RESUMEN
Tumor hypoxia is associated with therapy resistance and poor patient prognosis. Hypoxia-activated prodrugs, designed to selectively target hypoxic cells while sparing normal tissue, represent a promising treatment strategy. We report the pre-clinical efficacy of 1-methyl-2-nitroimidazole panobinostat (NI-Pano, CH-03), a novel bioreductive version of the clinically used lysine deacetylase inhibitor, panobinostat. NI-Pano was stable in normoxic (21% O2) conditions and underwent NADPH-CYP-mediated enzymatic bioreduction to release panobinostat in hypoxia (<0.1% O2). Treatment of cells grown in both 2D and 3D with NI-Pano increased acetylation of histone H3 at lysine 9, induced apoptosis, and decreased clonogenic survival. Importantly, NI-Pano exhibited growth delay effects as a single agent in tumor xenografts. Pharmacokinetic analysis confirmed the presence of sub-micromolar concentrations of panobinostat in hypoxic mouse xenografts, but not in circulating plasma or kidneys. Together, our pre-clinical results provide a strong mechanistic rationale for the clinical development of NI-Pano for selective targeting of hypoxic tumors.
Asunto(s)
Antineoplásicos/farmacología , Desarrollo de Medicamentos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Hipoxia/tratamiento farmacológico , Panobinostat/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/química , Hipoxia/metabolismo , Masculino , Ratones , Ratones Desnudos , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Panobinostat/síntesis química , Panobinostat/química , Células Tumorales CultivadasRESUMEN
The purinosome is a dynamic metabolic complex composed of enzymes responsible for de novo purine biosynthesis, whose formation has been associated with elevated purine demand. However, the physiological conditions that govern purinosome formation in cells remain unknown. Here, we report that purinosome formation is up-regulated in cells in response to a low-oxygen microenvironment (hypoxia). We demonstrate that increased purinosome assembly in hypoxic human cells requires the activation of hypoxia inducible factor 1 (HIF-1) and not HIF-2. Hypoxia-driven purinosome assembly was inhibited in cells lacking 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a single enzyme in de novo purine biosynthesis, and in cells treated with a small molecule inhibitor of ATIC homodimerization. However, despite the increase in purinosome assembly in hypoxia, we observed no associated increase in de novo purine biosynthesis in cells. Our results indicate that this was likely due to a reduction in mitochondrial one-carbon metabolism, resulting in reduced mitochondrion-derived one-carbon units needed for de novo purine biosynthesis. The findings of our study further clarify and deepen our understanding of purinosome formation by revealing that this process does not solely depend on cellular purine demand.
Asunto(s)
Transferasas de Hidroximetilo y Formilo/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Complejos Multienzimáticos/metabolismo , Nucleótido Desaminasas/metabolismo , Purinas/biosíntesis , Hipoxia de la Célula , Células HeLa , Humanos , Transferasas de Hidroximetilo y Formilo/genética , Factor 1 Inducible por Hipoxia/genética , Complejos Multienzimáticos/genética , Nucleótido Desaminasas/genéticaRESUMEN
Ligands for the bromodomain and extra-terminal domain (BET) family of bromodomains have shown promise as useful therapeutic agents for treating a range of cancers and inflammation. Here we report that our previously developed 3,5-dimethylisoxazole-based BET bromodomain ligand (OXFBD02) inhibits interactions of BRD4(1) with the RelA subunit of NF-κB, in addition to histone H4. This ligand shows a promising profile in a screen of the NCI-60 panel but was rapidly metabolised (t½â¯=â¯39.8â¯min). Structure-guided optimisation of compound properties led to the development of the 3-pyridyl-derived OXFBD04. Molecular dynamics simulations assisted our understanding of the role played by an internal hydrogen bond in altering the affinity of this series of molecules for BRD4(1). OXFBD04 shows improved BRD4(1) affinity (IC50â¯=â¯166â¯nM), optimised physicochemical properties (LEâ¯=â¯0.43; LLEâ¯=â¯5.74; SFIâ¯=â¯5.96), and greater metabolic stability (t½â¯=â¯388â¯min).
Asunto(s)
Proteínas Nucleares/química , Factores de Transcripción/química , Bioensayo , Western Blotting , Proteínas de Ciclo Celular , Cristalografía por Rayos X , Estabilidad de Medicamentos , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Concentración 50 Inhibidora , Ligandos , Luciferasas/química , Células MCF-7 , Simulación de Dinámica Molecular , Estructura Molecular , Relación Estructura-ActividadRESUMEN
With the increasing incidence of cancer worldwide, the need for specific, effective therapies is ever more urgent. One example of targeted cancer therapeutics is hypoxia-activated prodrugs (HAPs), also known as bioreductive prodrugs. These prodrugs are inactive in cells with normal oxygen levels but in hypoxic cells (with low oxygen levels) undergo chemical reduction to the active compound. Hypoxia is a common feature of solid tumors and is associated with a more aggressive phenotype and resistance to all modes of therapy. Therefore, the combination of radiation therapy and bioreductive drugs presents an attractive opportunity for synergistic effects, because the HAP targets the radiation-resistant hypoxic cells. Hypoxia-activated prodrugs have typically been precursors of DNA-damaging agents, but a new generation of molecularly targeted HAPs is emerging. By targeting proteins associated with tumorigenesis and survival, these compounds may result in greater selectivity over healthy tissue. We review the clinical progress of HAPs as adjuncts to radiation therapy and conclude that the use of HAPs alongside radiation is vastly underexplored at the clinical level.
Asunto(s)
Antineoplásicos/metabolismo , Terapia Molecular Dirigida/métodos , Proteínas de Neoplasias/efectos de los fármacos , Neoplasias/terapia , Profármacos/metabolismo , Tolerancia a Radiación/efectos de los fármacos , Hipoxia Tumoral/efectos de los fármacos , Antineoplásicos/química , Antineoplásicos/farmacología , Ensayos Clínicos como Asunto , Terapia Combinada/métodos , Humanos , Neoplasias/metabolismo , Fenotipo , Profármacos/química , Profármacos/farmacología , Tolerancia a Radiación/fisiología , Hipoxia Tumoral/fisiologíaRESUMEN
Azide-containing compounds have broad utility in organic synthesis and chemical biology. Their use as powerful tools for the labeling of biological systems in vitro has enabled insights into complex cellular functions. To date, fluorogenic azide-containing compounds have primarily been employed in the context of click chemistry and as sensitive functionalities for hydrogen sulfide detection. Here, we report an alternative use of this functionality: as fluorogenic probes for the detection of depleted oxygen levels (hypoxia). Oxygen is imperative to all life forms, and probes that enable quantification of oxygen tension are of high utility in many areas of biology. Here we demonstrate the ability of an azide-based dye to image hypoxia in a range of human cancer cell lines. We have found that cytochrome P450 enzymes are able to reduce these probes in an oxygen-dependent manner, while hydrogen sulfide does not play an important role in their reduction. These data indicate that the azide group is a new bioreductive functionality that can be employed in prodrugs and dyes. We have uncovered a novel mechanism for the cellular reduction of azides, which has implications for the use of click chemistry in hypoxia.
RESUMEN
The cellular response to hypoxia is orchestrated by HIF-1, a heterodimeric transcription factor composed of an α and a ß subunit that enables cell survival under low oxygen conditions by altering the transcription of over 300 genes. There is significant evidence that inhibition of HIF-1 would be beneficial for cancer therapy. We recently reported a cyclic hexapeptide that inhibits the HIF-1α/HIF-1ß protein-protein interaction in vitro and prevents HIF-1-mediated hypoxia-response signaling in cells. This cyclic peptide was identified from a library of 3.2 × 106 members generated using SICLOPPS split-intein mediated protein splicing. With a view to demonstrating the potential for encoding the production of a therapeutic agent in response to a disease marker, we have engineered human cells with an additional chromosomal control circuit that conditionally encodes the production of our cyclic peptide HIF-1 inhibitor. We demonstrate the conditional production of our HIF-1 inhibitor in response to hypoxia, and its inhibitory effect on HIF-1 dimerization and downstream hypoxia-response signaling. These engineered cells are used to illustrate the synthetic lethality of inhibiting HIF-1 dimerization and glycolysis in hypoxic cells. Our approach not only eliminates the need for the chemical synthesis and targeted delivery of our HIF-1 inhibitor to cells, it also demonstrates the wider possibility that the production machinery of other bioactive compounds may be incorporated onto the chromosome of human cells. This work demonstrates the potential of sentinel circuits that produce molecular modulators of cellular pathways in response to environmental or cellular disease stimuli.