Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
Proc Natl Acad Sci U S A ; 117(29): 16949-16960, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32616569

RESUMO

Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.


Assuntos
Anticorpos/química , Afinidade de Anticorpos , Arginase/imunologia , Regiões Determinantes de Complementaridade/química , Anticorpos/genética , Anticorpos/imunologia , Sítios de Ligação de Anticorpos , Regiões Determinantes de Complementaridade/imunologia , Humanos
2.
Toxicol Pathol ; 39(3): 516-23, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21441228

RESUMO

Autophagy is believed to be an important process during tumorgenesis, and in recent years it has been shown to be modulated in response to a number of conventional anticancer agents. Furthermore, the development of targeted small molecule inhibitors, such as those to the PI3K-AKT-mTOR pathway, has presented a molecular link between the disruption of this signalling cascade and the process of autophagy. The cellular consequence of stimulating or inhibiting autophagy in cancer cells is not completely understood, so it is important that this process be monitored, along with antiproliferative and apoptotic biomarkers, in the preclinical setting. The field of autophagy is still evolving, and there is a constantly changing set of criteria for the assessment of the process in cells, tissues, and organs. The gold standard technique for analyzing autophagy in mammalian cells remains transmission electron microscopy, which has many limitations and is often difficult to perform on in vivo tissue including human tumor xenografts. In order to monitor autophagy in human tumor xenogaft tissue, we have taken the approach to develop an immunohistochemical (IHC) method for the detection of the autophagosome-associated protein, microtubule-associated protein 1 light chain 3 (LC3), in human tumor xenografts. After synthesis, LC3 is cleaved to form LC3-I, and upon induction of autophagy, LC3-I is conjugated to the lipid phosphatidylethanolamine to form LC3-II, which is tightly bound to the membrane of the autophagosome. It is thought that detection of endogenous LC3-II by IHC could be difficult because of the relatively low level of expression of the protein. Here we present the validation of an IHC method to detect LC3 in human tumor xenografts that we believe is able to distinguish LC3-I from LC3-II. It is hoped that this assay can become a useful tool for the detection of autophagy in preclinical xenograft models and determine the effects of anticancer therapies on the autophagic process.


Assuntos
Autofagia/efeitos dos fármacos , Imuno-Histoquímica/métodos , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Imunofluorescência/métodos , Humanos , Immunoblotting/métodos , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/metabolismo , Transplante de Neoplasias , Transplante Heterólogo
3.
MAbs ; 12(1): 1801230, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32880207

RESUMO

Arginase 2 (ARG2) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine. The dysregulated expression of ARG2 within specific tumor microenvironments generates an immunosuppressive niche that effectively renders the tumor 'invisible' to the host's immune system. Increased ARG2 expression leads to a concomitant depletion of local L-arginine levels, which in turn leads to suppression of anti-tumor T-cell-mediated immune responses. Here we describe the isolation and characterization of a high affinity antibody (C0021158) that inhibits ARG2 enzymatic function completely, effectively restoring T-cell proliferation in vitro. Enzyme kinetic studies confirmed that C0021158 exhibits a noncompetitive mechanism of action, inhibiting ARG2 independently of L-arginine concentrations. To elucidate C0021158's inhibitory mechanism at a structural level, the co-crystal structure of the Fab in complex with trimeric ARG2 was solved. C0021158's epitope was consequently mapped to an area some distance from the enzyme's substrate binding cleft, indicating an allosteric mechanism was being employed. Following C0021158 binding, distinct regions of ARG2 undergo major conformational changes. Notably, the backbone structure of a surface-exposed loop is completely rearranged, leading to the formation of a new short helix structure at the Fab-ARG2 interface. Moreover, this large-scale structural remodeling at ARG2's epitope translates into more subtle changes within the enzyme's active site. An arginine residue at position 39 is reoriented inwards, sterically impeding the binding of L-arginine. Arg39 is also predicted to alter the pKA of a key catalytic histidine residue at position 160, further attenuating ARG2's enzymatic function. In silico molecular docking simulations predict that L-arginine is unable to bind effectively when antibody is bound, a prediction supported by isothermal calorimetry experiments using an L-arginine mimetic. Specifically, targeting ARG2 in the tumor microenvironment through the application of C0021158, potentially in combination with standard chemotherapy regimens or alternate immunotherapies, represents a potential new strategy to target immune cold tumors.


Assuntos
Afinidade de Anticorpos , Arginase/química , Anticorpos de Cadeia Única/química , Regulação Alostérica , Cristalografia por Raios X , Humanos
4.
Cancer Treat Rev ; 33(2): 203-12, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17210228

RESUMO

Lung cancer is the leading cause of cancer death worldwide. Despite the introduction of new agents and schedules, chemotherapy still obtains unsatisfactory overall response rates, rare complete remissions and responses of relatively short duration. The inhibitor of apoptosis proteins (IAPS) are a family of caspase inhibitors that selectively bind and inhibit caspases-3, -7, and -9. As caspase activation is central to apoptosis, novel therapeutic drugs that target IAPs enabling apoptosis to occur have potential as a treatment of malignancy. Several agents that target core components of the apoptotic signalling pathway are currently at an early stage of development. This review reports the progress being made in characterising the IAP family, with a focus on the available data relevant to the treatment of lung cancer.


Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/tratamento farmacológico , Inibidores de Caspase , Proteínas Inibidoras de Apoptose/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Humanos
5.
Br J Pharmacol ; 174(16): 2652-2661, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28556895

RESUMO

BACKGROUND AND PURPOSE: AZD8055 is a potent orally available mTOR kinase inhibitor with in vitro and in vivo antitumour activity against a range of tumour types. Preclinical studies showed that AZD8055 induced a dose-dependent pharmacodynamic effect in xenograft models in vivo, but a lack of understanding of the relative contributions of the maximum inhibition of the biomarkers and the duration of inhibition to the antitumour effect, limited the rational design of experiments to optimize the dose and schedules of treatment. EXPERIMENTAL APPROACH: In this study, a mathematical modelling approach was developed to relate pharmacodynamics and antitumour activity using preclinical data generated in mice bearing U87-MG xenografts. KEY RESULTS: Refinement and validation of the model was carried out in a panel of additional human tumour xenograft models with different growth rates and different sensitivity to AZD8055 (from partial growth inhibition to regression). Finally, the model was applied to accurately predict the efficacy of high, intermittent dosing schedules of AZD8055. CONCLUSIONS AND IMPLICATIONS: Overall, this new model linking pharmacokinetics, pharmacodynamic biomarkers and efficacy across several tumour xenografts with different sensitivity to AZD8055 was able to identify the optimal dose and route of administration to maximize the antitumour efficacy in preclinical models and its potential for translation into man.


Assuntos
Antineoplásicos , Modelos Biológicos , Morfolinas , Neoplasias/tratamento farmacológico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos Nus , Morfolinas/sangue , Morfolinas/farmacocinética , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Neoplasias/sangue , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Carga Tumoral
6.
Eur J Med Chem ; 112: 20-32, 2016 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-26874741

RESUMO

Deregulation of the receptor tyrosine kinase RET has been implicated in medullary thyroid cancer, a small percentage of lung adenocarcinomas, endocrine-resistant breast cancer and pancreatic cancer. There are several clinically approved multi-kinase inhibitors that target RET as a secondary pharmacology but additional activities, most notably inhibition of KDR, lead to dose-limiting toxicities. There is, therefore, a clinical need for more specific RET kinase inhibitors. Herein we report our efforts towards identifying a potent and selective RET inhibitor using vandetanib 1 as the starting point for structure-based drug design. Phenolic anilinoquinazolines exemplified by 6 showed improved affinities towards RET but, unsurprisingly, suffered from high metabolic clearance. Efforts to mitigate the metabolic liability of the phenol led to the discovery that a flanking substituent not only improved the hepatocyte stability, but could also impart a significant gain in selectivity. This culminated in the identification of 36; a potent RET inhibitor with much improved selectivity against KDR.


Assuntos
Piperidinas/química , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Quinazolinas/química , Quinazolinas/farmacologia , Animais , Linhagem Celular , Desenho de Fármacos , Humanos , Camundongos , Simulação de Acoplamento Molecular , Piperidinas/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Quinazolinas/farmacocinética
7.
Cancer Res ; 72(7): 1804-13, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22271687

RESUMO

The mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/AKT signaling pathways interact at multiple nodes in cancer, including at mTOR complexes, suggesting an increased likelihood of redundancy and innate resistance to any therapeutic effects of single pathway inhibition. In this study, we investigated the therapeutic effects of combining the MAPK extracellular signal-regulated kinase (MEK)1/2 inhibitor selumetinib (AZD6244) with the dual mTORC1 and mTORC2 inhibitor (AZD8055). Concurrent dosing in nude mouse xenograft models of human lung adenocarcinoma (non-small cell lung cancers) and colorectal carcinoma was well tolerated and produced increased antitumor efficacy relative to the respective monotherapies. Pharmacodynamic analysis documented reciprocal pathway inhibition associated with increased apoptosis and Bim expression in tumor tissue from the combination group, where key genes such as DUSP6 that are under MEK functional control were also modulated. Our work offers a strong rationale to combine selumetinib and AZD8055 in clinical trials as an attractive therapeutic strategy.


Assuntos
Apoptose/efeitos dos fármacos , Benzimidazóis/administração & dosagem , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Morfolinas/administração & dosagem , Neoplasias Experimentais/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Colorretais/tratamento farmacológico , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Mutação , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genética
8.
Oncol Rep ; 25(4): 1177-81, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21286665

RESUMO

Resistance to conventional chemotherapy is a major problem in several paediatric tumours. One explanation for this is that tumour cells are unable to engage apoptosis after cytotoxic drug-induced damage. Inhibitor of apoptosis proteins (IAPs) function by inhibiting both effector (9) and initiator (3 and 7) caspases. Repression of the widely expressed X-linked IAP (XIAP) by RNAi sensitises adult tumour cells to cytotoxics in vitro. Antisense oligonucleotide (ASO)-induced down-regulation of XIAP is effective at inducing cell death and delaying the growth of adult tumour cells as xenografts and these agents are currently in phase II clinical trials. The importance of XIAP in paediatric tumours has not been characterised but high expression correlates with poor survival in childhood AML. We have used the novel XIAP ASO (AEG35156) to evaluate the effects of down-regulation of XIAP in paediatric tumour cells. Here, we show that AEG35156 can down-regulate XIAP in a number of paediatric cell lines including models of osteosarcoma, rhabdomyosarcoma and Ewing's sarcoma. Cell death assays demonstrated a higher proportion of dead cells after XIAP down-regulation by ASO and these cells displayed increased levels of cleaved caspase-3 and cleaved PARP, showing cell death was due to apoptosis. In long-term clonogenic assays, XIAP ASO sensitised 791T osteosarcoma cells to doxorubicin, etoposide and vincristine. The work presented here suggests that AEG35156, as a monotherapy or in combination with cytotoxic agents, may be of benefit in the treatment of paediatric tumours.


Assuntos
Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Oligonucleotídeos/farmacologia , Osteossarcoma/patologia , Rabdomiossarcoma/patologia , Sarcoma de Ewing/patologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Adulto , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Western Blotting , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Criança , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Humanos , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/metabolismo , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/metabolismo , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco , Vincristina/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
9.
J Cell Sci ; 118(Pt 20): 4889-900, 2005 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16219694

RESUMO

Cenp-F is an unusual kinetochore protein in that it localizes to the nuclear matrix in interphase and the nuclear envelope at the G2/M transition; it is farnesylated and rapidly degraded after mitosis. We have recently shown that farnesylation of Cenp-F is required for G2/M progression, its localization to kinetochores, and its degradation. However, the role Cenp-F plays in mitosis has remained enigmatic. Here we show that, following repression of Cenp-F by RNA interference (RNAi), the processes of metaphase chromosome alignment, anaphase chromosome segregation and cytokinesis all fail. Although kinetochores attach to microtubules in Cenp-F-deficient cells, the oscillatory movements that normally occur following K-fibre formation are severely dampened. Consistently, inter-kinetochore distances are reduced. In addition, merotelic associations are observed, suggesting that whereas kinetochores can attach microtubules in the absence of Cenp-F, resolving inappropriate interactions is inhibited. Repression of Cenp-F does not appear to compromise the spindle checkpoint. Rather, the chromosome alignment defect induced by Cenp-F RNA interference is accompanied by a prolonged mitosis, indicating checkpoint activation. Indeed, the prolonged mitosis induced by Cenp-F RNAi is dependent on the spindle checkpoint kinase BubR1. Surprisingly, chromosomes in Cenp-F-deficient cells frequently show a premature loss of chromatid cohesion. Thus, in addition to regulating kinetochore-microtubule interactions, Cenp-F might be required to protect centromeric cohesion prior to anaphase commitment. Intriguingly, whereas most of the sister-less kinetochores cluster near the spindle poles, some align at the spindle equator, possibly through merotelic or lateral orientations.


Assuntos
Ciclo Celular , Centrômero/fisiologia , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Pareamento Cromossômico/fisiologia , Inativação Gênica , Fuso Acromático/fisiologia , Proteínas Cromossômicas não Histona/deficiência , Células HeLa , Humanos , Cinetocoros/metabolismo , Metáfase , Proteínas dos Microfilamentos , Mitose , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão , Transfecção , Tubulina (Proteína)/metabolismo
10.
J Cell Sci ; 117(Pt 8): 1577-89, 2004 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-15020684

RESUMO

During mitosis, the recruitment of spindle-checkpoint-associated proteins to the kinetochore occurs in a defined order. The protein kinase Bub1 localizes to the kinetochore very early during mitosis, followed by Cenp-F, BubR1, Cenp-E and finally Mad2. Using RNA interference, we have investigated whether this order of binding reflects a level of dependency in human somatic cells. Specifically, we show that Bub1 plays a key role in the assembly of checkpoint proteins at the kinetochore, being required for the subsequent localization of Cenp-F, BubR1, Cenp-E and Mad2. In contrast to studies in Xenopus, we also show that BubR1 is not required for kinetochore localization of Bub1. Repression of Bub1 increases the number of cells with lagging chromosomes at metaphase, suggesting that Bub1 plays a role in chromosome congression. However, repression of Bub1 does not appear to compromise spindle checkpoint function either during normal mitosis or in response to spindle damage. This raises the possibility that, in the absence of Bub1, other mechanisms contribute to spindle checkpoint function.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Humanos/fisiologia , Cinetocoros/metabolismo , Proteínas Quinases/metabolismo , Western Blotting , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Citometria de Fluxo , Células HeLa , Humanos , Imuno-Histoquímica , Proteínas Mad2 , Microscopia de Fluorescência , Mitose , Modelos Biológicos , Proteínas Serina-Treonina Quinases , Interferência de RNA , Proteínas Repressoras
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA