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1.
J Biol Chem ; 295(24): 8195-8203, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32350113

RESUMO

The Ser/Thr protein kinase MELK (maternal embryonic leucine zipper kinase) has been considered an attractive therapeutic target for managing cancer since 2005. Studies using expression analysis have indicated that MELK expression is higher in numerous cancer cells and tissues than in their normal, nonneoplastic counterparts. Further, RNAi-mediated MELK depletion impairs proliferation of multiple cancers, including triple-negative breast cancer (TNBC), and these growth defects can be rescued with exogenous WT MELK, but not kinase-dead MELK complementation. Pharmacological MELK inhibition with OTS167 (alternatively called OTSSP167) and NVS-MELK8a, among other small molecules, also impairs cancer cell growth. These collective results led to MELK being classified as essential for cancer proliferation. More recently, in 2017, the proliferation of TNBC and other cancer cell lines was reported to be unaffected by genetic CRISPR/Cas9-mediated MELK deletion, calling into question the essentiality of this kinase in cancer. To date, the requirement of MELK in cancer remains controversial, and mechanisms underlying the disparate growth effects observed with RNAi, pharmacological inhibition, and CRISPR remain unclear. Our objective with this review is to highlight the evidence on both sides of this controversy, to provide commentary on the purported requirement of MELK in cancer, and to emphasize the need for continued elucidation of the functions of MELK.


Assuntos
Neoplasias/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proliferação de Células , Humanos , Modelos Biológicos , Neoplasias/patologia
2.
J Biol Chem ; 295(8): 2359-2374, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31896573

RESUMO

The maternal embryonic leucine zipper kinase (MELK) has been implicated in the regulation of cancer cell proliferation. RNAi-mediated MELK depletion impairs growth and causes G2/M arrest in numerous cancers, but the mechanisms underlying these effects are poorly understood. Furthermore, the MELK inhibitor OTSSP167 has recently been shown to have poor selectivity for MELK, complicating the use of this inhibitor as a tool compound to investigate MELK function. Here, using a cell-based proteomics technique called multiplexed kinase inhibitor beads/mass spectrometry (MIB/MS), we profiled the selectivity of two additional MELK inhibitors, NVS-MELK8a (8a) and HTH-01-091. Our results revealed that 8a is a highly selective MELK inhibitor, which we further used for functional studies. Resazurin and crystal violet assays indicated that 8a decreases triple-negative breast cancer cell viability, and immunoblotting revealed that impaired growth is due to perturbation of cell cycle progression rather than induction of apoptosis. Using double-thymidine synchronization and immunoblotting, we observed that MELK inhibition delays mitotic entry, which was associated with delayed activation of Aurora A, Aurora B, and cyclin-dependent kinase 1 (CDK1). Following this delay, cells entered and completed mitosis. Using live-cell microscopy of cells harboring fluorescent proliferating cell nuclear antigen, we confirmed that 8a significantly and dose-dependently lengthens G2 phase. Collectively, our results provide a rationale for using 8a as a tool compound for functional studies of MELK and indicate that MELK inhibition delays mitotic entry, likely via transient G2/M checkpoint activation.


Assuntos
Espectrometria de Massas , Mitose , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Histonas/metabolismo , Humanos , Mitose/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/patologia
3.
Mol Cell Proteomics ; 16(4 suppl 1): S263-S276, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28237943

RESUMO

Human cytomegalovirus (HCMV) is a significant cause of disease in immune-compromised adults and immune naïve newborns. No vaccine exists to prevent HCMV infection, and current antiviral therapies have toxic side effects that limit the duration and intensity of their use. There is thus an urgent need for new strategies to treat HCMV infection. Repurposing existing drugs as antivirals is an attractive approach to limit the time and cost of new antiviral drug development. Virus-induced changes in infected cells are often driven by changes in cellular kinase activity, which led us to hypothesize that defining the complement of kinases (the kinome), whose abundance or expression is altered during infection would identify existing kinase inhibitors that could be repurposed as new antivirals. To this end, we applied a kinase capture technique, multiplexed kinase inhibitor bead-mass spectrometry (MIB-MS) kinome, to quantitatively measure perturbations in >240 cellular kinases simultaneously in cells infected with a laboratory-adapted (AD169) or clinical (TB40E) HCMV strain. MIB-MS profiling identified time-dependent increases and decreases in MIB binding of multiple kinases including cell cycle kinases, receptor tyrosine kinases, and mitotic kinases. Based on the kinome data, we tested the antiviral effects of kinase inhibitors and other compounds, several of which are in clinical use or development. Using a novel flow cytometry-based assay and a fluorescent reporter virus we identified three compounds that inhibited HCMV replication with IC50 values of <1 µm, and at doses that were not toxic to uninfected cells. The most potent inhibitor of HCMV replication was OTSSP167 (IC50 <1.2 nm), a MELK inhibitor, blocked HCMV early gene expression and viral DNA accumulation, resulting in a >3 log decrease in virus replication. These results show the utility of MIB-MS kinome profiling for identifying existing kinase inhibitors that can potentially be repurposed as novel antiviral drugs.


Assuntos
Antivirais/farmacologia , Citomegalovirus/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Células Cultivadas , Citomegalovirus/metabolismo , Reposicionamento de Medicamentos , Humanos , Espectrometria de Massas/métodos , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
4.
J Cell Biochem ; 118(11): 3595-3606, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28464261

RESUMO

The kinase enzymes within a cell, known collectively as the kinome, play crucial roles in many signaling pathways, including survival, motility, differentiation, stress response, and many more. Aberrant signaling through kinase pathways is often linked to cancer, among other diseases. A major area of scientific research involves understanding the relationships between kinases, their targets, and how the kinome adapts to perturbations of the cellular system. This review will discuss many of the current and developing methods for studying kinase activity, and evaluate their applications, advantages, and disadvantages. J. Cell. Biochem. 118: 3595-3606, 2017. © 2017 Wiley Periodicals, Inc.


Assuntos
Proteínas de Neoplasias/análise , Neoplasias/enzimologia , Proteínas Quinases/análise , Animais , Humanos
5.
Biochim Biophys Acta Gen Subj ; 1864(4): 129507, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31881245

RESUMO

BACKGROUND: Imatinib mesylate (imatinib) is the first-line treatment for newly diagnosed chronic myeloid leukemia (CML) due to its remarkable hematologic and cytogenetic responses. We previously demonstrated that the imatinib-resistant CML cells (Myl-R) contained elevated Lyn activity and intracellular creatine pools compared to imatinib-sensitive Myl cells. METHODS: Stable isotope metabolic labeling, media creatine depletion, and Na+/K+-ATPase inhibitor experiments were performed to investigate the origin of creatine pools in Myl-R cells. Inhibition and shRNA knockdown were performed to investigate the specific role of Lyn in regulating the Na+/K+-ATPase and creatine uptake. RESULTS: Inhibition of the Na+/K+-ATPase pump (ouabain, digitoxin), depletion of extracellular creatine or inhibition of Lyn kinase (ponatinib, dasatinib), demonstrated that enhanced creatine accumulation in Myl-R cells was dependent on uptake from the growth media. Creatine uptake was independent of the Na+/creatine symporter (SLC6A8) expression or de novo synthesis. Western blot analyses showed that phosphorylation of the Na+/K+-ATPase on Tyr 10 (Y10), a known regulatory phosphorylation site, correlated with Lyn activity. Overexpression of Lyn in HEK293 cells increased Y10 phosphorylation (pY10) of the Na+/K+-ATPase, whereas Lyn inhibition or shRNA knockdown reduced Na+/K+-ATPase pY10 and decreased creatine accumulation in Myl-R cells. Consistent with enhanced uptake in Myl-R cells, cyclocreatine (Ccr), a cytotoxic creatine analog, caused significant loss of viability in Myl-R compared to Myl cells. CONCLUSIONS: These data suggest that Lyn can affect creatine uptake through Lyn-dependent phosphorylation and regulation of the Na+/K+-ATPase pump activity. GENERAL SIGNIFICANCE: These studies identify kinase regulation of the Na+/K+-ATPase as pivotal in regulating creatine uptake and energy metabolism in cells.


Assuntos
Antineoplásicos/farmacologia , Creatina/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Quinases da Família src/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células Tumorais Cultivadas
6.
ACS Chem Biol ; 14(5): 1020-1029, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31021596

RESUMO

ONC201 is a first-in-class imipridone molecule currently in clinical trials for the treatment of multiple cancers. Despite enormous clinical potential, the mechanism of action is controversial. To investigate the mechanism of ONC201 and identify compounds with improved potency, we tested a series of novel ONC201 analogues (TR compounds) for effects on cell viability and stress responses in breast and other cancer models. The TR compounds were found to be ∼50-100 times more potent at inhibiting cell proliferation and inducing the integrated stress response protein ATF4 than ONC201. Using immobilized TR compounds, we identified the human mitochondrial caseinolytic protease P (ClpP) as a specific binding protein by mass spectrometry. Affinity chromatography/drug competition assays showed that the TR compounds bound ClpP with ∼10-fold higher affinity compared to ONC201. Importantly, we found that the peptidase activity of recombinant ClpP was strongly activated by ONC201 and the TR compounds in a dose- and time-dependent manner with the TR compounds displaying a ∼10-100 fold increase in potency over ONC201. Finally, siRNA knockdown of ClpP in SUM159 cells reduced the response to ONC201 and the TR compounds, including induction of CHOP, loss of the mitochondrial proteins (TFAM, TUFM), and the cytostatic effects of these compounds. Thus, we report that ClpP directly binds ONC201 and the related TR compounds and is an important biological target for this class of molecules. Moreover, these studies provide, for the first time, a biochemical basis for the difference in efficacy between ONC201 and the TR compounds.


Assuntos
Antineoplásicos/farmacologia , Endopeptidase Clp/antagonistas & inibidores , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia de Afinidade , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Ativação Enzimática , Técnicas de Silenciamento de Genes , Compostos Heterocíclicos de 4 ou mais Anéis/química , Humanos , Imidazóis , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Piridinas , Pirimidinas
7.
SLAS Discov ; 23(8): 850-861, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29742358

RESUMO

Continuous exposure of a pancreatic cancer cell line MIA PaCa-2 (MiaS) to gemcitabine resulted in the formation of a gemcitabine-resistant subline (MiaR). In an effort to discover kinase inhibitors that inhibited MiaR growth, MiaR cells were exposed to kinase inhibitors (PKIS-1 library) in a 384-well screening format. Three compounds (UNC10112721A, UNC10112652A, and UNC10112793A) were identified that inhibited the growth of MiaR cells by more than 50% (at 50 nM). Two compounds (UNC10112721A and UNC10112652A) were classified as cyclin-dependent kinase (CDK) inhibitors, whereas UNC10112793A was reported to be a PLK inhibitor. Dose-response experiments supported the efficacy of these compounds to inhibit growth and increase apoptosis in 2D cultures of these cells. However, only UNC10112721A significantly inhibited the growth of 3D spheroids composed of MiaR cells and GFP-tagged cancer-associated fibroblasts. Multiplexed inhibitor bead (MIB)-mass spectrometry (MS) kinome competition experiments identified CDK9, CLK1-4, DYRK1A, and CSNK1 as major kinase targets for UNC10112721A in MiaR cells. Another CDK9 inhibitor (CDK-IN-2) replicated the growth inhibitory effects of UNC10112721A, whereas inhibitors against the CLK, DYRK, or CSNK1 kinases had no effect. In summary, these studies describe a coordinated approach to discover novel kinase inhibitors, evaluate their efficacy in 3D models, and define their specificity against the kinome.


Assuntos
Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais , Linhagem Celular Tumoral , Sobrevivência Celular , Desoxicitidina/química , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Conformação Molecular , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Relação Estrutura-Atividade , Fluxo de Trabalho , Gencitabina
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