RESUMEN
Excessive blood vessel formation in the eye is implicated in wet age-related macular degeneration, proliferative diabetic retinopathy, neovascular glaucoma, and retinopathy of prematurity, which are major causes of blindness. Small molecule antiangiogenic drugs are strongly needed to supplement existing biologics. Homoisoflavonoids have been previously shown to have potent antiproliferative activities in endothelial cells over other cell types. Moreover, they demonstrated a strong antiangiogenic potential in vitro and in vivo in animal models of ocular neovascularization. Here, we tested the antiangiogenic activity of a group of naturally occurring homoisoflavonoids isolated from the family Hyacinthaceae and related synthetic compounds, chosen for synthesis based on structure-activity relationship observations. Several compounds showed interesting antiproliferative and antiangiogenic activities in vitro on retinal microvascular endothelial cells, a disease-relevant cell type, with the synthetic chromane, 46, showing the best activity (GI50 of 2.3 × 10-4 µM).
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Asparagaceae/química , Flavonoides/farmacología , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Estructura Molecular , Extractos Vegetales/química , Extractos Vegetales/farmacología , Raíces de Plantas/química , Neovascularización Retiniana/prevención & control , Relación Estructura-ActividadRESUMEN
Ocular neovascular diseases like wet age-related macular degeneration are a major cause of blindness. Novel therapies are greatly needed for these diseases. One appealing antiangiogenic target is reduction-oxidation factor 1-apurinic/apyrimidinic endonuclease 1 (Ref-1/APE1). This protein can act as a redox-sensitive transcriptional activator for nuclear factor (NF)-κB and other proangiogenic transcription factors. An existing inhibitor of Ref-1's function, APX3330, previously showed antiangiogenic effects. Here, we developed improved APX3330 derivatives and assessed their antiangiogenic activity. We synthesized APX2009 and APX2014 and demonstrated enhanced inhibition of Ref-1 function in a DNA-binding assay compared with APX3330. Both compounds were antiproliferative against human retinal microvascular endothelial cells (HRECs; GI50 APX2009: 1.1 µM, APX2014: 110 nM) and macaque choroidal endothelial cells (Rf/6a; GI50 APX2009: 26 µM, APX2014: 5.0 µM). Both compounds significantly reduced the ability of HRECs and Rf/6a cells to form tubes at mid-nanomolar concentrations compared with control, and both significantly inhibited HREC and Rf/6a cell migration in a scratch wound assay, reducing NF-κB activation and downstream targets. Ex vivo, APX2009 and APX2014 inhibited choroidal sprouting at low micromolar and high nanomolar concentrations, respectively. In the laser-induced choroidal neovascularization mouse model, intraperitoneal APX2009 treatment significantly decreased lesion volume by 4-fold compared with vehicle (P < 0.0001, ANOVA with Dunnett's post-hoc tests), without obvious intraocular or systemic toxicity. Thus, Ref-1 inhibition with APX2009 and APX2014 blocks ocular angiogenesis in vitro and ex vivo, and APX2009 is an effective systemic therapy for choroidal neovascularization in vivo, establishing Ref-1 inhibition as a promising therapeutic approach for ocular neovascularization.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Retina/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Humanos , Macaca , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Neovascularización Patológica/metabolismo , Retina/metabolismoRESUMEN
Bone is a common site of metastasis for breast, prostate, lung, kidney and other cancers. Bone metastases are incurable, and substantially reduce patient quality of life. To date, there exists no small-molecule therapeutic agent that can reduce tumor burden in bone. This is partly attributed to the lack of suitable in vitro assays that are good models of tumor growth in bone. Here, we take advantage of a novel ex vivo model of bone colonization to report a series of pyrrolopyrazolone small molecules that inhibit cancer cell invasion and ex vivo tumor growth in bone at single-digit micromolar concentration. We find that the compounds modulated the expression levels of genes associated with bone-forming osteoblasts, bone-destroying osteoclasts, cancer cell viability and metastasis. Our compounds provide chemical tools to uncover novel targets and pathways associated with bone metastasis, as well as for the development of compounds to prevent and reverse bone tumor growth in vivo.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Óseas/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Ratones , Estructura Molecular , Embarazo , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
The mitotic kinesin KIF14 has an essential role in the recruitment of proteins required for the final stages of cytokinesis. Genomic gain and/or overexpression of KIF14 has been documented in retinoblastoma and a number of other cancers, such as breast, lung and ovarian carcinomas, strongly suggesting its role as an oncogene. Despite evidence of oncogenic properties in vitro and in xenografts, Kif14's role in tumor progression has not previously been studied in a transgenic cancer model. Using a novel Kif14 overexpressing, simian virus 40 large T-antigen retinoblastoma (TAg-RB) double transgenic mouse model, we aimed to determine Kif14's role in promoting retinal tumor formation. Tumor initiation and development in double transgenics and control TAg-RB littermates were documented in vivo over a time course by optical coherence tomography, with subsequent ex vivo quantification of tumor burden. Kif14 overexpression led to an accelerated initiation of tumor formation in the TAg-RB model and a significantly decreased tumor doubling time (1.8 vs. 2.9 weeks). Moreover, overall percentage tumor burden was also increased by Kif14 overexpression. These data provide the first evidence that Kif14 can promote tumor formation in susceptible cells in vivo.
Asunto(s)
Cinesinas/biosíntesis , Neoplasias de la Retina/metabolismo , Retinoblastoma/metabolismo , Animales , Antígenos Virales de Tumores/biosíntesis , Procesos de Crecimiento Celular/genética , Modelos Animales de Enfermedad , Femenino , Cinesinas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias de la Retina/genética , Neoplasias de la Retina/patología , Retinoblastoma/genética , Retinoblastoma/patología , Virus 40 de los Simios/inmunologíaRESUMEN
A naturally occurring homoisoflavonoid, cremastranone (1) inhibited angiogenesis in vitro and in vivo. We developed an analogue SH-11037 (2) which is more potent than cremastranone in human retinal microvascular endothelial cells (HRECs) and blocks neovascularization in animal models. Despite their efficacy, the mechanism of these compounds is not yet fully known. In the course of building on a strong foundation of SAR and creating a novel chemical tool for target identification of homoisoflavonoid-binding proteins, various types of photoaffinity probes were designed and synthesized in which benzophenone and biotin were attached to homoisoflavanonoids using PEG linkers on either the C-3' or C-7 position. Notably, the photoaffinity probes linking on the phenol group of the C-3' position retain excellent activity of inhibiting retinal endothelial cell proliferation with up to 72nM of GI50.
Asunto(s)
Inhibidores de la Angiogénesis/síntesis química , Cromonas/síntesis química , Diseño de Fármacos , Isoflavonas/síntesis química , Isoflavonas/farmacología , Fenilalanina/análogos & derivados , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Proliferación Celular/efectos de los fármacos , Cromonas/química , Cromonas/farmacología , Humanos , Concentración 50 Inhibidora , Isoflavonas/química , Estructura Molecular , Fenilalanina/síntesis química , Fenilalanina/química , Fenilalanina/farmacología , Etiquetas de Fotoafinidad/químicaRESUMEN
Natural products are characterized by high chemical diversity and biochemical specificity; therefore, they are appealing as lead compounds for drug discovery. Given the importance of angiogenesis to many pathologies, numerous natural products have been explored as potential anti-angiogenic drugs. Ocular angiogenesis underlies blinding eye diseases such as retinopathy of prematurity (ROP) in children, proliferative diabetic retinopathy (DR) in adults of working age, and age-related macular degeneration (AMD) in the elderly. Despite the presence of effective therapy in many cases, these diseases are still a significant health burden. Anti-VEGF biologics are the standard of care, but may cause ocular or systemic side effects after intraocular administration and patients may be refractory. Many anti-angiogenic compounds inhibit tumor growth and metastasis alone or in combination therapy, but a more select subset of them has been tested in the context of ocular neovascular diseases. Here, we review the promise of natural products as anti-angiogenic agents, with a specific focus on retinal and choroidal neovascularization. The multifunctional curcumin and the chalcone isoliquiritigenin have demonstrated promising anti-angiogenic effects in mouse models of DR and choroidal neovascularization (CNV) respectively. The homoisoflavanone cremastranone and the flavonoid deguelin have been shown to inhibit ocular neovascularization in more than one disease model. The isoflavone genistein and the flavone apigenin on the other hand are showing potential in the prevention of retinal and choroidal angiogenesis with long-term administration. Many other products with anti-angiogenic potential in vitro such as the lactone withaferin A, the flavonol quercetin, and the stilbenoid combretastatin A4 are awaiting investigation in different ocular disease-relevant animal models. These natural products may serve as lead compounds for the design of more specific, efficacious, and affordable drugs with minimal side effects.
Asunto(s)
Productos Biológicos , Oftalmopatías/prevención & control , Neovascularización Patológica/prevención & control , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Oftalmopatías/patología , Humanos , Neovascularización Patológica/patologíaRESUMEN
An antiangiogenic homoisoflavanone, cremastranone, was synthesized for the first time. This scalable synthesis, which includes selective demethylation, could be used to develop lead molecules to treat angiogenesis-induced eye diseases. Synthetic cremastranone inhibited the proliferation, migration and tube formation ability of human retinal microvascular endothelial cells, important steps in pathological angiogenesis.
Asunto(s)
Inhibidores de la Angiogénesis/síntesis química , Inhibidores de la Angiogénesis/farmacología , Isoflavonas/síntesis química , Isoflavonas/farmacología , Proliferación Celular/efectos de los fármacos , Técnicas de Química Sintética , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , HumanosRESUMEN
Glucocorticoids (GCs) are essential steroid hormones that regulate numerous metabolic and homeostatic functions in almost all physiological systems. Synthetic glucocorticoids are among the most commonly prescribed drugs for the treatment of various conditions including autoimmune, allergic and inflammatory diseases. Glucocorticoids are mainly used for their potent anti-inflammatory and immunosuppressive activities mediated through signal transduction by their nuclear receptor, the glucocorticoid receptor (GR). Emerging evidence showing that diverse physiological and therapeutic actions of glucocorticoids are tissue-, cell-, and sex-specific, suggests more complex actions of glucocorticoids than previously anticipated. While several synthetic glucocorticoids are widely used in the ophthalmology clinic for the treatment of several ocular diseases, little is yet known about the mechanism of glucocorticoid signaling in different layers of the eye. GR has been shown to be expressed in different cell types of the eye such as cornea, lens, and retina, suggesting an important role of GR signaling in the physiology of these ocular tissues. In this review, we provide an update on the recent findings from in vitro and in vivo studies reported in the last 5â¯yearsâ¯that aim at understanding the role of GR signaling specifically in the eye. Advances in studying the physiological effects of glucocorticoids in the eye are vital for the elaboration of optimized and targeted GC therapies with potent anti-inflammatory potential while minimizing adverse effects.
Asunto(s)
Ojo/citología , Receptores de Glucocorticoides/metabolismo , Transducción de Señal , Animales , Glucocorticoides/metabolismo , HumanosRESUMEN
The KIF14 locus is gained and overexpressed in various malignancies, with prognostic relevance. Its protein product, a mitotic kinesin, accelerates growth of normal mammary epithelial cells in vitro and retinoblastoma tumours in a mouse model, while KIF14 knockdown blocks growth of brain, liver, ovarian, breast, prostate, and other tumour cells and xenografts. However, the tumour-initiating effects of Kif14 overexpression have not been studied. We aged a cohort of Kif14-overexpressing transgenic mice and wild-type littermates and documented survival, cause of death, and tumour burden. The Kif14 transgene was expressed in all tissues examined, and was associated with increased proliferation marker expression. Neither mouse weights nor overall survival differed between genotypes. However, Kif14 transgenic mice showed a higher incidence of fatal lymphomas (73 vs. 50%, p = 0.03, Fisher's exact test), primarily follicular and diffuse B-cell lymphomas. Non-tumour findings included a bilateral ballooning degeneration of lens in 12% of Kif14 transgenic mice but no wild-type mice (p = 0.02). Overall, this work reveals a novel association of Kif14 overexpression with lymphoma but suggests that Kif14 does not have as prominent a role in initiating cancer in other cell types as it does in accelerating tumour development in response to other oncogenic insults.
Asunto(s)
Biomarcadores de Tumor/genética , Carcinogénesis/genética , Cinesinas/genética , Linfoma de Células B/genética , Linfoma Folicular/genética , Animales , Regulación Neoplásica de la Expresión Génica/genética , Genotipo , Humanos , Linfoma de Células B/patología , Linfoma Folicular/patología , Ratones , Pronóstico , Carga Tumoral/genéticaRESUMEN
The standard-of-care therapeutics for the treatment of ocular neovascular diseases like wet age-related macular degeneration (AMD) are biologics targeting vascular endothelial growth factor signaling. There are currently no FDA approved small molecules for treating these blinding eye diseases. Therefore, therapeutic agents with novel mechanisms are critical to complement or combine with existing approaches. Here, we identified soluble epoxide hydrolase (sEH), a key enzyme for epoxy fatty acid metabolism, as a target of an antiangiogenic homoisoflavonoid, SH-11037. SH-11037 inhibits sEH in vitro and in vivo and docks to the substrate binding cleft in the sEH hydrolase domain. sEH levels and activity are up-regulated in the eyes of a choroidal neovascularization (CNV) mouse model. sEH is overexpressed in human wet AMD eyes, suggesting that sEH is relevant to neovascularization. Known sEH inhibitors delivered intraocularly suppressed CNV. Thus, by dissecting a bioactive compound's mechanism, we identified a new chemotype for sEH inhibition and characterized sEH as a target for blocking the CNV that underlies wet AMD.
Asunto(s)
Cromonas/farmacología , Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/metabolismo , Terapia Molecular Dirigida/métodos , Fenilalanina/análogos & derivados , Degeneración Macular Húmeda/metabolismo , Inhibidores de la Angiogénesis/farmacología , Animales , Sitios de Unión , Neovascularización Coroidal/tratamiento farmacológico , Neovascularización Coroidal/metabolismo , Cromonas/química , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Epóxido Hidrolasas/química , Ácidos Grasos Insaturados/metabolismo , Humanos , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Fenilalanina/química , Fenilalanina/farmacología , Dominios Proteicos , Proteómica/métodos , Degeneración Macular Húmeda/patologíaRESUMEN
Ocular neovascularization underlies major blinding eye diseases such as "wet" age-related macular degeneration (AMD). Despite the successes of treatments targeting the vascular endothelial growth factor (VEGF) pathway, resistant and refractory patient populations necessitate discovery of new therapeutic targets. Using a forward chemical genetic approach, we identified the heme synthesis enzyme ferrochelatase (FECH) as necessary for angiogenesis in vitro and in vivo FECH is overexpressed in wet AMD eyes and murine choroidal neovascularization; siRNA knockdown of Fech or partial loss of enzymatic function in the Fechm1Pas mouse model reduces choroidal neovascularization. FECH depletion modulates endothelial nitric oxide synthase function and VEGF receptor 2 levels. FECH is inhibited by the oral antifungal drug griseofulvin, and this compound ameliorates choroidal neovascularization in mice when delivered intravitreally or orally. Thus, FECH inhibition could be used therapeutically to block ocular neovascularization.
Asunto(s)
Ferroquelatasa/metabolismo , Degeneración Macular/patología , Neovascularización Patológica/fisiopatología , Neovascularización Retiniana/fisiopatología , Animales , Humanos , RatonesRESUMEN
Ocular neovascularisation underlies blinding eye diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. These diseases cause irreversible vision loss, and provide a significant health and economic burden. Biologics targeting vascular endothelial growth factor (VEGF) are the major approach for treatment. However, up to 30% of patients are non-responsive to these drugs and they are associated with ocular and systemic side effects. Therefore, there is a need for small molecule ocular angiogenesis inhibitors to complement existing therapies. We examined the safety and therapeutic potential of SH-11037, a synthetic derivative of the antiangiogenic homoisoflavonoid cremastranone, in models of ocular neovascularisation. SH-11037 dose-dependently suppressed angiogenesis in the choroidal sprouting assay ex vivo and inhibited ocular developmental angiogenesis in zebrafish larvae. Additionally, intravitreal SH-11037 (1 µM) significantly reduced choroidal neovascularisation (CNV) lesion volume in the laser-induced CNV mouse model, comparable to an anti-VEGF antibody. Moreover, SH-11037 synergised with anti-VEGF treatments in vitro and in vivo. Up to 100 µM SH-11037 was not associated with signs of ocular toxicity and did not interfere with retinal function or pre-existing retinal vasculature. SH-11037 is thus a safe and effective treatment for murine ocular neovascularisation, worthy of further mechanistic and pharmacokinetic evaluation.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Anticuerpos Neutralizantes/farmacología , Coroides/efectos de los fármacos , Neovascularización Coroidal/prevención & control , Cromonas/farmacología , Fenilalanina/análogos & derivados , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Bioensayo , Coroides/irrigación sanguínea , Coroides/patología , Neovascularización Coroidal/genética , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Combinación de Medicamentos , Sinergismo Farmacológico , Femenino , Expresión Génica , Humanos , Inyecciones Intravítreas , Larva/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Fenilalanina/farmacología , Retina/efectos de los fármacos , Retina/patología , Técnicas de Cultivo de Tejidos , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Pez CebraRESUMEN
PURPOSE: Therapeutic efficacy is routinely assessed by measurement of lesion size using flatmounted choroids and confocal microscopy in the laser-induced choroidal neovascularization (L-CNV) rodent model. We investigated whether optical coherence tomography (OCT) quantification, using an ellipsoid volume measurement, was comparable to standard ex vivo evaluation methods for this model and whether this approach could be used to monitor treatment-related lesion changes. METHODS: Bruch's membrane was ruptured by argon laser in the dilated eyes of C57BL/6J mice, followed by intravitreal injections of anti-VEGF164 or vehicle, or no injection. In vivo OCT images were acquired using Micron III or InVivoVue systems at 7, 10, and/or 14 days post-laser and neovascular lesion volume was calculated as an ellipsoid. Subsequently, lesion volume was compared to that calculated from confocal Z-stack images of agglutinin-stained choroidal flatmounts. RESULTS: Ellipsoid volume measurement of orthogonal 2-dimensional OCT images obtained from different imaging systems correlated with ex vivo lesion volumes for L-CNV (Spearman's ρ=0.82, 0.75, and 0.82 at days 7, 10, and 14, respectively). Ellipsoid volume calculation allowed temporal monitoring and evaluation of CNV lesions in response to antivascular endothelial growth factor treatment. CONCLUSIONS: Ellipsoid volume measurements allow rapid, quantitative use of OCT for the assessment of CNV lesions in vivo. This novel method can be used with different OCT imaging systems with sensitivity to distinguish between treatment conditions. It may serve as a useful adjunct to the standard ex vivo confocal quantification, to assess therapeutic efficacy in preclinical models of CNV, and in models of other ocular diseases.
Asunto(s)
Neovascularización Coroidal/patología , Tomografía de Coherencia Óptica/métodos , Animales , Lámina Basal de la Coroides/cirugía , Coroides/metabolismo , Neovascularización Coroidal/fisiopatología , Modelos Animales de Enfermedad , Femenino , Angiografía con Fluoresceína/métodos , Inyecciones Intravítreas , Coagulación con Láser/instrumentación , Coagulación con Láser/métodos , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal/métodos , Reproducibilidad de los Resultados , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Eye diseases characterized by excessive angiogenesis such as wet age-related macular degeneration, proliferative diabetic retinopathy, and retinopathy of prematurity are major causes of blindness. Cremastranone is an antiangiogenic, naturally occurring homoisoflavanone with efficacy in retinal and choroidal neovascularization models and antiproliferative selectivity for endothelial cells over other cell types. We undertook a cell-based structure-activity relationship study to develop more potent cremastranone analogues, with improved antiproliferative selectivity for retinal endothelial cells. Phenylalanyl-incorporated homoisoflavonoids showed improved activity and remarkable selectivity for retinal microvascular endothelial cells. A lead compound inhibited angiogenesis in vitro without inducing apoptosis and had efficacy in the oxygen-induced retinopathy model in vivo.