RESUMEN
Seven new peptaibols named tolypocladamides A-G have been isolated from an extract of the fungus Tolypocladium inflatum, which inhibits the interaction between Raf and oncogenic Ras in a cell-based high-throughput screening assay. Each peptaibol contains 11 amino acid residues, an octanoyl or decanoyl fatty acid chain at the N-terminus, and a leucinol moiety at the C-terminus. The peptaibol sequences were elucidated on the basis of 2D NMR and mass spectral fragmentation analyses. Amino acid configurations were determined by advanced Marfey's analyses. Tolypocladamides A-G caused significant inhibition of Ras/Raf interactions with IC50 values ranging from 0.5 to 5.0 µM in a nanobioluminescence resonance energy transfer (NanoBRET) assay; however, no interactions were observed in a surface plasmon resonance assay for binding of the compounds to wild type or G12D mutant Ras constructs or to the Ras binding domain of Raf. NCI 60 cell line testing was also conducted, and little panel selectivity was observed.
Asunto(s)
Antineoplásicos , Hypocreales , Aminoácidos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Hypocreales/química , Peptaiboles/farmacologíaRESUMEN
RAF kinases are essential effectors of RAS, but how RAS binding initiates the conformational changes needed for autoinhibited RAF monomers to form active dimers has remained unclear. Here, we present cryo-electron microscopy structures of full-length BRAF complexes derived from mammalian cells: autoinhibited, monomeric BRAF:14-3-32:MEK and BRAF:14-3-32 complexes, and an inhibitor-bound, dimeric BRAF2:14-3-32 complex, at 3.7, 4.1, and 3.9 Å resolution, respectively. In both autoinhibited, monomeric structures, the RAS binding domain (RBD) of BRAF is resolved, revealing that the RBD forms an extensive contact interface with the 14-3-3 protomer bound to the BRAF C-terminal site and that key basic residues required for RBD-RAS binding are exposed. Moreover, through structure-guided mutational studies, our findings indicate that RAS-RAF binding is a dynamic process and that RBD residues at the center of the RBD:14-3-3 interface have a dual function, first contributing to RAF autoinhibition and then to the full spectrum of RAS-RBD interactions.
Asunto(s)
Microscopía por Crioelectrón/métodos , Mutación , Neoplasias/patología , Multimerización de Proteína , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Animales , Línea Celular , Humanos , Ratones , Neoplasias/genética , Neoplasias/metabolismo , Conformación Proteica , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/químicaRESUMEN
Activating mutations in RAS are found in approximately 30% of human cancers, resulting in the delivery of a persistent signal to critical downstream effectors that drive tumorigenesis. RAS-driven malignancies respond poorly to conventional cancer treatments and inhibitors that target RAS directly are limited; therefore, the identification of new strategies and/or drugs to disrupt RAS signaling in tumor cells remains a pressing therapeutic need. Taking advantage of the live-cell bioluminescence resonance energy transfer (BRET) methodology, we describe the development of a NanoBRET screening platform to identify compounds that modulate binding between activated KRAS and the CRAF kinase, an essential effector of RAS that initiates ERK cascade signaling. Using this strategy, libraries containing synthetic compounds, targeted inhibitors, purified natural products, and natural product extracts were evaluated. These efforts resulted in the identification of compounds that inhibit RAS/RAF binding and in turn suppress RAS-driven ERK activation, but also compounds that have the deleterious effect of enhancing the interaction to upregulate pathway signaling. Among the inhibitor hits identified, the majority were compounds derived from natural products, including ones reported to alter KRAS nanoclustering (ophiobolin A), to impact RAF function (HSP90 inhibitors and ROS inducers) as well as some with unknown targets and activities. These findings demonstrate the potential for this screening platform in natural product drug discovery and in the development of new therapeutic agents to target dysregulated RAS signaling in human disease states such as cancer.
Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Fibroblastos/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-raf/antagonistas & inhibidores , Proteínas ras/agonistas , Proteínas ras/antagonistas & inhibidores , Animales , Fibroblastos/metabolismo , Humanos , Ligandos , Nanotecnología/métodos , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas ras/metabolismoRESUMEN
Two new cyclic depsipeptides named swinhopeptolides A (1) and B (2) have been isolated from the marine sponge Theonella swinhoei cf. verrucosa, collected from Papua New Guinea. They each contain 11 diverse amino acid residues and 13-carbon polyketide moieties attached at the N-terminus. Compounds 1 and 2 each exist as two conformers in DMSO-d6 due to cis/trans isomerism of the proline residue, and their structures were successfully assigned by extensive NMR analyses complemented by chemical degradation and derivatization studies. Swinhopeptolide B (2) contains a previously undescribed 2,6,8-trimethyldeca-(2E,4E,6E)-trienoic acid moiety N-linked to a terminal serine residue. Swinhopeptolides A (1) and B (2) showed significant inhibition of the Ras/Raf signaling pathway with IC50 values of 5.8 and 8.5 µM, respectively.
Asunto(s)
Depsipéptidos/farmacología , Proteínas Proto-Oncogénicas c-raf/antagonistas & inhibidores , Theonella/química , Proteínas ras/antagonistas & inhibidores , Aminoácidos/química , Animales , Depsipéptidos/química , Depsipéptidos/aislamiento & purificación , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Papúa Nueva Guinea , Poríferos/química , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas ras/metabolismoRESUMEN
BACKGROUND: Multi-drug resistance (MDR) develops because cancer cells evade toxicity of several structurally unrelated drugs. Besides other mechanisms, MDR is linked to the overexpression of ATP Binding Cassette (ABC), transporters, among which ABCB1 is the best characterized one. Since overactivation of PI3K/Akt/mTOR plays a pivotal role in the growth of human cancers, we hypothesized whether dual PI3K and mTOR inhibitor, BEZ235 (BEZ, dactolisib) reverses resistance to doxorubicin (DOX). METHODS: Ovarian (A2780) and pancreatic (MiaPaca2) cancer cells were used to generate DOX-resistant clones by overexpressing ABCB1 or stepwise treatment of DOX. Intracellular accumulation of DOX was measured by flow cytometry after treatment with BEZ. RESULTS: BEZ treatment caused an increase in intracellular levels of DOX which was almost identical to the naïve parental cell lines. BEZ was found to be a weak substrate for ABCB1 as demonstrated by minimal increase in ATPase activity. BEZ treatment caused a dose-dependent decrease in cell viability in combination with DOX, which was associated with an increase in cleaved PARP expression in the drug resistant clones. CONCLUSIONS: These results suggest that BEZ is a non-substrate inhibitor of ABCB1 and is able to effectively re-sensitize cells overexpressing ABCB1 to the effects of DOX. GENERAL SIGNIFICANCE: Dual PI3 Kinase/mTOR inhibitor, BEZ, has the potential to reverse MDR in cancer patients.
Asunto(s)
Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Serina-Treonina Quinasas TOR/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Línea Celular Tumoral , Doxorrubicina/efectos adversos , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Imidazoles/farmacología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Quinolinas/farmacologíaRESUMEN
The Ras GTPases are frequently mutated in human cancer, and, although the Raf kinases are essential effectors of Ras signaling, the tumorigenic properties of specific Ras-Raf complexes are not well characterized. Here, we examine the ability of individual Ras and Raf proteins to interact in live cells using bioluminescence resonance energy transfer (BRET) technology. We find that C-Raf binds all mutant Ras proteins with high affinity, whereas B-Raf exhibits a striking preference for mutant K-Ras. This selectivity is mediated by the acidic, N-terminal segment of B-Raf and requires the K-Ras polybasic region for high-affinity binding. In addition, we find that C-Raf is critical for mutant H-Ras-driven signaling and that events stabilizing B-Raf/C-Raf dimerization, such as Raf inhibitor treatment or certain B-Raf mutations, can allow mutant H-Ras to engage B-Raf with increased affinity to promote tumorigenesis, thus revealing a previously unappreciated role for C-Raf in potentiating B-Raf function.
Asunto(s)
Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Neoplasias/enzimología , Quinasas raf/metabolismo , Proteínas ras/metabolismo , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Masculino , Ratones , Mutación , Células 3T3 NIH , Neoplasias/genética , Neoplasias/patología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal/genética , Esferoides Celulares , Quinasas raf/genética , Proteínas ras/genéticaRESUMEN
The Raf protein kinases are key intermediates in cellular signal transduction, functioning as direct effectors of the Ras GTPases and as the initiating kinases in the ERK cascade. In human cancer, Raf activity is frequently dysregulated due to mutations in the Raf family member B-Raf or to alterations in upstream Raf regulators, including Ras and receptor tyrosine kinases. First-generation Raf inhibitors, such as vemurafenib and dabrafenib, have yielded dramatic responses in malignant melanomas containing B-Raf mutations; however, their overall usefulness has been limited by both intrinsic and acquired drug resistance. In particular, issues related to the dimerisation of the Raf kinases can impact the efficacy of these compounds and are a primary cause of drug resistance. Here, we will review the importance of Raf dimerisation in cell signalling as well as its effects on Raf inhibitor therapy, and we will present the new strategies that are being pursued to overcome the 'Raf Dimer Dilemma'.
Asunto(s)
Neoplasias/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Quinasas raf/química , Resistencia a Antineoplásicos , Humanos , Imidazoles/farmacología , Imidazoles/uso terapéutico , Modelos Moleculares , Terapia Molecular Dirigida , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Oximas/farmacología , Oximas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Multimerización de Proteína , Transducción de Señal , Vemurafenib/farmacología , Vemurafenib/uso terapéutico , Quinasas raf/antagonistas & inhibidores , Quinasas raf/genéticaRESUMEN
Ras pathway signaling plays a critical role in cell growth control and is often upregulated in human cancer. The Raf kinases selectively interact with GTP-bound Ras and are important effectors of Ras signaling, functioning as the initiating kinases in the ERK cascade. Here, we identify a route for the phospho-inhibition of Ras/Raf/MEK/ERK pathway signaling that is mediated by the stress-activated JNK cascade. We find that key Ras pathway components, the RasGEF Sos1 and the Rafs, are phosphorylated on multiple S/TP sites in response to JNK activation and that the hyperphosphorylation of these sites renders the Rafs and Sos1 unresponsive to upstream signals. This phospho-regulatory circuit is engaged by cancer therapeutics, such as rigosertib and paclitaxel/Taxol, that activate JNK through mitotic and oxidative stress as well as by physiological regulators of the JNK cascade and may function as a signaling checkpoint to suppress the Ras pathway during conditions of cellular stress.
Asunto(s)
Glicina/análogos & derivados , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Paclitaxel , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Sulfonas , Activación Enzimática/efectos de los fármacos , Glicina/farmacocinética , Glicina/farmacología , Células HeLa , Humanos , Estrés Oxidativo , Paclitaxel/farmacocinética , Paclitaxel/farmacología , Fosforilación , Sulfonas/farmacocinética , Sulfonas/farmacología , Proteínas ras/metabolismoRESUMEN
Pancreatic cancer has the lowest 5-year survival rate of all major cancers despite decades of effort to design and implement novel, more effective treatment options. In this study, we tested whether the dual phosphoinositide 3-kinase/mechanistic target of rapamycin inhibitor BEZ235 (BEZ) potentiates the antitumor effects of doxorubicin (DOX) against pancreatic cancer. Cotreatment of BEZ235 with DOX resulted in dose-dependent inhibition of the phosphoinositide 3-kinase/mechanistic target of rapamycin survival pathway, which corresponded with an increase in poly ADP ribose polymerase cleavage. Moreover, BEZ cotreatment significantly improved the effects of DOX toward both cell viability and cell death in part through reduced Bcl-2 expression and increased expression of the shorter, more cytotoxic forms of BIM. BEZ also facilitated intracellular accumulation of DOX, which led to enhanced DNA damage and reactive oxygen species generation. Furthermore, BEZ in combination with gemcitabine reduced MiaPaca2 cell proliferation but failed to increase reactive oxygen species generation or BIM expression, resulting in reduced necrosis and apoptosis. Treatment with BEZ and DOX in mice bearing tumor xenographs significantly repressed tumor growth as compared with BEZ, DOX, or gemcitabine. Additionally, in contrast to the enhanced expression seen in MiaPaca2 cells, BEZ and DOX cotreatment reduced BIM expression in H9C2 cardiomyocytes. Also, the Bcl-2/Bax ratio was increased, which was associated with a reduction in cell death. In vivo echocardiography showed decreased cardiac function with DOX treatment, which was not improved by combination treatment with BEZ. Thus, we propose that combining BEZ with DOX would be a better option for patients than current standard of care by providing a more effective tumor response without the associated increase in toxicity.
Asunto(s)
Antibióticos Antineoplásicos/efectos adversos , Doxorrubicina/farmacología , Imidazoles/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Quinolinas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Antibióticos Antineoplásicos/farmacología , Apoptosis , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Cardiotoxicidad , Supervivencia Celular , Doxorrubicina/efectos adversos , Sinergismo Farmacológico , Femenino , Células HCT116 , Humanos , Imidazoles/uso terapéutico , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Desnudos , Miocitos Cardíacos/efectos de los fármacos , Neoplasias Pancreáticas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Quinolinas/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Diabetic patients suffer augmented severity of myocardial infarction. Excessive activation of the mammalian target of rapamycin (mTOR) and decreased activation of STAT3 are implicated in diabetic complications. Considering the potent cardioprotective effect of mTOR inhibitor, rapamycin, we hypothesized that reperfusion therapy with rapamycin would reduce infarct size in the diabetic hearts through STAT3 signaling. Hearts from adult male db/db or wild type (WT) C57 mice were isolated and subjected to 30 min of normothermic global ischemia and 60 min of reperfusion in Langendorff mode. Rapamycin (100 nM) was infused at the onset of reperfusion. Myocardial infarct size (IS) was significantly reduced in rapamycin-treated mice (13.3 ± 2.4 %) compared to DMSO vehicle control (35.9 ± 0.9 %) or WT mice (27.7 ± 1.1 %). Rapamycin treatment restored phosphorylation of STAT3 and enhanced AKT phosphorylation (target of mTORC2), but significantly reduced ribosomal protein S6 phosphorylation (target of mTORC1) in the diabetic heart. To determine the cause and effect relationship of STAT3 in cardioprotection, inducible cardiac-specific STAT3-deficient (MCM TG:STAT3(flox/flox)) and WT mice (MCM TG:STAT3(flox/flox)) were made diabetic by feeding high fat diet (HFD). Rapamycin given at reperfusion reduced IS in WT mice but not in STAT3-deficient mice following I/R. Moreover, cardiomyocytes isolated from HFD-fed WT mice showed resistance against necrosis (trypan blue staining) and apoptosis (TUNEL assay) when treated with rapamycin during reoxygenation following simulated ischemia. Such protection was absent in cardiomyocytes from HFD-fed STAT3-deficient mice. STAT3 signaling plays critical role in reducing IS and attenuates cardiomyocyte death following reperfusion therapy with rapamycin in diabetic heart.
Asunto(s)
Cardiomiopatías Diabéticas/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Factor de Transcripción STAT3/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Western Blotting , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Cardiomiopatías Diabéticas/patología , Modelos Animales de Enfermedad , Inmunosupresores/farmacología , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Daño por Reperfusión Miocárdica/etiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sirolimus/farmacologíaRESUMEN
The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with clinically relevant chemotherapies to kill gastrointestinal/genitourinary cancer cells. In bladder cancer cells, regardless of H-RAS mutational status, at clinically achievable doses, PDE5 inhibitors interacted in a greater than additive fashion with doxorubicin/mitomycin C/gemcitabine/cisplatin/paclitaxel to cause cell death. In pancreatic tumor cells expressing mutant active K-RAS, PDE5 inhibitors interacted in a greater than additive fashion with doxorubicin/gemcitabine/paclitaxel to cause cell death. The most potent PDE5 inhibitor was sildenafil. Knock down of PDE5 expression recapitulated the combination effects of PDE5 inhibitor drugs with chemotherapy drugs. Expression of cellular FLICE-like inhibitory protein-short did not significantly inhibit chemotherapy lethality but did significantly reduce enhanced killing in combination with sildenafil. Overexpression of B-cell lymphoma-extra large suppressed individual and combination drug toxicities. Knock down of CD95 or Fas-associated death domain protein suppressed drug combination toxicity. Combination toxicity was also abolished by necrostatin or receptor interacting protein 1 knock down. Treatment with PDE5 inhibitors and chemotherapy drugs promoted autophagy, which was maximal at â¼24 hour posttreatment, and 3-methyl adenine or knock down of Beclin1 suppressed drug combination lethality by â¼50%. PDE5 inhibitors enhanced and prolonged the induction of DNA damage as judged by Comet assays and γhistone 2AX (γH2AX) and checkpoint kinase 2 (CHK2) phosphorylation. Knock down of ataxia telangiectasia mutated suppressed γH2AX and CHK2 phosphorylation and enhanced drug combination lethality. Collectively our data demonstrate that the combination of PDE5 inhibitors with standard of care chemotherapy agents for gastrointestinal/genitourinary cancers represents a novel modality.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Gastrointestinales/tratamiento farmacológico , Inhibidores de Fosfodiesterasa 5/farmacología , Neoplasias Urogenitales/tratamiento farmacológico , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Quinasa de Punto de Control 2/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Neoplasias Gastrointestinales/metabolismo , Histonas/metabolismo , Humanos , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/metabolismo , Ratones , Fosforilación/efectos de los fármacos , Ratas , Neoplasias Urogenitales/metabolismo , Receptor fas/metabolismoRESUMEN
BACKGROUND: Insulin resistance impairs nitric oxide (NO) bioavailability and obesity promotes a state of chronic inflammation and damages the vascular endothelium. Phosphodiesterase-5 inhibitors restore NO signaling and may reduce circulating inflammatory markers, and improve metabolic parameters through a number of mechanisms. We hypothesized that daily administration of the PDE-5 inhibitor, tadalafil (TAD) will attenuate inflammation, improve fasting plasma glucose and triglyceride levels, body weight, and reduce infarct size after ischemia/reperfusion injury in obese, diabetic mice. METHODS: Twenty leptin receptor null (db/db) mice underwent treatment with TAD (1 mg/Kg) or 10% DMSO for 28 days. Body weight and fasting plasma glucose levels were determined weekly. Upon completion, hearts were isolated and subjected to 30 min global ischemia followed by 60 min reperfusion in a Langendorff model. Plasma samples were taken for cytokine analysis and fasting triglyceride levels. Infarct size was measured using computer morphometry of tetrazolium stained sections. Additionally, ventricular cardiomyocytes were isolated and subjected to 40 min of simulated ischemia and reoxygenation. Necrosis was determined using trypan blue exclusion and LDH release assay and apoptosis was assessed by TUNEL assay after 1 h or 18 h of reoxygenation, respectively. RESULTS: Treatment with TAD caused a reduction in infarct size in the diabetic heart (23.2 ± 1.5 vs. 47.8 ± 3.7%, p<0.01, n = 6/group), reduced fasting glucose levels (292 ± 31.8 vs. 511 ± 19.3 mg/dL, p<0.001) and fasting triglycerides (43.3 ± 21 vs. 129.7 ± 29 mg/dL, p<0.05) as compared to DMSO, however body weight was not significantly reduced. Circulating tumor necrosis factor-α and interleukin-1ß were reduced after treatment compared to control (257 ± 16.51 vs. 402.3 ± 17.26 and 150.8 ± 12.55 vs. 264 ± 31.85 pg/mL, respectively; P<0.001) Isolated cardiomyocytes from TAD-treated mice showed reduced apoptosis and necrosis. CONCLUSION: We have provided the first evidence that TAD therapy ameliorates circulating inflammatory cytokines and chemokines in a diabetic animal model while improving fasting glucose levels and reducing infarct size following ischemia-reperfusion injury in the heart.
Asunto(s)
Carbolinas/farmacología , Diabetes Mellitus Experimental/tratamiento farmacológico , Infarto del Miocardio/tratamiento farmacológico , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Necrosis/tratamiento farmacológico , Obesidad/tratamiento farmacológico , Inhibidores de Fosfodiesterasa/farmacología , Animales , Apoptosis/efectos de los fármacos , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Carbolinas/uso terapéutico , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Esquema de Medicación , Ayuno , Interleucina-1beta/sangre , Masculino , Ratones , Ratones Noqueados , Infarto del Miocardio/complicaciones , Infarto del Miocardio/metabolismo , Daño por Reperfusión Miocárdica/complicaciones , Daño por Reperfusión Miocárdica/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Necrosis/complicaciones , Necrosis/metabolismo , Obesidad/complicaciones , Obesidad/metabolismo , Inhibidores de Fosfodiesterasa/uso terapéutico , Receptores de Leptina/genética , Tadalafilo , Triglicéridos/sangre , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Hemiasterlins are cytotoxic tripeptides with antimicrotubule activity originally isolated from marine sponges. We have developed new hemiasterlin derivatives BF65 and BF78 that are highly potent to induce cancer cell death in the low nanomolar range. Examination of their mechanisms of cell cycle arrest and disruption of microtubules revealed an unusual characteristic in addition to anti-tubulin effect. Immunofluorescence staining revealed that A549 lung carcinoma cells treated with BF65 or BF78 exhibited both monopolar and multipolar mitotic spindles. Centrosomes were separated with short spindle microtubules in cells with multipolar spindles. In vitro tubulin polymerization assay confirmed that both BF65 and BF78 were highly potent to inhibit tubulin polymerization. These two compounds induced the formation of monoastral spindles suggesting that they might be inhibitors of mitotic kinesins such as KSP/Eg5. However, kinetic measurement of microtubule activated kinesin ATPase activity demonstrated that unlike the positive control monastrol, neither BF65 nor BF78 suppressed KSP/Eg5 activity. Hence the effect may be a variant form of tubulin inhibition. Similar to vinca alkaloids, BF compounds synergized with a colchicine site microtubule inhibitor stilbene 5c both in vitro and in vivo, which may provide a potential drug combination in the future clinical application.
Asunto(s)
Antineoplásicos/farmacología , Oligopéptidos/farmacología , Polimerizacion/efectos de los fármacos , Estilbenos/farmacología , Moduladores de Tubulina/farmacología , Tubulina (Proteína)/metabolismo , Animales , Antineoplásicos/química , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Sinergismo Farmacológico , Técnica del Anticuerpo Fluorescente , Humanos , Cinesinas/metabolismo , Ratones , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Oligopéptidos/química , Transducción de Señal/efectos de los fármacos , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A representative series of structural analogues of the antimitotic tripeptides hemiasterlins have been synthesized. The key-step of this synthetic strategy consists of an Ag(2)O-promoted nucleophilic substitution on a common precursor, a chiral non-racemic 2-bromoacyl derivative. Simple variation of nucleophile substituents allows a rapid and stereocontrolled development of new series of derivatives. Some reported compounds showed potent biological activity as growth inhibitors of cancer cell lines and tubulin polymerization inhibitors.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Animales , Antineoplásicos/química , Línea Celular Tumoral , Humanos , Estructura Molecular , Oligopéptidos/química , RatasRESUMEN
Colchicine site tubulin inhibitors are currently developed as vascular disrupting agents (VDAs). However, they were found to have cardiotoxicity in clinical trials. To overcome the problem, we developed a stilbene derivative, cis-3, 4', 5-trimethoxy-3'-aminostilbene (stilbene 5c), which is highly potent and has no bone marrow and cardiac toxicity in mice. Here we attempt to optimize stilbene 5c using computer-based drug design and synthesize derivatives with benzimidazole or indole group. Biological evaluation showed that they are weaker than stilbene 5c without better water solubility. Alternative approach was thus adopted to make prodrugs of stilbene 5c. A water-soluble prodrug PD7 was synthesized by addition of a morpholino group with carbamate linkage to the amino group of stilbene 5c. In vitro studies show that PD7 induces mitotic arrest and disrupts microtubule similar to stilbene 5c. The cell signaling events in Cdc2, p53, Akt, and aurora kinase are similar in cells treated with stilbene 5c, CA4 or PD7, suggesting that they share the same mechanism. Although PD7 is less effective than stilbene 5c in vitro, the biological activity of PD7 as a single agent is similar to that of stilbene 5c. Combination of PD7 with VEGF inhibitor bevacizumab significantly enhances the therapeutic efficacy of PD7 in mouse xenograft model. These data suggest that PD7 could be a good candidate for further pre-clinical and clinical development as a new VDA for cancer therapy.