RESUMO
The accurate experimental estimation of protein-ligand systems' residence time (τ) has become very relevant in drug design projects due to its importance in the last stages of refinement of the drug's pharmacodynamics and pharmacokinetics. It is now well-known that it is not sufficient to estimate the affinity of a protein-drug complex in the thermodynamic equilibrium process in in vitro experiments (closed systems), where the concentrations of the drug and protein remain constant. On the contrary, it is mandatory to consider the conformational dynamics of the system in terms of the binding and unbinding processes between protein and drugs in in vivo experiments (open systems), where their concentrations are in constant flux. This last model has been proven to dictate much of several drugs' pharmacological activities in vivo. At the atomistic level, molecular dynamics simulations can explain why some drugs are more effective than others or unveil the molecular aspects that make some drugs work better in one molecular target. Here, the protein kinases Aurora A/B, complexed with its inhibitor Danusertib, were studied using conventional and enhanced molecular dynamics (MD) simulations to estimate the dissociation paths and, therefore, the computational τ values and their comparison with experimental ones. Using classical molecular dynamics (cMD), three differential residues within the Aurora A/B active site, which seems to play an essential role in the observed experimental Danusertib's residence time against these kinases, were characterized. Then, using WT-MetaD, the relative Danusertib's residence times against Aurora A/B kinases were measured in a nanosecond time scale and were compared to those τ values observed experimentally. In addition, the potential dissociation paths of Danusertib in Aurora A and B were characterized, and differences that might be explained by the differential residues in the enzyme's active sites were found. In perspective, it is expected that this computational protocol can be applied to other protein-ligand complexes to understand, at the molecular level, the differences in residence times and amino acids that may contribute to it.
Assuntos
Aurora Quinase A , Aurora Quinase B , Simulação de Dinâmica Molecular , Aurora Quinase B/metabolismo , Aurora Quinase B/química , Aurora Quinase B/antagonistas & inibidores , Aurora Quinase A/metabolismo , Aurora Quinase A/química , Aurora Quinase A/antagonistas & inibidores , Pirazóis/química , Pirazóis/metabolismo , Conformação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/metabolismo , Ligação Proteica , Humanos , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacologia , TermodinâmicaRESUMO
Myeloproliferative neoplasms (MPN) are consolidated as a relevant group of diseases derived from the malfunction of the hematopoiesis process and have as a particular attribute the increased proliferation of myeloid lineage. Among these, chronic neutrophilic leukemia (CNL) is distinguished, caused by the T618I mutation of the CSF3R gene, a trait that generates ligand-independent receptor activation and downstream JAK2/STAT signaling. Previous studies reported that mutations in BCR::ABL1 and JAK2V617F increased the expression of the aurora kinase A (AURKA) and B (AURKB) in Ba/F3 cells and their pharmacological inhibition displays antineoplastic effects in human BCR::ABL1 and JAK2V617F positive cells. Delimiting the current scenario, aspects related to the AURKA and AURKB as a potential target in CSF3RT618I-driven models is little known. In the present study, the cellular and molecular effects of pharmacological inhibitors of aurora kinases, such as aurora A inhibitor I, AZD1152-HQPA, and reversine, were evaluated in Ba/F3 expressing the CSF3RT618I mutation. AZD1152-HQPA and reversine demonstrated antineoplastic potential, causing a decrease in cell viability, clonogenicity, and proliferative capacity. At molecular levels, all inhibitors reduced histone H3 phosphorylation, aurora A inhibitor I and reversine reduced STAT5 phosphorylation, and AZD1152-HQPA and reversine induced PARP1 cleavage and γH2AX expression. Reversine more efficiently modulated genes associated with cell cycle and apoptosis compared to other drugs. In summary, our findings shed new insights into the use of AURKB inhibitors in the context of CNL.
Assuntos
Antineoplásicos , Aurora Quinase A , Humanos , Aurora Quinase A/metabolismo , Quinazolinas/farmacologia , Organofosfatos/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Receptores de Fator Estimulador de ColôniasRESUMO
In equatorial Brazil, the association of Burkitt lymphoma and Epstein-Barr virus manifests at high rates. Here, we report, for the first time, amplifications of aurora kinase genes (AURKA/B) in a patient with a history of periodontal abscess and the presence of a remaining nodule, diagnosed with Burkitt lymphoma and Epstein-Barr virus, and /HIV positive. The patient was a 38-year-old man who presented with a 2-week-old severe jaw pain and a 3-day-old severe bilateral headache. He had a history of human papilloma virus. Interphase FISH analysis showed AURKA and AURKB amplification. The patient's condition worsened, progressing to death a month after the initial care. Changes in the MYCC and AURKA pathways are directly associated with genomic instability. Thus, MYCC rearrangements and higher expression of AURKA/B may be associated with therapy resistance, highlighting the importance of AURKA/B evaluation in Burkitt lymphoma.
Assuntos
Linfoma de Burkitt , Infecções por Vírus Epstein-Barr , Síndromes de Imunodeficiência , Masculino , Humanos , Adulto , Linfoma de Burkitt/complicações , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Herpesvirus Humano 4/metabolismo , Aurora Quinase B/genética , Aurora Quinase B/metabolismoRESUMO
Gastric cancer is one of the most common and deadly types of cancer in the world, and poor prognosis with treatment failure is widely reported in the literature. In this context, kinases have been considered a relevant choice for targeted therapy in gastric cancer. Here, we explore the antiproliferative and antimigratory effects of the AURKA inhibitor and the prognostic and therapeutic value as a biomarker of gastric cancer. A total of 145 kinase inhibitors were screened to evaluate the cytotoxic or cytostatic effects in the gastric cancer cell line. Using the Alamar Blue assay, flow cytometry, quantitative polymerase chain reaction, and observation of caspase 3/7 activity and cell migration, we investigated the antiproliferative, proapoptotic, and antimigratory effects of the AURKA inhibitor. Moreover, AURKA overexpression was evaluated in the gastric cell lines and the gastric tumor tissue. Out of the 145 inhibitors, two presented the highest antiproliferative effect. Both molecules can induce apoptosis by the caspases 3/7 pathway in addition to inhibiting cancer cell migration, mainly the AURKA inhibitor. Moreover, molecular docking analysis revealed that GW779439X interacts in the active site of the AURKA enzyme with similar energy as a well-described inhibitor. Our study identified AURKA overexpression in the gastric cancer cell line and gastric tumor tissue, revealing that its overexpression in patients with cancer is correlated with low survival. Therefore, it is feasible to suggest AURKA as a potential marker of gastric cancer, besides providing robust information for diagnosis and estimated survival of patients. AURKA can be considered a new molecular target used in the prognosis and therapy of gastric cancer.
Assuntos
Aurora Quinase A/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Piridazinas/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Apoptose , Aurora Quinase A/metabolismo , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Simulação de Acoplamento Molecular , Prognóstico , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/patologia , Taxa de SobrevidaRESUMO
Aurora kinases (AURKA and AURKB) are mitotic kinases with an important role in the regulation of several mitotic events, and in hematological malignancies, AURKA and AURKB hyperexpression are found in patients with cytogenetic abnormalities presenting a unfavorable prognosis. The aim of this study was evaluated the mRNA expression profile of pediatric Acute Lymphoblastic Leukaemia (ALL) patients and the efficacy of two AURKA and AURKB designed inhibitors (GW809897X and GW806742X) in a leukemia cell line as a potential novel therapy for ALL patients. Cellular experiments demonstrated that both inhibitors induced cell death with caspase activation and cell cycle arrest, however only the GW806742X inhibitor decreased with more efficacy AURKA and AURKB expression in K-562 leukemia cells. In ALL patients both AURKA and AURKB showed a significant overexpression, when compared to health controls. Moreover, AURKB expression level was significant higher than AURKA in patients, and predicted a poorer prognosis with significantly lower survival rates. No differences were found in AURKA and AURKB expression between gene fusions, immunophenotypic groups, white blood cells count, gender or age. In summary, the results in this study indicates that the AURKA and AURKB overexpression are important findings in pediatric ALL, and designed inhibitor, GW806742X tested in vitro were able to effectively inhibit the gene expression of both aurora kinases and induce apoptosis in K-562 cells, however our data clearly shown that AURKB proves to be a singular finding and potential prognostic biomarker that may be used as a promising therapeutic target to those patients.
Assuntos
Aurora Quinase A/metabolismo , Aurora Quinase B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Biomarcadores Tumorais/metabolismo , Brasil/epidemiologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Lactente , Células K562 , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Mapas de Interação de ProteínasRESUMO
Primary effusion lymphoma (PEL) is an aggressive malignancy with poor prognosis even under chemotherapy. Kaposi sarcoma-associated herpesvirus (KSHV), one of the human oncogenic viruses, is the principal causative agent. Currently, there is no specific treatment for PEL; therefore, developing new therapies is of great importance. Sphingolipid metabolism plays an important role in determining the fate of tumor cells. Our previous studies have demonstrated that there is a correlation between sphingolipid metabolism and KSHV+ tumor cell survival. To further develop sphingolipid metabolism-targeted therapy, after screening a series of newly synthesized ceramide analogs, here, we have identified compounds with effective anti-PEL activity. These compounds induce significant PEL apoptosis, cell-cycle arrest, and intracellular ceramide production through regulation of ceramide synthesizing or ceramide metabolizing enzymes and dramatically suppress tumor progression without visible toxicity in vivo. These new compounds also increase viral lytic gene expression in PEL cells. Our comparative transcriptomic analysis revealed their mechanisms of action for inducing PEL cell death and identified a subset of novel cellular genes, including AURKA and CDCA3, controlled by sphingolipid metabolism, and required for PEL survival with functional validation. These data provide the framework for the development of promising sphingolipid-based therapies against this virus-associated malignancy.
Assuntos
Aurora Quinase A/metabolismo , Ceramidas/farmacologia , Herpesvirus Humano 8/patogenicidade , Linfoma de Efusão Primária/tratamento farmacológico , Sarcoma de Kaposi/complicações , Esfingolipídeos/farmacologia , Animais , Apoptose , Aurora Quinase A/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular , Ceramidas/química , Feminino , Perfilação da Expressão Gênica , Humanos , Linfoma de Efusão Primária/etiologia , Linfoma de Efusão Primária/metabolismo , Linfoma de Efusão Primária/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Sarcoma de Kaposi/virologia , Células Tumorais Cultivadas , Replicação Viral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Oncogenic KRAS mutations are found in over 90% of pancreatic ductal adenocarcinomas (PDACs). As yet, however, no effective therapies are available for KRAS-induced malignancies. Therefore, research aimed at the identification of KRAS targets with therapeutic potential is warranted. Our goal was to investigate Aurora A (AURKA) and targeting protein for Xklp2 (TPX2) as potential therapeutic targets in PDAC. METHODS: AURKA and TPX2 expression was assessed using RNAseq and qRT-PCR in PDAC patient samples and matched non-tumor pancreatic tissues. Publicly available PDAC datasets were used to investigate associations of AURKA and TPX2 expression levels with patient survival and the presence of KRAS mutations. Next, we used an Aurora kinase inhibitor, or KRAS, AURKA and TPX2 targeting using RNA interference in KRAS-mutant PDAC cells and, subsequently, analyzed their clonogenic and anchorage-independent growth and migration. RESULTS: We found that relative to matched non-tumor tissues, PDAC tumors displayed significantly higher expression levels of AURKA and TPX2. In addition, we found that AURKA and TPX2 were co-expressed in PDAC datasets, and that high expression levels of AURKA and TPX2 were associated with a shorter patient survival and with the presence of oncogenic KRAS mutations. In addition, we found that siRNA-mediated KRAS targeting in KRAS-mutant PDAC cells reduced AURKA and TPX2 expression. Furthermore, targeting AURKA or TPX2 in KRAS-mutant PDAC cells reduced their clonogenic and anchorage-independent growth, as well their migration. CONCLUSIONS: From our data we conclude that AURKA and TPX2 may act as KRAS biomarkers in PDAC that can predict a worse prognosis, and that AURKA or TPX2 targeting in PDAC cells may reduce their transformed phenotype. These results indicate that AURKA and TPX2 may serve as promising targets to be explored for KRAS-mutant PDAC therapy.
Assuntos
Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Terapia de Alvo Molecular , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Aurora Quinase A/antagonistas & inibidores , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Estimativa de Kaplan-Meier , Mutação/genética , Oncogenes , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fenótipo , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/metabolismoRESUMO
JAK2/STAT signaling participates in the Ph-negative myeloproliferative neoplasms (MPN) pathophysiology and has been targeted by ruxolitinib, a JAK1/2 inhibitor. In the present study, the impact of ruxolitinib treatment on cytoskeleton-related genes expression was explored. In SET2 cells, AURKA and AURKB expression/activity were downregulated in a dose- and time-dependent manner by ruxolitinib. Reversine, a multikinase inhibitor selective for aurora kinases, reduced cell viability in a dose- and/or time-dependent manner in JAK2V617F cells. Reversine significantly increased apoptosis and mitotic catastrophe, and reduced cell proliferation and clonogenic capacity in SET2 and HEL cells. In the molecular scenario, reversine induced DNA damage and apoptosis markers, as well as, reduced AURKA and AURKB expression/activity. In SET2 cells, reversine modulated the expression of 32 out of 84 apoptosis-related genes investigated, including downregulation of antiapoptotic (BCL2, BCL2L1, and BIRC5) and upregulation of proapoptotic (BIK, BINP3, and BNIP3L) genes. Synergism experiments indicated that low dose of reversine had a potentiating effect under ruxolitinib treatment at low doses in SET2 cells. In summary, our exploratory study establishes new targets, related to the regulation of the cellular cytoskeleton, for potential pharmacological intervention in MPN. These findings indicate that AURKA and AURKB participate in the JAK2/STAT signaling pathway and contribute to the MPN phenotype.
Assuntos
Apoptose , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Janus Quinase 2/metabolismo , Morfolinas/farmacologia , Mutação , Transtornos Mieloproliferativos/patologia , Purinas/farmacologia , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Biomarcadores Tumorais/genética , Ciclo Celular , Proliferação de Células , Humanos , Janus Quinase 2/genética , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Inibidores de Proteínas Quinases , Células Tumorais CultivadasRESUMO
In the present investigation, trans-chalcone and licochalcone A were tested against MCF-7 and BT-20 breast cancer cell lines for anti-tumor activity. We found that both chalcones down regulated important genes associated to cancer development and inhibited cell migration of metastatic cells (BT-20). Finally, we observed that licochalcone A reduces the MDR-1 protein, while both chalcones suppress the AURKA protein in a dose-dependent manner. In conclusion, we observed the trans-chalcone and licochalcone A affected the cell viability of breast cancer cell lines MCF-7 and BT-20 and presents anti-metastatic and anti-resistance potential, by the repression of AUKA and MDR-1 proteins.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Aurora Quinase A/antagonistas & inibidores , Chalconas/farmacologia , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chalconas/química , Feminino , Perfilação da Expressão Gênica , Humanos , Estrutura Molecular , Metástase Neoplásica , Estadiamento de NeoplasiasRESUMO
Precise regulation of kinetochore-microtubule attachments is essential for successful chromosome segregation. Central to this regulation is Aurora B kinase, which phosphorylates kinetochore substrates to promote microtubule turnover. A critical target of Aurora B is the N-terminal "tail" domain of Hec1, which is a component of the NDC80 complex, a force-transducing link between kinetochores and microtubules. Although Aurora B is regarded as the "master regulator" of kinetochore-microtubule attachment, other mitotic kinases likely contribute to Hec1 phosphorylation. In this study, we demonstrate that Aurora A kinase regulates kinetochore-microtubule dynamics of metaphase chromosomes, and we identify Hec1 S69, a previously uncharacterized phosphorylation target site in the Hec1 tail, as a critical Aurora A substrate for this regulation. Additionally, we demonstrate that Aurora A kinase associates with inner centromere protein (INCENP) during mitosis and that INCENP is competent to drive accumulation of the kinase to the centromere region of mitotic chromosomes. These findings reveal that both Aurora A and B contribute to kinetochore-microtubule attachment dynamics, and they uncover an unexpected role for Aurora A in late mitosis.