RESUMO
The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, dihydroorotate dehydrogenase (DHODH), and the blockage of uridine transport into cells. These findings hold a 3-fold significance: first, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; second, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibition as this is a major contributor to the mechanism of action of the most widely used tenovins; and finally, that small changes in the structure of a small molecule can lead to a dramatic change in the target profile of the molecule even when the phenotypic readout remains static.
Assuntos
Acetanilidas/farmacologia , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Polifarmacologia , Sirtuína 1/antagonistas & inibidores , Tioureia/análogos & derivados , Proteína Supressora de Tumor p53/metabolismo , Autofagia , Proliferação de Células , Di-Hidro-Orotato Desidrogenase , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Tioureia/farmacologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
The interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among co-factor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fosfolipídeos/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Di-Hidro-Orotato Desidrogenase , Elétrons , Humanos , Ligantes , Simulação de Dinâmica Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Fosfolipídeos/química , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Tenovin-6 is the most studied member of a family of small molecules with antitumour activity in vivo. Previously, it has been determined that part of the effects of tenovin-6 associate with its ability to inhibit SirT1 and activate p53. However, tenovin-6 has also been shown to modulate autophagic flux. Here we show that blockage of autophagic flux occurs in a variety of cell lines in response to certain tenovins, that autophagy blockage occurs regardless of the effect of tenovins on SirT1 or p53, and that this blockage is dependent on the aliphatic tertiary amine side chain of these molecules. Additionally, we evaluate the contribution of this tertiary amine to the elimination of proliferating melanoma cells in culture. We also demonstrate that the presence of the tertiary amine is sufficient to lead to death of tumour cells arrested in G1 phase following vemurafenib treatment. We conclude that blockage of autophagic flux by tenovins is necessary to eliminate melanoma cells that survive B-Raf inhibition and achieve total tumour cell kill and that autophagy blockage can be achieved at a lower concentration than by chloroquine. This observation is of great relevance as relapse and resistance are frequently observed in cancer patients treated with B-Raf inhibitors.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Benzamidas/farmacologia , Indóis/farmacologia , Melanoma/genética , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/farmacologia , Antineoplásicos/química , Benzamidas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Melanoma/tratamento farmacológico , Estrutura Molecular , Mutação , Sirtuínas/genética , Proteína Supressora de Tumor p53/genética , VemurafenibRESUMO
The original PDF version of this Article listed the authors as "Marcus J.G.W. Ladds," where it should have read "Marcus J. G. W. Ladds, Ingeborg M. M. van Leeuwen, Catherine J. Drummond et al.#".Also in the PDF version, it was incorrectly stated that "Correspondence and requests for materials should be addressed to S. Lín.", instead of the correct "Correspondence and requests for materials should be addressed to S. Laín."This has been corrected in the PDF version of the Article. The HTML version was correct from the time of publication.
RESUMO
The development of non-genotoxic therapies that activate wild-type p53 in tumors is of great interest since the discovery of p53 as a tumor suppressor. Here we report the identification of over 100 small-molecules activating p53 in cells. We elucidate the mechanism of action of a chiral tetrahydroindazole (HZ00), and through target deconvolution, we deduce that its active enantiomer (R)-HZ00, inhibits dihydroorotate dehydrogenase (DHODH). The chiral specificity of HZ05, a more potent analog, is revealed by the crystal structure of the (R)-HZ05/DHODH complex. Twelve other DHODH inhibitor chemotypes are detailed among the p53 activators, which identifies DHODH as a frequent target for structurally diverse compounds. We observe that HZ compounds accumulate cancer cells in S-phase, increase p53 synthesis, and synergize with an inhibitor of p53 degradation to reduce tumor growth in vivo. We, therefore, propose a strategy to promote cancer cell killing by p53 instead of its reversible cell cycle arresting effect.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Indazóis/farmacologia , Neoplasias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Di-Hidro-Orotato Desidrogenase , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteólise/efeitos dos fármacos , Proteína Supressora de Tumor p53/genéticaRESUMO
Colorectal cancer (CRC) is one of the best characterised cancers, with extensive data documenting the sequential gene mutations that underlie its development. Complementary datasets are also being generated describing changes in protein and RNA expression, tumour biology and clinical outcome. Both the quantity and the variety of information are inexorably increasing and there is now an accompanying need to integrate these highly disparate datasets. In this article we aim to explain why we believe that mathematical modelling represents a natural tool or language with which to integrate these data and, in so doing, to provide insight into CRC.
Assuntos
Neoplasias Colorretais/patologia , Neoplasias Colorretais/fisiopatologia , Modelos Teóricos , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células , Neoplasias Colorretais/etiologia , Humanos , Transdução de Sinais/fisiologia , Processos EstocásticosRESUMO
The availability of multiple carbon/energy sources, as is common in wastewater treatment plants, often enhances the biodegradation of recalcitrant compounds. In this paper, we classify and model different modes of multiple substrate utilization in a systematic way, using the concept of synthesizing unit. According to this concept, substrates can be substitutable or complementary; their uptake (or processing) can be sequential or parallel. We show how the different modes of multiple substrate interaction can be described by a single general model. From the general model, we derive simple expressions for co-metabolism of substrates that are not structurally analogous. Both the general and the specific co-metabolism model have the advantage that they can be used in combination with any microbial growth model. To test the co-metabolism model's realism, we confront it with experimental data. The results attained with the co-metabolism model support that the general model constitutes a useful framework for modeling aspects of multiple substrate utilization.
Assuntos
Carbono/metabolismo , Modelos Teóricos , Eliminação de Resíduos Líquidos , Poluentes da Água/metabolismo , Bactérias , Biodegradação Ambiental , Biomassa , Relação Estrutura-AtividadeRESUMO
In their natural environment microorganisms encounter changes in substrate availability, involving either nutrient concentrations or nutrient types. They have to adapt to the new conditions in order to survive. We present a model for slow microbial adaptation, involving the synthesis of new enzymes, in response to changes in the availability of substitutable substrates. The model is based on reciprocal (or mutual) inhibition of expression of both the substrate-specific carriers and the associated assimilatory machinery. The inhibition kinetics is derived from the kinetics of synthesizing units. An interesting property of the adaptation model is that the presence of a single limiting resource results in a constant maximum specific substrate consumption rate for fully adapted microorganisms. Because the maximum specific consumption rate is not a function of substrate concentration, for growth on one substrate, the Monod and Pirt models for instance are still valid. Other adaptation models known to us do not fulfil this property. The simplest version of our model describes adaptation during diauxic growth, using only one preference parameter and one initial condition. The applicability of the model is exemplified by fitting it to published data from diauxic growth experiments.
Assuntos
Modelos Teóricos , Microbiologia da Água , Micronutrientes/metabolismo , Dinâmica Populacional , Eliminação de Resíduos LíquidosRESUMO
Acetylation of C-terminal lysine residues in the p53 tumor suppressor is associated with increased stability and transcription factor activity. The function, protein level, and acetylation of p53 are downregulated by mdm2, which in its turn is inhibited by the p14(ARF) tumor suppressor. Here, we show that p14(ARF) increases the level of p53 acetylated at lysine 382 in a nuclear chromatin-rich fraction. Unexpectedly, this accumulation of p53AcK382 is dramatically enhanced in the presence of ectopic mdm2. In light of these observations, we propose that p14(ARF) increases the binding of p53-mdm2 complexes to chromatin, thereby limiting the access of protein deacetylases to p53. Supporting this notion, we show that p53AcK382 can be deacetylated in the cytoplasm and that sirtuin SirT2 catalyzes this reaction. These results help understand why inhibition of both SirT1 and SirT2 is needed to achieve effective activation of p53 by small-molecule sirtuin inhibitors.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Sirtuína 2/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Linhagem Celular Tumoral , Humanos , Modelos Biológicos , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Sirtuína 2/genética , Proteína Supressora de Tumor p53/química , UbiquitinaçãoRESUMO
While small-molecule inhibitors of class I/II histone deacetylases (HDAC) have been approved for cancer treatment, inhibitors of the sirtuins (a family of class III HDACs) still require further validation and optimization to enter clinical trials. Recent studies show that tenovin-6, a small-molecule inhibitor of sirtuins SirT1 and SirT2, reduces tumor growth in vivo and eliminates leukemic stem cells in a murine model for chronic myelogenous leukemia. Here, we describe a tenovin analogue, tenovin-D3, that preferentially inhibits sirtuin SirT2 and induces predicted phenotypes for SirT2 inhibition. Unlike tenovin-6 and in agreement with its weak effect on SirT1 (a p53 deacetylase), tenovin-D3 fails to increase p53 levels or transcription factor activity. However, tenovin-D3 promotes expression of the cell-cycle regulator and p53 target p21(WAF1/CIP1) (CDKN1A) in a p53-independent manner. Structure-activity relationship studies strongly support that the ability of tenovin-D3 to inhibit SirT2 contributes to this p53-independent induction of p21. The ability of tenovin-D3 to increase p21 mRNA and protein levels is shared with class I/II HDAC inhibitors currently used in the clinic and therefore suggests that SirT2 inhibition and class I/II HDAC inhibitors have similar effects on cell-cycle progression.
Assuntos
Anilidas/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sirtuína 2/antagonistas & inibidores , Tioureia/análogos & derivados , Proteína Supressora de Tumor p53/metabolismo , Anilidas/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzamidas/química , Benzamidas/farmacologia , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/farmacologia , Humanos , Tioureia/química , Tioureia/farmacologia , Transcrição Gênica , Proteína Supressora de Tumor p53/genéticaRESUMO
Cyclotherapy is a promising endeavor to improve cancer treatment by tackling the dose-limiting side effects of chemotherapy, especially for cancers harboring mutations in the TP53 tumor suppressor. In this particular context, pretreatment with a p53 activator halts proliferation in healthy tissue, while leaving the p53-deficient tumor susceptible to conventional chemotherapy.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Pharmacological activation of wild-type p53 has been found to protect normal cells in culture from cytotoxicity and nuclear aberrations caused by conventional cancer therapeutics. Hence, small-molecule p53 activators could have clinical benefits as chemoprotectants for cancer patients bearing p53-mutant tumors. We have evaluated 16 p53-based cyclotherapy regimes combining p53 activators tenovin-6, leptomycin B, nutlin-3 and low dose actinomycin D, with clinically utilized chemotherapeutic agents (S- and M-phase poisons), vinblastine, vinorelbine, cytosine arabinoside and gemcitabine. All the p53 activators induce reversible cell-cycle arrest in primary human fibroblasts and protect them from both S- and M-phase poisons. Furthermore, studies with p53-mutant cancer cell lines show that nutlin-3 and low dose actinomycin D do not affect the sensitivity of these cells to any of the chemotherapeutics tested. Thus, these two small molecules could be suitable choices for cyclotherapy regimes involving S- or M-phase poisons. In contrast, pre-incubation of p53-mutant cells with tenovin-6 or leptomycin B reduces the efficacy of vinca alkaloids, suggesting that these p53 activators could be effective as chemoprotectants if combined with S- but not M-phase poisons. Discrepancies were observed between the levels of protection detected immediately after treatment and following recovery in fresh medium. This highlights the need to assess both short- and long-term effects when evaluating compounds as potential chemoprotectants for cancer therapy.
Assuntos
Antineoplásicos/efeitos adversos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzamidas/farmacologia , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Citarabina/farmacologia , Dactinomicina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Ácidos Graxos Insaturados/farmacologia , Fibroblastos/efeitos dos fármacos , Células HCT116 , Humanos , Imidazóis/farmacologia , Piperazinas/farmacologia , Proteína Supressora de Tumor p53/genética , Vimblastina/análogos & derivados , Vimblastina/farmacologia , Alcaloides de Vinca/farmacologia , VinorelbinaRESUMO
Recent advances in the field of pharmacological activation of the p53 tumor suppressor are beginning to be translated into the clinic. In addition, small molecules that activate p53 through established mechanisms of action are proving invaluable tools for basic research. Here we analyze and compare the effects of nutlin-3, tenovin-6 and low doses of actinomycin-D on p53 and its main negative regulator, mdm2. We reveal striking differences in the speed at which these compounds increase p53 protein levels, with nutlin-3 having a substantial impact within minutes. We also show that nutlin-3 is very effective at increasing the synthesis of mdm2 mRNA, mdm2 being not only a modulator of p53 but also a transcriptional target. In addition, we show that nutlin-3 stabilizes mdm2's conformation and protects mdm2 from degradation. These strong effects of nutlin-3 on mdm2 correlate with a remarkable rate of recovery of p53 levels upon removal of the compound. We discuss the potential application of our results as molecular signatures to assess the on-target effects of small-molecule mdm2 inhibitors. To conclude, we discuss the implications of our observations for using small-molecule p53 activators to reduce the growth of tumors retaining wild-type p53 or to protect normal tissues against the undesired side effects of conventional chemotherapy.
Assuntos
Dactinomicina/farmacologia , Imidazóis/farmacologia , Piperazinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Humanos , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Mensageiro/metabolismoRESUMO
Ageing is a complex multifactorial process involving a progressive physiological decline that, ultimately, leads to the death of an organism. It involves multiple changes in many components that play fundamental roles under healthy and pathological conditions. Simultaneously, every organism undergoes accumulative 'wear and tear' during its lifespan, which confounds the effects of the ageing process. The scenario is complicated even further by the presence of both age-dependent and age-independent competing causes of death. Various manipulations have been shown to interfere with the ageing process. Calorie restriction, for example, has been reported to increase the lifespan of a wide range of organisms, which suggests a strong relation between energy metabolism and ageing. Such a link is also supported within the main theories for ageing: the free radical hypothesis, for instance, links oxidative damage production directly to energy metabolism. The Dynamic Energy Budgets (DEB) theory, which characterizes the uptake and use of energy by living organisms, therefore constitutes a useful tool for gaining insight into the ageing process. Here we compare the existing DEB-based modelling approaches and, then, discuss how new biological evidence could be incorporated within a DEB framework.
Assuntos
Envelhecimento/metabolismo , Metabolismo Energético , Modelos Biológicos , Animais , Evolução Biológica , Restrição Calórica , Radicais Livres/metabolismo , HumanosRESUMO
p53-Based cyclotherapy is proving to be a promising approach to palliate undesired effects of chemotherapy in patients with tumours carrying p53 mutations. For example, pre-treatment of cell cultures with Nutlin-3, a highly-selective inhibitor of the p53-mdm2 interaction, has been successfully used as a cytostatic agent to protect normal cells, but not p53-defective cells, from subsequent treatment with mitotic poisons or S-phase specific drugs. Here we sought to evaluate whether low doses of Actinomycin D (LDActD), a clinically-approved drug and potent p53 activator, could substitute Nutlin-3 in p53-based cyclotherapy. We found that pre-treatment with LDActD before adding the aurora kinase inhibitor VX-680 protects normal fibroblasts from polyploidy and nuclear morphology abnormalities induced by VX-680. However, and although to a lower extent than normal fibroblasts, tumour cell lines bearing p53 mutations were also protected by LDActD (but not Nutlin-3) from VX-680-induced polyploidy. We also report that a difference between the response of p53 wild-type cells and p53-defective cells to the LDActD/VX-680 sequential combination is that only the former fail to enter S-phase and therefore accumulate in G1/G0. We propose that drugs that incorporate into DNA during S-phase may perform better as second drugs than mitotic poisons in cyclotherapy approaches using LDActD as a cytostatic agent.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Dactinomicina/administração & dosagem , Genes p53/fisiologia , Neoplasias/tratamento farmacológico , Piperazinas/administração & dosagem , Aneugênicos/administração & dosagem , Aneugênicos/farmacologia , Aneuploidia , Aurora Quinases , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Relação Dose-Resposta a Droga , Técnicas de Inativação de Genes , Células HCT116 , Humanos , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Neoplasias/genética , Neoplasias/patologia , Piperazinas/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidoresRESUMO
Wnt signalling is involved in a wide range of physiological and pathological processes. The presence of an extracellular Wnt stimulus induces cytoplasmic stabilisation and nuclear translocation of beta-catenin, a protein that also plays an essential role in cadherin-mediated adhesion. Two main hypotheses have been proposed concerning the balance between beta-catenin's adhesive and transcriptional functions: either beta-catenin's fate is determined by competition between its binding partners, or Wnt induces folding of beta-catenin into a conformation allocated preferentially to transcription. The experimental data supporting each hypotheses remain inconclusive. In this paper we present a new mathematical model of the Wnt pathway that incorporates beta-catenin's dual function. We use this model to carry out a series of in silico experiments and compare the behaviour of systems governed by each hypothesis. Our analytical results and model simulations provide further insight into the current understanding of Wnt signalling and, in particular, reveal differences in the response of the two modes of interaction between adhesion and signalling in certain in silico settings. We also exploit our model to investigate the impact of the mutations most commonly observed in human colorectal cancer. Simulations show that the amount of functional APC required to maintain a normal phenotype increases with increasing strength of the Wnt signal, a result which illustrates that the environment can substantially influence both tumour initiation and phenotype.