RESUMEN
UNLABELLED: We have identified previously undiscovered BRAF in-frame deletions near the αC-helix region of the kinase domain in pancreatic, lung, ovarian, and thyroid cancers. These deletions are mutually exclusive with KRAS mutations and occur in 4.21% of KRAS wild-type pancreatic cancer. siRNA knockdown in cells harboring BRAF deletions showed that the MAPK activity and cell growth are BRAF dependent. Structurally, the BRAF deletions are predicted to shorten the ß3/αC-helix loop and hinder its flexibility by locking the helix in the active αC-helix-in conformation that favors dimer formation. Expression of L485-P490-deleted BRAF is able to transform NIH/3T3 cells in a BRAF dimer-dependent manner. BRAF homodimer is confirmed to be the dominant RAF dimer by proximity ligation assays in BRAF deletion cells, which are resistant to the BRAF inhibitor vemurafenib and sensitive to LY3009120, a RAF dimer inhibitor. In tumor models with BRAF deletions, LY3009120 has shown tumor growth regression, whereas vemurafenib is inactive. SIGNIFICANCE: This study discovered oncogenic BRAF deletions with a distinct activation mechanism dependent on the BRAF dimer formation in tumor cells. LY3009120 is active against these cells and represents a potential treatment option for patients with cancer with these BRAF deletions, or other atypical BRAF mutations where BRAF functions as a dimer.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Eliminación de Gen , Compuestos de Fenilurea/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Multimerización de Proteína , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Pirimidinas/farmacología , Animales , Antineoplásicos/química , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Expresión Génica Ectópica , Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Modelos Moleculares , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas B-raf/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Compuestos de Fenilurea/farmacología , Isoformas de Proteínas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Pirimidinas/farmacología , Proteínas ras/genética , Línea Celular Tumoral , Dimerización , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Mutación/efectos de los fármacos , Mutación/genética , Neoplasias/genética , Fosforilación/efectos de los fármacos , Fosforilación/genética , Isoformas de Proteínas/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-raf/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
The RAS-RAF-MEK-MAPK cascade is an essential signaling pathway, with activation typically mediated through cell surface receptors. The kinase inhibitors vemurafenib and dabrafenib, which target oncogenic BRAF V600E, have shown significant clinical efficacy in melanoma patients harboring this mutation. Because of paradoxical pathway activation, both agents were demonstrated to promote growth and metastasis of tumor cells with RAS mutations in preclinical models and are contraindicated for treatment of cancer patients with BRAF WT background, including patients with KRAS or NRAS mutations. In order to eliminate the issues associated with paradoxical MAPK pathway activation and to provide therapeutic benefit to patients with RAS mutant cancers, we sought to identify a compound not only active against BRAF V600E but also wild type BRAF and CRAF. On the basis of its superior in vitro and in vivo profile, compound 13 was selected for further development and is currently being evaluated in phase I clinical studies.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Compuestos de Fenilurea/química , Compuestos de Fenilurea/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Pirimidinas/química , Pirimidinas/farmacología , Proteínas ras/metabolismo , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Línea Celular Tumoral/efectos de los fármacos , Técnicas de Química Sintética , Perros , Femenino , Semivida , Humanos , Masculino , Ratones Desnudos , Terapia Molecular Dirigida , Mutación , Compuestos de Fenilurea/síntesis química , Compuestos de Fenilurea/farmacocinética , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Pirimidinas/síntesis química , Pirimidinas/farmacocinética , Ratas Sprague-Dawley , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/genéticaRESUMEN
B-RAF selective inhibitors, including vemurafenib, were recently developed as effective therapies for melanoma patients with B-RAF V600E mutation. However, most patients treated with vemurafenib eventually develop resistance largely due to reactivation of MAPK signaling. Inhibitors of MAPK signaling, including MEK1/2 inhibitor trametinib, failed to show significant clinical benefit in patients with acquired resistance to vemurafenib. Here, we describe that cell lines with acquired resistance to vemurafenib show reactivation of MAPK signaling and upregulation of cyclin D1 and are sensitive to inhibition of LY2835219, a selective inhibitor of cyclin-dependent kinase (CDK) 4/6. LY2835219 was demonstrated to inhibit growth of melanoma A375 tumor xenografts and delay tumor recurrence in combination with vemurafenib. Furthermore, we developed an in vivo vemurafenib-resistant model by continuous administration of vemurafenib in A375 xenografts. Consistently, we found that MAPK is reactivated and cyclin D1 is elevated in vemurafenib-resistant tumors, as well as in the resistant cell lines derived from these tumors. Importantly, LY2835219 exhibited tumor growth regression in a vemurafenib-resistant model. Mechanistic analysis revealed that LY2835219 induced apoptotic cell death in a concentration-dependent manner in vemurafenib-resistant cells whereas it primarily mediated cell-cycle G1 arrest in the parental cells. Similarly, RNAi-mediated knockdown of cyclin D1 induced significantly higher rate of apoptosis in the resistant cells than in parental cells, suggesting that elevated cyclin D1 activity is important for the survival of vemurafenib-resistant cells. Altogether, we propose that targeting cyclin D1-CDK4/6 signaling by LY2835219 is an effective strategy to overcome MAPK-mediated resistance to B-RAF inhibitors in B-RAF V600E melanoma.
Asunto(s)
Aminopiridinas/farmacología , Bencimidazoles/farmacología , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Resistencia a Antineoplásicos , Femenino , Humanos , Indoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Desnudos , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Transfección , Regulación hacia Arriba/efectos de los fármacos , Vemurafenib , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
DNA-dependent RNA polymerase II (RNAP II) largest subunit RPB1 C-terminal domain (CTD) kinases, including CDK9, are serine/threonine kinases known to regulate transcriptional initiation and elongation by phosphorylating Ser 2, 5, and 7 residues on CTD. Given the reported dysregulation of these kinases in some cancers, we asked whether inhibiting CDK9 may induce stress response and preferentially kill tumor cells. Herein, we describe a potent CDK9 inhibitor, LY2857785, that significantly reduces RNAP II CTD phosphorylation and dramatically decreases MCL1 protein levels to result in apoptosis in a variety of leukemia and solid tumor cell lines. This molecule inhibits the growth of a broad panel of cancer cell lines, and is particularly efficacious in leukemia cells, including orthotopic leukemia preclinical models as well as in ex vivo acute myeloid leukemia and chronic lymphocytic leukemia patient tumor samples. Thus, inhibition of CDK9 may represent an interesting approach as a cancer therapeutic target, especially in hematologic malignancies.
Asunto(s)
Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Quinasa 9 Dependiente de la Ciclina/genética , Ciclohexilaminas/administración & dosificación , Indazoles/administración & dosificación , Leucemia/tratamiento farmacológico , Línea Celular Tumoral , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Humanos , Leucemia/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/biosíntesis , Fosforilación/efectos de los fármacos , Serina/metabolismoRESUMEN
p38α mitogen-activated protein kinase (MAPK) is activated in cancer cells in response to environmental factors, oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of substrates, including MAPKAP-K2 (MK2), and regulates the production of cytokines in the tumor microenvironment, such as TNF-α, interleukin-1ß (IL-1ß), IL-6, and CXCL8 (IL-8). p38α MAPK is highly expressed in human cancers and may play a role in tumor growth, invasion, metastasis, and drug resistance. LY2228820 dimesylate (hereafter LY2228820), a trisubstituted imidazole derivative, is a potent and selective, ATP-competitive inhibitor of the α- and ß-isoforms of p38 MAPK in vitro (IC(50) = 5.3 and 3.2 nmol/L, respectively). In cell-based assays, LY2228820 potently and selectively inhibited phosphorylation of MK2 (Thr334) in anisomycin-stimulated HeLa cells (at 9.8 nmol/L by Western blot analysis) and anisomycin-induced mouse RAW264.7 macrophages (IC(50) = 35.3 nmol/L) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc ≤ 10 µmol/L. LY2228820 also reduced TNF-α secretion by lipopolysaccharide/IFN-γ-stimulated macrophages (IC(50) = 6.3 nmol/L). In mice transplanted with B16-F10 melanoma, tumor phospho-MK2 (p-MK2) was inhibited by LY2228820 in a dose-dependent manner [threshold effective dose (TED)(70) = 11.2 mg/kg]. Significant target inhibition (>40% reduction in p-MK2) was maintained for 4 to 8 hours following a single 10 mg/kg oral dose. LY2228820 produced significant tumor growth delay in multiple in vivo cancer models (melanoma, non-small cell lung cancer, ovarian, glioma, myeloma, breast). In summary, LY2228820 is a p38 MAPK inhibitor, which has been optimized for potency, selectivity, drug-like properties (such as oral bioavailability), and efficacy in animal models of human cancer.
Asunto(s)
Imidazoles/farmacología , Neoplasias/tratamiento farmacológico , Piridinas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Animales , Anisomicina/farmacología , Sitios de Unión , Western Blotting , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Imidazoles/química , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Ratones , Estructura Molecular , Neoplasias/genética , Neoplasias/metabolismo , Fosforilación/efectos de los fármacos , Piridinas/química , Interferencia de ARN , Resultado del Tratamiento , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
LY2334737, an oral prodrug of gemcitabine, is cleaved in vivo, releasing gemcitabine and valproic acid. Oral dosing of mice results in absorption of intact prodrug with slow systemic hydrolysis yielding higher plasma levels of LY2334737 than gemcitabine and prolonged gemcitabine exposure. Antitumor activity was evaluated in human colon and lung tumor xenograft models. The dose response for efficacy was examined using 3 metronomic schedules, once-a-day dosing for 14 doses, every other day for 7 doses, and once a day for 7 doses, 7 days rest, followed by an additional 7 days of once-a-day dosing. These schedules gave significant antitumor activity and were well tolerated. Oral gavage of 6 mg/kg LY2334737 daily for 21 days gave equivalent activity to i.v. 240 mg/kg gemcitabine. HCl administered once a week for 3 weeks to mice bearing a patient mesothelioma tumor PXF 1118 or a non-small cell lung cancer tumor LXFE 937. The LXFE 397 tumor possessed elevated expression of the equilibrative nucleoside transporter-1 (ENT1) important for gemcitabine uptake but not prodrug uptake and responded significantly better to treatment with LY2334737 than gemcitabine (P ≤ 0.001). In 3 colon xenografts, antitumor activity of LY2334737 plus a maximally tolerated dose of capecitabine, an oral prodrug of 5-fluorouracil, was significantly greater than either monotherapy. During treatment, the expression of carboxylesterase 2 (CES2) and concentrative nucleoside transporter-3 was induced in HCT-116 tumors; both are needed for the activity of the prodrugs. Thus, metronomic oral low-dose LY2334737 is efficacious, well tolerated, and easily combined with capecitabine for improved efficacy. Elevated CES2 or ENT1 expression may enhance LY2334737 tumor response.
Asunto(s)
Desoxicitidina/análogos & derivados , Desoxiuridina/análogos & derivados , Profármacos/administración & dosificación , Administración Metronómica , Administración Oral , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/química , Desoxicitidina/farmacología , Desoxiuridina/administración & dosificación , Desoxiuridina/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Expresión Génica , Células HCT116 , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Profármacos/química , Ácido Valproico/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Oncogenic B-RAF V600E mutation is found in 50% of melanomas and drives MEK/ERK pathway and cancer progression. Recently, a selective B-RAF inhibitor, vemurafenib (PLX4032), received clinical approval for treatment of melanoma with B-RAF V600E mutation. However, patients on vemurafenib eventually develop resistance to the drug and demonstrate tumor progression within an average of 7 months. Recent reports indicated that multiple complex and context-dependent mechanisms may confer resistance to B-RAF inhibition. In the study described herein, we generated B-RAF V600E melanoma cell lines of acquired-resistance to vemurafenib, and investigated the underlying mechanism(s) of resistance. Biochemical analysis revealed that MEK/ERK reactivation through Ras is the key resistance mechanism in these cells. Further analysis of total gene expression by microarray confirmed a significant increase of Ras and RTK gene signatures in the vemurafenib-resistant cells. Mechanistically, we found that the enhanced activation of fibroblast growth factor receptor 3 (FGFR3) is linked to Ras and MAPK activation, therefore conferring vemurafenib resistance. Pharmacological or genetic inhibition of the FGFR3/Ras axis restored the sensitivity of vemurafenib-resistant cells to vemurafenib. Additionally, activation of FGFR3 sufficiently reactivated Ras/MAPK signaling and conferred resistance to vemurafenib in the parental B-RAF V600E melanoma cells. Finally, we demonstrated that vemurafenib-resistant cells maintain their addiction to the MAPK pathway, and inhibition of MEK or pan-RAF activities is an effective therapeutic strategy to overcome acquired-resistance to vemurafenib. Together, we describe a novel FGFR3/Ras mediated mechanism for acquired-resistance to B-RAF inhibition. Our results have implications for the development of new therapeutic strategies to improve the outcome of patients with B-RAF V600E melanoma.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Indoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/enzimología , Mutación Missense , Proteínas Proto-Oncogénicas B-raf/metabolismo , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Sulfonamidas/farmacología , Proteínas ras/metabolismo , Sustitución de Aminoácidos , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Humanos , Sistema de Señalización de MAP Quinasas/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Vemurafenib , Proteínas ras/genéticaRESUMEN
Prasugrel, a thienopyridine ADP receptor antagonist, is an orally administered prodrug requiring in vivo metabolism to form the active metabolite that irreversibly inhibits platelet activation and aggregation mediated by the P2Y12[sub 12] receptor. A comprehensive nonclinical safety assessment including genotoxicity and carcinogenicity studies supported the chronic use of prasugrel in patients with atherothrombotic disease. In addition, a special assessment of the potential for prasugrel to enhance tumor growth was undertaken to address regulatory concerns relating to increases in human cancers. Prasugrel demonstrated no evidence of genotoxicity and was not oncogenic in a 2-year rat carcinogenicity study. In the 2-year mouse study, an increase in hepatocellular adenomas was considered secondary to enzyme induction and not relevant to human safety. Further, the absence of any increase in common background tumors at any other organ site in either rodent study indicated a lack of tumor promoting activity (apart from the CYP450 induction-related increase in mouse liver tumors). Cell culture studies with 3 human tumor cell lines (lung, colon, prostate) demonstrated that exposure of serum-starved cells to prasugrel's active and major circulating human metabolites does not increase cell proliferation relative to starved cells stimulated to proliferate by addition of 10% FBS. Prasugrel also did not increase tumor growth relative to vehicle controls in nude mice implanted with 3 human tumor cell lines. Thus, traditional genotoxicity and 2-year bioassays as well as specially designed tumor growth enhancement studies in human tumor cell lines and mouse xenograft models clearly demonstrated prasugrel's lack of tumorigenic potential.
Asunto(s)
Plaquetas/efectos de los fármacos , Carcinógenos/toxicidad , Piperazinas/efectos adversos , Inhibidores de Agregación Plaquetaria/efectos adversos , Tiofenos/efectos adversos , Adenoma de Células Hepáticas/patología , Animales , Plaquetas/metabolismo , Carcinógenos/administración & dosificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos ICR , Piperazinas/administración & dosificación , Inhibidores de Agregación Plaquetaria/administración & dosificación , Clorhidrato de Prasugrel , Ratas , Ratas Endogámicas F344 , Factores de Riesgo , Tiofenos/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
LY2457546 is a potent and orally bioavailable inhibitor of multiple receptor tyrosine kinases involved in angiogenic and tumorigenic signalling. In biochemical and cellular assays, LY2457546 demonstrates potent activity against targets that include VEGFR2 (KDR), PDGFRß, FLT-3, Tie-2 and members of the Eph family of receptors. With activities against both Tie2 and Eph receptors, LY2457546 possesses an activity profile that distinguishes it from multikinase inhibitors. When compared head to head with sunitinib, LY2457546 was more potent for inhibition of endothelial tube formation in an in vitro angiogenesis co-culture model with an intermittent treatment design. In vivo, LY2457546 inhibited VEGF-driven autophosphorylation of lung KDR in the mouse and rat in a dose and concentration dependent manner. LY2457546 was well tolerated and exhibited efficacy in a 13762 syngeneic rat mammary tumor model in both once and twice daily continuous dosing schedules and in mouse human tumor xenograft models of lung, colon, and prostate origin. Additionally, LY2457546 caused complete regression of well-established tumors in an acute myelogenous leukemia (AML) FLT3-ITD mutant xenograft tumor model. The observed efficacy that was displayed by LY2457546 in the AML FLT3-ITD mutant tumor model was superior to sunitinib when both were evaluated using equivalent doses normalized to in vivo inhibition of pKDR in mouse lung. LY2457546 was well tolerated in non-clinical toxicology studies conducted in rats and dogs. The majority of the toxicities observed were similar to those observed with other multi-targeted anti-angiogenic kinase inhibitors (MAKs) and included bone marrow hypocellularity, hair and skin depigmentation, cartilage dysplasia and lymphoid organ degeneration and necrosis. Thus, the unique spectrum of target activity, potent in vivo anti-tumor efficacy in a variety of rodent and human solid tumor models, exquisite potency against a clinically relevant model of AML, and non-clinical safety profile justify the advancement of LY2457546 into clinical testing.
Asunto(s)
Acetanilidas/uso terapéutico , Inhibidores de la Angiogénesis/uso terapéutico , Neoplasias Experimentales/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/uso terapéutico , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Acetanilidas/síntesis química , Acetanilidas/farmacología , Inhibidores de la Angiogénesis/síntesis química , Inhibidores de la Angiogénesis/farmacología , Animales , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Perros , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Desnudos , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/síntesis química , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
The design, synthesis, and biological characterization of an orally active prodrug (3) of gemcitabine are described. Additionally, the identification of a novel co-crystal solid form of the compound is presented. Valproate amide 3 is orally bioavailable and releases gemcitabine into the systemic circulation after passing through the intestinal mucosa. The compound has entered clinical trials and is being evaluated as a potential new anticancer agent.
Asunto(s)
Antineoplásicos/química , Desoxicitidina/análogos & derivados , Profármacos/química , Profármacos/farmacología , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Línea Celular Tumoral , Transformación Celular Neoplásica , Neoplasias del Colon/tratamiento farmacológico , Cristalización , Cristalografía por Rayos X , Citidina/química , Desoxicitidina/administración & dosificación , Desoxicitidina/química , Desoxicitidina/farmacología , Humanos , Ratones , Modelos Moleculares , Conformación Molecular , Profármacos/administración & dosificación , Profármacos/síntesis química , Solubilidad , GemcitabinaRESUMEN
BACKGROUND: Tumor angiogenesis is a highly regulated process involving intercellular communication as well as the interactions of multiple downstream signal transduction pathways. Disrupting one or even a few angiogenesis pathways is often insufficient to achieve sustained therapeutic benefits due to the complexity of angiogenesis. Targeting multiple angiogenic pathways has been increasingly recognized as a viable strategy. However, translation of the polypharmacology of a given compound to its antiangiogenic efficacy remains a major technical challenge. Developing a global functional association network among angiogenesis-related genes is much needed to facilitate holistic understanding of angiogenesis and to aid the development of more effective anti-angiogenesis therapeutics. RESULTS: We constructed a comprehensive gene functional association network or interactome by transcript profiling an in vitro angiogenesis model, in which human umbilical vein endothelial cells (HUVECs) formed capillary structures when co-cultured with normal human dermal fibroblasts (NHDFs). HUVEC competence and NHDF supportiveness of cord formation were found to be highly cell-passage dependent. An enrichment test of Biological Processes (BP) of differentially expressed genes (DEG) revealed that angiogenesis related BP categories significantly changed with cell passages. Built upon 2012 DEGs identified from two microarray studies, the resulting interactome captured 17226 functional gene associations and displayed characteristics of a scale-free network. The interactome includes the involvement of oncogenes and tumor suppressor genes in angiogenesis. We developed a network walking algorithm to extract connectivity information from the interactome and applied it to simulate the level of network perturbation by three multi-targeted anti-angiogenic kinase inhibitors. Simulated network perturbation correlated with observed anti-angiogenesis activity in a cord formation bioassay. CONCLUSION: We established a comprehensive gene functional association network to model in vitro angiogenesis regulation. The present study provided a proof-of-concept pilot of applying network perturbation analysis to drug phenotypic activity assessment.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Modelos Biológicos , Neovascularización Patológica/genética , Inhibidores de Proteínas Quinasas/farmacología , Algoritmos , Bioensayo , Comunicación Celular/genética , Técnicas de Cocultivo , Dermis/citología , Células Endoteliales/citología , Fibroblastos/citología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neovascularización Patológica/tratamiento farmacológico , Análisis de Secuencia por Matrices de Oligonucleótidos , Oncogenes , Fenotipo , Venas Umbilicales/citologíaRESUMEN
The interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment induces proliferation and survival of MM cells, as well as osteoclastogenesis. This study investigated the therapeutic potential of novel p38 mitogen-activated protein kinase (p38MAPK) inhibitor LY2228820 (LY) in MM. Although cytotoxicity against MM cell lines was modest, LY significantly enhanced the toxicity of bortezomib by down-regulating bortezomib-induced heat shock protein 27 phosphorylation. LY inhibited interleukin-6 secretion from long term cultured-BM stromal cells and BM mononuclear cells (BMMNCs) derived from MM patients in remission. LY also inhibited macrophage inflammatory protein-1alpha secretion from patient MM cells and BMMNCs as well as normal CD14 positive osteoclast precursor cells. Moreover, LY significantly inhibited in vitro osteoclastogenesis from CD14 positive cells induced by macrophage-colony stimulating factor and soluble receptor activator of nuclear factor-kappaB ligand. Finally, LY also inhibited in vivo osteoclatogenesis in a severe combined immunodeficiency mouse model of human MM. These results suggest that LY represents a promising novel targeted approach to improve MM patient outcome both by enhancing the effect of bortezomib and by reducing osteoskeletal events.
Asunto(s)
Ácidos Borónicos/farmacología , Imidazoles/farmacología , Mieloma Múltiple/patología , Osteoclastos/efectos de los fármacos , Pirazinas/farmacología , Piridinas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Antineoplásicos , Bortezomib , Muerte Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Proteínas de Choque Térmico/metabolismo , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones SCID , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Trasplante de Neoplasias , Fosforilación/efectos de los fármacos , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
Multidrug resistance protein-5 (MRP5, ABCC5) is a member of the ATP-binding cassette transporter superfamily that effluxes a broad range of natural and xenobiotic compounds such as cyclic GMP, antiviral compounds, and cancer chemotherapeutic agents including nucleoside-based drugs, antifolate agents and platinum compounds. In cellular assays, MRP5 transfectants are less fluorescent after incubation with 5-chloromethylfluorescein diacetate (CMFDA). The present study examines the uptake of a close fluorescent analog, carboxydichlorofluorescein (CDCF), and drug substrates into inside-out membrane vesicles prepared from MRP transfected cells. MRP5-mediated uptake of CDCF was ATP-dependent and GSH-independent and possessed a Km of 12 microM and a Vmax of 56 pmol/min/mg prot. Comparison of kinetic parameters with drug substrates such as methotrexate (MTX), pemetrexed (Alimta), and the metabolite of 5-fluorouracil, 5-fluorodeoxyuridine monophosphate (5-FdUMP) (Km values of 0.3-1.3 mM) indicated that MRP5 has a 25-100-fold higher affinity for CDCF than for these drugs and that they share a common transport binding site. In addition, the potency of MRP5 inhibitors such as probenecid, MK571, and the phosphodiesterase 5 inhibitors correlated well between the uptake of CDCF and MTX. A survey of CDCF uptake by other MRPs revealed that MRP2 (ABCC2) also demonstrated ATP-dependent uptake with a Km of 19 microM and Vmax of 95.5 pmol/min/mg prot, while MRP1 (ABCC1) and MRP4 (ABCC4) had little to no uptake. Taken together, these data indicate that CDCF is a useful fluorescent drug surrogate with which to measure ATP-dependent MRP5-mediated transport.
Asunto(s)
Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , 3',5'-GMP Cíclico Fosfodiesterasas , Antimetabolitos Antineoplásicos/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5 , Glutamatos/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Células HeLa , Humanos , Cinética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Metotrexato/metabolismo , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Pemetrexed , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Probenecid/farmacología , Propionatos/farmacología , Quinolinas/farmacología , Reproducibilidad de los Resultados , Coloración y Etiquetado/métodos , TransfecciónRESUMEN
Structure-activity relationship (SAR) studies on the tricyclic isoxazole series of MRP1 modulators have resulted in the identification of potent and selective inhibitors containing cyclohexyl-based linkers. These studies ultimately identified compound 21b, which reverses drug resistance to MRP1 substrates, such as doxorubicin, in HeLa-T5 cells (EC(50)=0.093microM), while showing no inherent cytotoxicity. Additionally, 21b inhibits ATP-dependent, MRP1-mediated LTC(4) uptake into membrane vesicles prepared from the MRP1-overexpressing HeLa-T5 cells (EC(50)=0.064microM) and shows selectivity (1115-fold) against the related transporter, P-glycoprotein, in HL60/Adr and HL60/Vinc cells. Finally, when dosed in combination with the oncolytic MRP1 substrate vincristine, 21b showed tumor regression and growth delay in MRP1-overexpressing tumors in vivo.
Asunto(s)
Isoxazoles/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Células HeLa , Humanos , Isoxazoles/síntesis química , Cinética , Conformación Molecular , Relación Estructura-ActividadRESUMEN
Our study examines the ability of LY335979 (Zosuquidar trihydrochloride) to modulate 3 distinct ABC transporters that are mechanisms of drug resistance: P-glycoprotein (Pgp, ABCB1), multidrug resistance associated protein (MRP1, ABCC2) and breast cancer resistance protein (BCRP, ABCG2). Pgp-mediated resistance can be modulated by coadministration with the highly potent, selective inhibitor, LY335979. Modulation of resistance by mitoxantrone and vinorelbine, 2 drugs used to treat certain solid tumors, was examined in a 3-day cytotoxicity assay using a panel of HL60 leukemia cell lines or MCF-7 breast cancer transfectants. LY335979, at 0.5 microM, substantially reversed mitoxantrone resistance and fully reversed vinorelbine resistance of Pgp-expressing HL60/Vinc cells. However, LY335979 did not modulate drug resistance in the MRP1-expressing HL60/ADR or drug-sensitive parental HL60 cells. To ascertain if LY335979 modulates BCRP-mediated drug resistance, the sensitivity of 26-fold mitoxantrone resistant, BCRP-transfected MCF-7 cells was evaluated. Addition of 5 microM LY335979, a concentration approximately 100-fold higher than the affinity of Pgp, had little to no effect on the BCRP transfectant. [(125)I]Iodomycin photolabeled Pgp in CEM/VLB(100) membranes and was inhibited by 5 microM LY335979 and GF120918. No photolabeling of MRP or BCRP occurred in H69AR or MCF-7/BCRP membranes, respectively. These results further demonstrate that LY335979 is highly specific for Pgp and does not modulate MRP1- or BCRP-mediated resistance and can be used in combination with mitoxantrone and vinorelbine in tumor cells.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Neoplasias de la Mama/metabolismo , Dibenzocicloheptenos/farmacología , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Quinolinas/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Doxorrubicina/farmacología , Femenino , Viabilidad Fetal/efectos de los fármacos , Humanos , Immunoblotting , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas de Neoplasias/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
Tricyclic isoxazoles were identified from a screen as a novel class of selective multidrug resistance protein (MRP1) inhibitors. From a screen lead, SAR efforts resulted in the preparation of LY 402913 (9h), which inhibits MRP1 and reverses drug resistance to MRP1 substrates, such as doxorubicin, in HeLa-T5 cells (EC(50)=0.90 microM), while showing no inherent cytotoxicity. Additionally, LY 402913 inhibits ATP-dependent, MRP1-mediated LTC(4) uptake into membrane vesicles prepared from the MRP1-overexpressing HeLa-T5 cells (EC(50)=1.8 microM). LY 402913 also shows selectivity ( approximately 22-fold) against the related transporter, P-glycoprotein, in HL60/Adr and HL60/Vinc cells. Finally, when dosed in combination with the oncolytic MRP1 substrate vincristine, LY 402913 delays the growth of MRP1-overexpressing tumors in vivo.
Asunto(s)
Isoxazoles/síntesis química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos Fitogénicos/farmacología , Transporte Biológico Activo/efectos de los fármacos , División Celular/efectos de los fármacos , Doxorrubicina/farmacología , Resistencia a Medicamentos , Sinergismo Farmacológico , Compuestos Heterocíclicos con 3 Anillos/síntesis química , Compuestos Heterocíclicos con 3 Anillos/química , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Isoxazoles/química , Isoxazoles/farmacología , Leucotrieno C4/metabolismo , Relación Estructura-Actividad , Células Tumorales Cultivadas , Vincristina/farmacologíaRESUMEN
U-937 human leukemia cells were selected for resistance to doxorubicin in the presence or absence of a specific drug modulator that inhibits the activity of P-glycoprotein (Pgp), encoded by the multidrug-resistance gene (MDR1). Parental cells expressed low basal levels of the multidrug-resistance-associated gene (MRP1) and major vault protein (MVP) mRNAs and no MDR1 mRNA. Two doxorubicin-resistant cell lines were selected. Both drug-resistant cell lines upregulated the MVP mRNA level 1.5-fold within 1 cell passage. The MVP mRNA level continued to increase over time as the doxorubicin selection pressure was increased. MVP protein levels generally paralleled the mRNA levels. The 2 high molecular weight vault protein mRNAs were always expressed at constitutive levels. Fully formed vault particles consisting of the MVP, the 2 high molecular weight proteins and the vault RNA assembled and accumulated to increased levels in drug-selected cells. MVP induction is therefore the rate-limiting step for vault particle formation in U-937 cells. By passage 25 and thereafter, the selected cells were resistant to doxorubicin, etoposide, mitoxantrone and 5-fluorouracil by a pathway that was independent of MDR1, MRP1, MRP2 and breast cancer resistance protein. In summary, U-937 doxorubicin-selected cells are programmed to rapidly upregulate MVP mRNA levels, to accumulate vault particles and to become multidrug resistant.