RESUMEN
The bromodomain-containing protein 4 (BRD4), which is a key epigenetic regulator in cancer, has emerged as an attractive target for the treatment of melanoma. In this study, we investigate 7-phenoxy-benzimidazole derivative 12, which is a novel BRD4 inhibitor for the treatment of melanoma, by performing scaffold hopping on the previously reported benzimidazole derivative 1. Despite their good oral and intravenous exposure, the compounds obtained by modifying derivate 1 exhibit mutagenicity, which was confirmed by the positive Ames test results. Based on our hypothesis that the cause of the Ames test positivity is the metabolic intermediates generated from those chemical series, we implemented a scaffold hopping strategy to avoid the N-benzyl moiety by relocating the substituent groups to preserve the essential interaction. Based on this strategy, we successfully obtained compound 12; the Ames test results of this compound were negative. Notably, compound 12 not only exhibited a favorable pharmacokinetic (PK) profile but also significant tumor growth inhibition in a mouse melanoma xenograft model, indicating its potential as a therapeutic agent for the treatment of melanoma.
Asunto(s)
Antineoplásicos , Bencimidazoles , Proteínas de Ciclo Celular , Melanoma , Factores de Transcripción , Animales , Bencimidazoles/química , Bencimidazoles/farmacología , Bencimidazoles/síntesis química , Ratones , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/síntesis química , Melanoma/tratamiento farmacológico , Melanoma/patología , Humanos , Administración Oral , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Relación Estructura-Actividad , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Estructura Molecular , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Relación Dosis-Respuesta a Droga , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Proteínas que Contienen BromodominioRESUMEN
The epigenetic regulation of the protein bromodomain-containing protein 4 (BRD4) has emerged as a compelling target for cancer treatment. In this study, we outline the discovery of a novel BRD4 inhibitor for melanoma therapy. Our initial finding was that benzimidazole derivative 1, sourced from our library, was a powerful BRD4 inhibitor. However, it exhibited a poor pharmacokinetic (PK) profile. To address this, we conducted a scaffold-hopping procedure with derivative 1, which resulted in the creation of benzimidazolinone derivative 5. This new derivative displayed an improved PK profile. To further enhance the BRD4 inhibitory activity, we attempted to introduce hydrogen bond acceptors. This indeed improved the activity, but at the cost of decreased membrane permeability. Our search for a potent inhibitor with desirable permeability led to the development of tricyclic 18. This compound demonstrated powerful inhibitory activity and a favorable PK profile. More significantly, tricyclic 18 showed antitumor efficacy in a mouse melanoma xenograft model, suggesting that it holds potential as a therapeutic agent for melanoma treatment.
Asunto(s)
Melanoma , Proteínas Nucleares , Animales , Ratones , Humanos , Epigénesis Genética , Factores de Transcripción , Melanoma/tratamiento farmacológico , Permeabilidad de la Membrana Celular , Proteínas de Ciclo CelularRESUMEN
Asthma is airway inflammatory diseases caused by the activation of group 2 innate lymphoid cells (ILC2s) and type 2 helper T (TH2) cells. Cysteine proteases allergen cause tissue damage to airway epithelial cells and activate ILC2-mediated type 2 airway inflammation. FK506 is an immunosuppressive agent against calcium-dependent NFAT activation that is also effective against asthmatic inflammation. However, the effects of FK506 on cysteine protease allergen-mediated airway inflammation remain unclear. In this study, we investigated the suppressive effects of FK506 on airway inflammation. FK506 had a partial inhibitory effect on ILC2-dependent eosinophil inflammation and a robust inhibitory effect on T cell-dependent eosinophil inflammation in a cysteine protease-induced mouse asthma model. The infiltration of T1/ST2+ CD4 T cells in the lungs contributed to the persistence of eosinophil infiltration in the airway; FK506 completely inhibited the infiltration of T1/ST2+ CD4 T cells. In the initial phase, FK506 treatment targeted lung ILC2 activation induced by leukotriene B4 (LTB4)-mediated calcium signaling, but not IL-33 signaling. FK506 also inhibited the IL-13-dependent accumulation of T1/ST2+ CD4 T cells in the lungs of the later responses. These results indicated that FK506 potently suppressed airway inflammation by targeting ILC2 activation and T1/ST2+ CD4 T cell accumulation.
Asunto(s)
Asma , Proteasas de Cisteína , Eosinofilia , Neumonía , Ratones , Animales , Inmunidad Innata , Tacrolimus/farmacología , Tacrolimus/uso terapéutico , Proteína 1 Similar al Receptor de Interleucina-1 , Linfocitos , Asma/tratamiento farmacológico , Neumonía/tratamiento farmacológico , Alérgenos , Inflamación/tratamiento farmacológicoRESUMEN
Recently air pollutants and irritants have been labelled as possible exogenous risk factors for allergic disorders. Although the underlying causes of allergic disorders such as atopic dermatitis and asthma remain unclear, the T helper type 2 (Th2) cell-mediated allergic inflammatory cascade may contribute to their pathogenesis. In the last decade, it has been documented that one of the candidates for triggering Th2 commitment is thymic stromal lymphopoietin (TSLP), the expression of which is up-regulated in the lesions of allergic patients. Here, we describe TSLP function in a fluorescein isothiocyanate (FITC) -induced contact hypersensitivity (CHS) model. A cytokine profile indicated that the model was dominantly mediated by the Th2 milieu. Interestingly, TSLP was increased in the skin during the sensitization phase when stimulated by a solvent, dibutyl phthalate (DBP), but not by FITC hapten or another solvent, acetone. Ear swelling in FITC-induced CHS was totally abrogated by removing DBP from the sensitization or elicitation phase, and was restored by complementary injection of TSLP. Inversely, the ear swelling was suppressed by injection of small interfering RNA against TSLP during the sensitization phase, which was concomitant with decreasing expression of interleukin-4 at the swollen skin site. Taken together, DBP-induced TSLP during the sensitization phase plays a role in establishing FITC-induced CHS and may be one of the causes of Th2 commitment in the model, suggesting that certain environmental toxins, such as DBP, may endow pro-allergic and atopic predisposition in humans or animals.
Asunto(s)
Citocinas/biosíntesis , Dermatitis Alérgica por Contacto/inmunología , Dibutil Ftalato/farmacología , Fluoresceína-5-Isotiocianato/farmacología , Células Th2/efectos de los fármacos , Alérgenos/inmunología , Animales , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Citocinas/genética , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Dibutil Ftalato/química , Modelos Animales de Enfermedad , Femenino , Fluoresceína-5-Isotiocianato/química , Silenciador del Gen , Inmunoglobulina E/sangre , Interleucina-4/inmunología , Interleucina-4/metabolismo , Ratones , Ratones Endogámicos BALB C , ARN Interferente Pequeño/genética , Piel/efectos de los fármacos , Piel/inmunología , Solventes/química , Solventes/farmacología , Células Th2/inmunología , Linfopoyetina del Estroma TímicoAsunto(s)
Cadherinas/antagonistas & inhibidores , Cadherinas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , ARN Interferente Pequeño/genética , Secuencia de Bases , Cadherinas/fisiología , Agregación Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Humanos , TransfecciónAsunto(s)
Uniones Adherentes/metabolismo , Cadherinas/metabolismo , Queratinocitos/metabolismo , Uniones Adherentes/ultraestructura , Animales , Anticuerpos/inmunología , Cadherinas/genética , Cadherinas/inmunología , Humanos , Queratinocitos/ultraestructura , ARN Mensajero/genética , ARN Mensajero/metabolismo , ConejosRESUMEN
T-cadherin is a glycosyl-phosphatidylinositol (GPI) anchored cadherin molecule. We previously reported that T-cadherin is normally expressed on the basal keratinocytes of the epidermis and is down-regulated in cutaneous squamous cell carcinoma (SCC). We found that expression of T-cadherin in cutaneous squamous carcinoma cells regulated level of surface beta1 integrin, which functioned as extracellular matrix (ECM) receptor. Involvement of T-cadherin in beta1 integrin trafficking was studied using three different stable cell lines with cytomegalovirus (CMV)-driven over-expression, tetracycline (Tet)-inducible expression and RNAi-mediated suppressed expression of T-cadherin. Pulse-chase analysis using a cholesterol-depleting reagent and a tyrosine kinase inhibitor showed that beta1 integrin mainly internalized via caveolae. Over-expression of T-cadherin suppressed the internalization of both beta1 integrin and cholera toxin (CTX), a marker of caveolae-mediated endocytosis. By Western blot analysis of tyrosine-kinase target molecules, we demonstrated a reduced level of EGF receptor (EGFR)-phosphorylation in T-cadherin over-expressing cells. In addition, studies using EGF and EGFR specific inhibitors revealed that EGFR activation stimulated beta1 integrin internalization. Taking these results together, T-cadherin may modulate cell-matrix adhesion in basal keratinocytes as well as invasive potency in SCC by regulating surface level of beta1 integrin.
Asunto(s)
Cadherinas/biosíntesis , Carcinoma de Células Escamosas/metabolismo , Regulación Neoplásica de la Expresión Génica , Regulación de la Expresión Génica , Integrina beta1/metabolismo , Neoplasias Cutáneas/metabolismo , Cadherinas/metabolismo , Adhesión Celular , Línea Celular , Receptores ErbB/metabolismo , Matriz Extracelular/metabolismo , Glicosilfosfatidilinositoles/química , Humanos , Queratinocitos/metabolismo , Interferencia de ARN , Transducción de SeñalRESUMEN
T-cadherin is a unique member of the cadherin superfamily that lacks the transmembrane and cytoplasmic domains, and is instead linked to the cell membrane via a glycosyl-phosphatidylinositol anchor. We previously reported that T-cadherin was specifically expressed on the basal keratinocytes of the epidermis, and the expression of T-cadherin was significantly reduced in invasive cutaneous squamous cell carcinoma (SCC) and in the lesional skin of psoriasis vulgaris. In this study, to obtain an insight into the role of T-cadherin in keratinocytes, we used transfection methods and examined the effect of overexpression or knockdown of T-cadherin in immortalized keratinocyte cell lines derived from SCC. T-cadherin overexpressed cells showed clearly reduced cell proliferation, but the influence of cell-cell adhesiveness and cell mobility was not detected. Using a tetracycline-regulated expression system, we also confirmed that the suppression of cell proliferation was dependent on the expression level of T-cadherin. Cell cycle analysis demonstrated that over expression of T-cadherin induced a delay in the G(2)/M phase. Our findings suggest that T-cadherin acts as an endogenous negative regulator of keratinocyte proliferation and its inactivation is the cause for keratinocyte hyperproliferation in SCC or in psoriasis vulgaris.