produced optimized scaffolds, which manifest midnanomolar activity against a panel of cancer cell lines and serve as lead compounds that manifest their activities through Hsp90 inhibition.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Cumarínicos/química , Desenho de Fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Novobiocina/análogos & derivados , Antibacterianos/química , Antibacterianos/farmacologia , Antineoplásicos/química , Carboidratos/química , Linhagem Celular Tumoral , Cumarínicos/farmacologia , Feminino , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Masculino , Estrutura Molecular , Terapia de Alvo Molecular , Novobiocina/química , Novobiocina/farmacologia , Relação Estrutura-Atividade
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