RESUMO
Radiation sensitivity varies greatly between tissues. The transcription factor p53 mediates the response to radiation; however, the abundance of p53 protein does not correlate well with the extent of radiosensitivity across tissues. Given recent studies showing that the temporal dynamics of p53 influence the fate of cultured cells in response to irradiation, we set out to determine the dynamic behavior of p53 and its impact on radiation sensitivity in vivo. We find that radiosensitive tissues show prolonged p53 signaling after radiation, while more resistant tissues show transient p53 activation. Sustaining p53 using a small molecule (NMI801) that inhibits Mdm2, a negative regulator of p53, reduced viability in cell culture and suppressed tumor growth. Our work proposes a mechanism for the control of radiation sensitivity and suggests tools to alter the dynamics of p53 to enhance tumor clearance. Similar approaches can be used to enhance killing of cancer cells or reduce toxicity in normal tissues following genotoxic therapies.
Assuntos
Tolerância a Radiação , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Distribuição Tecidual/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos da radiação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hdm2 (human MDM2, human double minuteâ 2 homologue) counteracts p53 function by direct binding to p53 and by ubiquitin-dependent p53 protein degradation. Activation of p53 by inhibitors of the p53-Hdm2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. In addition, HdmX (human MDMX, human MDM4) was also identified as an important therapeutic target to efficiently reactivate p53, and it is likely that dual inhibition of Hdm2 and HdmX is beneficial. Herein we report four new X-ray structures for Hdm2 and five new X-ray structures for HdmX complexes, involving different classes of synthetic compounds (including the worldwide highest resolutions for Hdm2 and HdmX, at 1.13 and 1.20â Å, respectively). We also reveal the key additive 18-crown-ether, which we discovered to enable HdmX crystallization and show its stabilization of various Lys residues. In addition, we report the previously unpublished details of X-ray structure determinations for eight further Hdm2 complexes, including the clinical trial compounds NVP-CGM097 and NVP-HDM201. An analysis of all compound binding modes reveals new and deepened insight into the possible adaptations and structural states of Hdm2 (e.g., flip of F55, flip of Y67, reorientation of H96) and HdmX (e.g., flip of H55, dimer induction), enabling key binding interactions for different compound classes. To facilitate comparisons, we used the same numbering for Hdm2 (as in Q00987) and HdmX (as in O15151, but minus 1). Taken together, these structural insights should prove useful for the design and optimization of further selective and/or dual Hdm2/HdmX inhibitors.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Compostos Heterocíclicos/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , Cristalografia por Raios X , Compostos Heterocíclicos/química , Humanos , Ligação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-mdm2/químicaRESUMO
Small molecule inhibitors of the p53-MDM2 protein complex are under intense investigation in clinical trials as anti-cancer agents, including our first generation inhibitor NVP-CGM097. We recently described the rational design of a novel pyrazolopyrrolidinone core as a new lead structure and now we report on the synthesis and optimization of this to provide a highly potent lead compound. This new compound displayed excellent oral efficacy in our preclinical mechanistic in vivo model and marked a significant milestone towards the identification of our second generation clinical candidate NVP-HDM201.
Assuntos
Antineoplásicos/farmacologia , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Pirazóis/farmacologia , Pirrolidinonas/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Cães , Haplorrinos , Humanos , Masculino , Camundongos , Microssomos Hepáticos/metabolismo , Pirazóis/síntese química , Pirazóis/química , Pirazóis/farmacocinética , Pirrolidinonas/síntese química , Pirrolidinonas/química , Pirrolidinonas/farmacocinética , Ratos Sprague-Dawley , EstereoisomerismoRESUMO
Activation of p53 by inhibitors of the p53-MDM2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. Here, we report distinct mechanisms by which the novel, potent, and selective inhibitor of the p53-MDM2 interaction HDM201 elicits therapeutic efficacy when applied at various doses and schedules. Continuous exposure of HDM201 led to induction of p21 and delayed accumulation of apoptotic cells. By comparison, high-dose pulses of HDM201 were associated with marked induction of PUMA and a rapid onset of apoptosis. shRNA screens identified PUMA as a mediator of the p53 response specifically in the pulsed regimen. Consistent with this, the single high-dose HDM201 regimen resulted in rapid and marked induction of PUMA expression and apoptosis together with downregulation of Bcl-xL in vivo Knockdown of Bcl-xL was identified as the top sensitizer to HDM201 in vitro, and Bcl-xL was enriched in relapsing tumors from mice treated with intermittent high doses of HDM201. These findings define a regimen-dependent mechanism by which disruption of MDM2-p53 elicits therapeutic efficacy when given with infrequent dosing. In an ongoing HDM201 trial, the observed exposure-response relationship indicates that the molecular mechanism elicited by pulse dosing is likely reproducible in patients. These data support the clinical comparison of daily and intermittent regimens of p53-MDM2 inhibitors.Significance: Pulsed high doses versus sustained low doses of the p53-MDM2 inhibitor HDM201 elicit a proapoptotic response from wild-type p53 cancer cells, offering guidance to current clinical trials with this and other drugs that exploit the activity of p53. Cancer Res; 78(21); 6257-67. ©2018 AACR.
Assuntos
Antineoplásicos/administração & dosagem , Imidazóis/administração & dosagem , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Pirimidinas/administração & dosagem , Pirróis/administração & dosagem , Proteína Supressora de Tumor p53/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Apoptose , Área Sob a Curva , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imidazóis/farmacologia , Estimativa de Kaplan-Meier , Dose Máxima Tolerável , Camundongos , Transplante de Neoplasias , Pirimidinas/farmacologia , Pirróis/farmacologia , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , Proteína bcl-X/metabolismoRESUMO
As a result of our persistent efforts to discover novel inhibitors of the p53-MDM2 protein-protein interaction useful for the treatment of cancer, the potent and selective MDM2 inhibitors NVP-CGM097 and NVP-HDM201 with excellent in vitro and in vivo profile were selected as clinical candidates and are currently in phase 1 clinical development. This short review article provides a summary of the program history, the applied pharmacophore model and the discovery story of these novel p53-MDM2 inhibitor investigational drugs.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Ensaios Clínicos Fase I como Assunto , Humanos , Proteínas Proto-Oncogênicas c-mdm2/química , Proteína Supressora de Tumor p53/químicaRESUMO
Misdirected catalytic activity of histone methyltransferase Dot1L is believed to be causative for a subset of highly aggressive acute leukemias. Targeting the catalytic domain of Dot1L represents a potential therapeutic approach for these leukemias. In the context of a comprehensive Dot1L hit finding strategy, a knowledge-based virtual screen of the Dot1L SAM binding pocket led to the discovery of 2, a non-nucleoside fragment mimicking key interactions of SAM bound to Dot1L. Fragment linking of 2 and 3, an induced back pocket binder identified in earlier studies, followed by careful ligand optimization led to the identification of 7, a highly potent, selective and structurally novel Dot1L inhibitor.
RESUMO
Oncogenic MLL fusion proteins aberrantly recruit Dot1L, a histone methyltransferase, to ectopic loci, leading to local hypermethylation of H3K79 and misexpression of HoxA genes driving MLL-rearranged leukemias. Inhibition of the methyltransferase activity of Dot1L in this setting is predicted to reverse aberrant H3K79 methylation, leading to repression of leukemogenic genes and tumor growth inhibition. In the context of our Dot1L drug discovery program, high-throughput screening led to the identification of 2, a weak Dot1L inhibitor with an unprecedented, induced pocket binding mode. A medicinal chemistry campaign, strongly guided by structure-based consideration and ligand-based morphing, enabled the discovery of 12 and 13, potent, selective, and structurally completely novel Dot1L inhibitors.
RESUMO
The p53-MDM2 interaction is an anticancer drug target under investigation in the clinic. Our compound NVP-CGM097 is one of the small molecule inhibitors of this protein-protein interaction currently evaluated in cancer patients. As part of our effort to identify new classes of p53-MDM2 inhibitors that could lead to additional clinical candidates, we report here the design of highly potent inhibitors having a pyrazolopyrrolidinone core structure. The conception of these new inhibitors originated in a consideration on the MDM2 bound conformation of the dihydroisoquinolinone class of inhibitors to which NVP-CGM097 belongs. This work forms the foundation of the discovery of HDM201, a second generation p53-MDM2 inhibitor that recently entered phase I clinical trial.
Assuntos
Descoberta de Drogas , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Conformação Molecular , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
As a result of our efforts to discover novel p53:MDM2 protein-protein interaction inhibitors useful for treating cancer, the potent and selective MDM2 inhibitor NVP-CGM097 (1) with an excellent in vivo profile was selected as a clinical candidate and is currently in phase 1 clinical development. This article provides an overview of the discovery of this new clinical p53:MDM2 inhibitor. The following aspects are addressed: mechanism of action, scientific rationale, binding mode, medicinal chemistry, pharmacokinetic and pharmacodynamic properties, and in vivo pharmacology/toxicology in preclinical species.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Isoquinolinas/síntese química , Isoquinolinas/farmacologia , Piperazinas/síntese química , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Animais , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Ensaios Clínicos Fase I como Assunto , Descoberta de Drogas , Humanos , Isoquinolinas/farmacocinética , Piperazinas/farmacocinética , Ratos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Blocking the interaction between the p53 tumor suppressor and its regulatory protein MDM2 is a promising therapeutic concept under current investigation in oncology drug research. We report here the discovery of the first representatives of a new class of small molecule inhibitors of this protein-protein interaction: the dihydroisoquinolinones. Starting from an initial hit identified by virtual screening, a derivatization program has resulted in compound 11, a low nanomolar inhibitor of the p53-MDM2 interaction showing significant cellular activity. Initially based on a binding mode hypothesis, this effort was then guided by a X-ray co-crystal structure of MDM2 in complex with one of the synthesized analogs. The X-ray structure revealed an unprecedented binding mode for p53-MDM2 inhibitors.
Assuntos
Isoquinolinas/química , Isoquinolinas/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Cristalografia por Raios X , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/antagonistas & inibidoresRESUMO
Biomarkers for patient selection are essential for the successful and rapid development of emerging targeted anti-cancer therapeutics. In this study, we report the discovery of a novel patient selection strategy for the p53-HDM2 inhibitor NVP-CGM097, currently under evaluation in clinical trials. By intersecting high-throughput cell line sensitivity data with genomic data, we have identified a gene expression signature consisting of 13 up-regulated genes that predicts for sensitivity to NVP-CGM097 in both cell lines and in patient-derived tumor xenograft models. Interestingly, these 13 genes are known p53 downstream target genes, suggesting that the identified gene signature reflects the presence of at least a partially activated p53 pathway in NVP-CGM097-sensitive tumors. Together, our findings provide evidence for the use of this newly identified predictive gene signature to refine the selection of patients with wild-type p53 tumors and increase the likelihood of response to treatment with p53-HDM2 inhibitors, such as NVP-CGM097.
Assuntos
Biomarcadores/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Isoquinolinas/farmacologia , Neoplasias/tratamento farmacológico , Seleção de Pacientes , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Transferência Ressonante de Energia de Fluorescência , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
An efficient and tunable 176-550 nm source based on the emission of resonant dispersive radiation from ultrafast solitons at 800 nm is demonstrated in a gas-filled hollow-core photonic crystal fiber (PCF). By careful optimization and appropriate choice of gas, informed by detailed numerical simulations, we show that bright, high quality, localized bands of UV light (relative widths of a few percent) can be generated at all wavelengths across this range. Pulse energies of more than 75 nJ in the deep-UV, with relative bandwidths of ~3%, are generated from pump pulses of a few µJ. Excellent agreement is obtained between numerical and experimental results. The effects of positive and negative axial pressure gradients are also experimentally studied, and the coherence of the deep-UV dispersive wave radiation numerically investigated.
Assuntos
Gases/química , Iluminação/instrumentação , Refratometria/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Raios Ultravioleta , VácuoRESUMO
Eph receptor tyrosine kinases and their ligands (ephrins) have a pivotal role in the homeostasis of many adult organs and are widely expressed in the kidney. Glomerular diseases beginning with mesangiolysis can recover, with podocytes having a critical role in this healing process. We studied here the role of Eph signaling in glomerular disease recovery following mesangiolytic Thy1.1 nephritis in rats. EphB4 and ephrinBs were expressed in healthy glomerular podocytes and were upregulated during Thy1.1 nephritis, with EphB4 strongly phosphorylated around day 9. Treatment with NPV-BHG712, an inhibitor of EphB4 phosphorylation, did not cause glomerular changes in control animals. Nephritic animals treated with vehicle did not have morphological evidence of podocyte injury or loss; however, application of this inhibitor to nephritic rats induced glomerular microaneurysms, podocyte damage, and loss. Prolonged NPV-BHG712 treatment resulted in increased albuminuria and dysregulated mesangial recovery. Additionally, NPV-BHG712 inhibited capillary repair by intussusceptive angiogenesis (an alternative to sprouting angiogenesis), indicating a previously unrecognized role of podocytes in regulating intussusceptive vessel splitting. Thus, our results identify EphB4 signaling as a pathway allowing podocytes to survive transient capillary collapse during glomerular disease.
Assuntos
Glomerulonefrite/metabolismo , Glomérulos Renais/metabolismo , Podócitos/metabolismo , Receptor EphB4/metabolismo , Transdução de Sinais , Cicatrização , Albuminúria/imunologia , Albuminúria/metabolismo , Albuminúria/patologia , Animais , Anticorpos Monoclonais , Apoptose , Capilares/imunologia , Capilares/metabolismo , Capilares/patologia , Linhagem Celular , Modelos Animais de Doenças , Glomerulonefrite/imunologia , Glomerulonefrite/patologia , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/imunologia , Glomérulos Renais/patologia , Masculino , Camundongos , Neovascularização Fisiológica , Fosforilação , Podócitos/efeitos dos fármacos , Podócitos/imunologia , Podócitos/patologia , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Antígenos Thy-1/imunologia , Fatores de Tempo , Fator de Crescimento Transformador beta1/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
We show theoretically that the photoionization process in a hollow-core photonic crystal fiber filled with a Raman-inactive noble gas leads to a constant acceleration of solitons in the time domain with a continuous shift to higher frequencies, limited only by ionization loss. This phenomenon is opposite to the well-known Raman self-frequency redshift of solitons in solid-core glass fibers. We also predict the existence of unconventional long-range nonlocal soliton interactions leading to spectral and temporal soliton clustering. Furthermore, if the core is filled with a Raman-active molecular gas, spectral transformations between redshifted, blueshifted, and stabilized solitons can take place in the same fiber.
RESUMO
EphB4 and its cognitive ligand ephrinB2 play an important role in embryonic vessel development and vascular remodeling. In addition, several reports suggest that this receptor ligand pair is also involved in pathologic vessel formation in adults including tumor angiogenesis. Eph/ephrin signaling is a complex phenomena characterized by receptor forward signaling through the tyrosine kinase of the receptor and ephrin reverse signaling through various protein-protein interaction domains and phosphorylation motifs of the ephrin ligands. Therefore, interfering with EphR/ephrin signaling by the means of targeted gene ablation, soluble receptors, dominant negative mutants or antisense molecules often does not allow to discriminate between inhibition of Eph/ephrin forward and reverse signaling. We developed a specific small molecular weight kinase inhibitor of the EphB4 kinase, NVP-BHG712, which inhibits EphB4 kinase activity in the low nanomolar range in cellular assays showed high selectivity for targeting the EphB4 kinase when profiled against other kinases in biochemical as well as in cell based assays. Furthermore, NVP-BHG712 shows excellent pharmacokinetic properties and potently inhibits EphB4 autophosphorylation in tissues after oral administration. In vivo, NVP-BHG712 inhibits VEGF driven vessel formation, while it has only little effects on VEGF receptor (VEGFR) activity in vitro or in cellular assays. The data shown here suggest a close cross talk between the VEGFR and EphR signaling during vessel formation. In addition to its established function in vascular remodeling and endothelial arterio-venous differentiation, EphB4 forward signaling appears to be an important mediator of VEGF induced angiogenesis since inhibition of EphB4 forward signaling is sufficient to inhibit VEGF induced angiogenesis.
Assuntos
Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Neovascularização Patológica/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Receptor EphB4/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacocinética , Animais , Bioensaio , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Neovascularização Patológica/enzimologia , Tamanho do Órgão/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Pirazóis/química , Pirazóis/farmacocinética , Pirimidinas/química , Pirimidinas/farmacocinética , Receptor EphB4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Solubilidade/efeitos dos fármacosRESUMO
A substantial number of experimental and epidemiological studies support an important role for the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) pathway in the biology of human cancers. Components of this signaling cascade have been found to be deregulated in a wide range of solid tumors and hematologic malignancies, and extensive anti-cancer therapeutic programs are now devoted to the identification of agents that specifically block this molecular pathway. This article focuses on the current knowledge of the alterations of the PI3K/PKB pathway in cancer cells and ongoing drug discovery efforts to therapeutically target it. Particular emphasis is placed on medicinal chemistry activities to identify and develop compounds able to modulate the kinase activity of its main molecular components.