RESUMO
Quinoid inhibitors of Cdc25B were designed based on the Linear Combination of Atomic Potentials (LCAP) methodology. In contrast to a published hypothesis, the biological activities and hydrogen peroxide generation in reducing media of three synthetic models did not correlate with the quinone half-wave potential, E(1/2).
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Quinonas/síntese química , Quinonas/farmacologia , Fosfatases cdc25/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Química Combinatória , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Humanos , Peróxido de Hidrogênio , Estrutura Molecular , Oxirredução/efeitos dos fármacos , Quinonas/química , Relação Estrutura-Atividade , Especificidade por Substrato , Fosfatases cdc25/metabolismoRESUMO
Mitogen-activated protein kinase phosphatase (MKP)-1 is a dual-specificity phosphatase that negatively regulates the activity of mitogen-activated kinases and that is overexpressed in human tumors. Contemporary studies suggest that induction of MKP-1 during chemotherapy may limit the efficacy of clinically used antineoplastic agents. Thus, MKP-1 is a rational target to enhance anticancer drug activity, but suitable small-molecule inhibitors of MKP-1 are currently unavailable. Here, we have used a high-content, multiparameter fluorescence-based chemical complementation assay for MKP activity in intact mammalian cells to evaluate the cellular MKP-1 and MKP-3 inhibitory activities of four previously described, quinone-based, dual-specificity phosphatase inhibitors, that is, NSC 672121, NSC 95397, DA-3003-1 (NSC 663284), and JUN-1111. All compounds induced formation of reactive oxygen species in mammalian cells, but only one (NSC 95397) inhibited cellular MKP-1 and MKP-3 with an IC(50) of 13 mumol/L. Chemical induction of MKP-1 by dexamethasone protected cells from paclitaxel-induced apoptosis but had no effect on NSC 95397. NSC 95397 phenocopied the effects of MKP-1 small inhibitory RNA by reversing the cytoprotective effects of dexamethasone in paclitaxel-treated cells. Isobologram analysis revealed synergism between paclitaxel and NSC 95397 only in the presence of dexamethasone. The data show the power of a well-defined cellular assay for identifying cell-active inhibitors of MKPs and support the hypothesis that small-molecule inhibitors of MKP-1 may be useful as antineoplastic agents under conditions of high MKP-1 expression.
Assuntos
Dexametasona/antagonistas & inibidores , Dexametasona/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fosfatases da Proteína Quinase Ativada por Mitógeno/antagonistas & inibidores , Naftoquinonas/farmacologia , Paclitaxel/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Fosfatase 1 de Especificidade Dupla/antagonistas & inibidores , Fosfatase 1 de Especificidade Dupla/metabolismo , Fosfatase 6 de Especificidade Dupla/antagonistas & inibidores , Fosfatase 6 de Especificidade Dupla/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Naftoquinonas/administração & dosagem , Paclitaxel/administração & dosagem , Quinonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais CultivadasRESUMO
We report here the miniaturization, development, and implementation of a homogeneous 384-well fluorescence intensity high-throughput screening (HTS) assay for identifying mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) dual-specificity phosphatase inhibitors. As part of the National Institutes of Health (NIH) Molecular Libraries Screening Center Network (MLSCN), the MKP-1 assay was utilized to screen an NIH diversity library of 65,239 compounds for inhibitors of MKP-1 activity at 10 microM and was also used to confirm the concentration dependence of active agents identified in the primary screen. We observed 100 (0.15%) compounds that inhibited MKP-1 in vitro by > or =50% at 10 microM in the primary assay, and 46 of the 100 compounds were confirmed as concentration-dependent inhibitors of MKP-1 with 50% inhibitory concentration (IC(50)) values of <50 microM; four exhibited IC(50) values <1.0 microM, six produced IC(50) values in the 1-10 microM range, and 36 produced IC(50) values in the 10-50 microM range. A clustering and classification analysis of the compound structures of the 46 confirmed MKP-1 inhibitors produced 29 singleton structures and seven clusters of related structures. Some MKP-1 inhibitors were members of structural classes or contained substructure pharmacophores that previously were reported to inhibit either MKP-1 or other protein tyrosine phosphatases, validating the HTS assay. Importantly, we have identified several attractive and novel MKP-1 inhibitor structures that warrant further investigation as potential probes to study the biology of MKP-1 and its role in controlling the amplitude and/or duration of MAPK signaling, cell survival, and tumor progression.