RESUMEN
The anti-apoptotic protein MCL-1 is a key regulator of cancer cell survival and a known resistance factor for small-molecule BCL-2 family inhibitors such as ABT-263 (navitoclax), making it an attractive therapeutic target. However, directly inhibiting this target requires the disruption of high-affinity protein-protein interactions, and therefore designing small molecules potent enough to inhibit MCL-1 in cells has proven extremely challenging. Here, we describe a series of indole-2-carboxylic acids, exemplified by the compound A-1210477, that bind to MCL-1 selectively and with sufficient affinity to disrupt MCL-1-BIM complexes in living cells. A-1210477 induces the hallmarks of intrinsic apoptosis and demonstrates single agent killing of multiple myeloma and non-small cell lung cancer cell lines demonstrated to be MCL-1 dependent by BH3 profiling or siRNA rescue experiments. As predicted, A-1210477 synergizes with the BCL-2/BCL-XL inhibitor navitoclax to kill a variety of cancer cell lines. This work represents the first description of small-molecule MCL-1 inhibitors with sufficient potency to induce clear on-target cellular activity. It also demonstrates the utility of these molecules as chemical tools for dissecting the basic biology of MCL-1 and the promise of small-molecule MCL-1 inhibitors as potential therapeutics for the treatment of cancer.
Asunto(s)
Compuestos de Anilina/farmacología , Apoptosis/efectos de los fármacos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Neoplasias/patología , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfonamidas/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Ácidos Carboxílicos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Indoles/farmacología , Proteínas de la Membrana/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
Non-small-cell lung cancer (NSCLC) is the most deadly type of cancer in the United States and worldwide. Although new therapy is available, the survival rate of NSCLC patients remains low. One hallmark of cancer cells is defects in the apoptotic cell death program. In this study, we investigate the role of B-cell lymphoma 2 (Bcl-2) family members Bcl-2, Bcl-x(L) and Mcl-1, known to regulate cell survival and death, in a panel of fourteen NSCLC cell lines. NSCLC cell lines express high levels of Mcl-1 and Bcl-x(L), but not Bcl-2. Silencing the expression of Mcl-1 with small interfering RNA (siRNA) oligonucleotides potently killed a subgroup of NSCLC cell lines. In contrast, Bcl-x(L) siRNA had no effect in these lines unless Mcl-1 siRNA was also introduced. Interestingly, high MCL1 to BCL-xl messenger RNA determines whether the cells depend on Mcl-1 for survival. We further investigated the role of Mcl-1 in NSCLC cells using a Mcl-1-dependent cell line, H23. The expression of a complementary DNA containing only the coding region of MCL1 rescued H23 cells from the toxicity of a 3' untranslated region (UTR) targeting Mcl-1 siRNA but not a siRNA targeting the coding region of MCL1. Furthermore, we show that Mcl-1 sequesters the BH3-only protein Noxa and Bim and the apoptotic effector Bak. Not surprisingly, Noxa, Bim, or Bak knockdown partially rescued H23 cells from toxicity mediated by Mcl-1 siRNA to different degrees. Collectively, our results indicate that targeting Mcl-1 may improve therapy for a subset of NSCLC patients.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Línea Celular Tumoral , Humanos , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
Platelets are relatively short-lived, anucleated cells that are essential for proper hemostasis. The regulation of platelet survival in the circulation remains poorly understood. The process of platelet activation and senescence in vivo is associated with processes similar to those observed during apoptosis in nucleated cells, including loss of mitochondrial membrane potential, caspase activation, phosphatidylserine (PS) externalization, and cell shrinkage. ABT-737, a potent antagonist of Bcl-2, Bcl-X(L), and Bcl-w, induces apoptosis in nucleated cells dependent on these proteins for survival. In vivo, ABT-737 induces a reduction of circulating platelets that is maintained during drug therapy, followed by recovery to normal levels within several days after treatment cessation. Whole body scintography utilizing ([111])Indium-labeled platelets in dogs shows that ABT-737-induced platelet clearance is primarily mediated by the liver. In vitro, ABT-737 treatment leads to activation of key apoptotic processes including cytochrome c release, caspase-3 activation, and PS externalization in isolated platelets. Despite these changes, ABT-737 is ineffective in promoting platelet activation as measured by granule release markers and platelet aggregation. Taken together, these data suggest that ABT-737 induces an apoptosis-like response in platelets that is distinct from platelet activation and results in enhanced clearance in vivo by the reticuloendothelial system.
Asunto(s)
Plaquetas/citología , Plaquetas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Plaquetas/efectos de los fármacos , Separación Celular , Supervivencia Celular/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Perros , Relación Dosis-Respuesta a Droga , Exocitosis/efectos de los fármacos , Citometría de Flujo , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Nitrofenoles/farmacología , Fosfatidilserinas/metabolismo , Piperazinas/farmacología , Agregación Plaquetaria/efectos de los fármacos , Recuento de Plaquetas , Sulfonamidas/farmacologíaRESUMEN
The preparation and characterization of a series of selective glucocorticoid receptor modulators are described. The preliminary structure-activity relationship of nonaromatic C-5 substitution on the tetracyclic quinoline core showed a preference for small lipophilic side chains. Proper substitution at this position maintained the transcriptional repression of proinflammatory transcription factors while diminishing the transcriptional activation activity of the ligand/glucocorticoid receptor complex. The optimal compounds described in this study were the allyl analogue 18 and cyclopentyl analogue 32. These candidates showed slightly less potent, highly efficacious E-selectin repression with significantly reduced levels of glucocorticoid response element activation in reporter gene assays vs prednisolone. Allyl analogue 18 was evaluated in vivo. An oral dose of 18 showed an ED(50) = 1.7 mg/kg as compared to 1.2 mg/kg for prednisolone in the Sephadex-induced pulmonary eosinophilia model and an ED(50) = 15 mg/kg vs 4 mg/kg for prednisolone in the carrageenan-induced paw edema model.
Asunto(s)
Benzopiranos/síntesis química , Quinolinas/síntesis química , Receptores de Glucocorticoides/efectos de los fármacos , Animales , Benzopiranos/química , Benzopiranos/farmacología , Unión Competitiva , Carragenina , Línea Celular , Chlorocebus aethiops , Depresión Química , Selectina E/genética , Selectina E/metabolismo , Edema/inducido químicamente , Edema/patología , Eosinófilos/patología , Genes Reporteros , Humanos , Insectos , Luciferasas/genética , Luciferasas/metabolismo , Masculino , FN-kappa B/genética , FN-kappa B/metabolismo , Neumonía/patología , Quinolinas/química , Quinolinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Glucocorticoides/genética , Elementos de Respuesta , Relación Estructura-Actividad , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
A novel class of functional ligands for the human glucocorticoid receptor is described. Substituents in the C-10 position of the tetracyclic core are essential for glucocorticoid receptor (GR) selectivity versus other steroid receptors. The C-5 position is derivatized with meta-substituted aromatic groups, resulting in analogues with a high affinity for GR (K(i) = 2.4-9.3 nM) and functional activity comparable to prednisolone in reporter gene assays of glucocorticoid-mediated gene transcription. The biological activity of these novel quinolines was also prednisolone-equivalent in whole cell assays of glucocorticoid function, and compound 13 was similar to prednisolone (po ED(50) = 2.8 mpk for 13 vs ED(50) = 1.2 mpk for prednisolone) in a rodent model of asthma (sephadex-induced eosinophil influx).
Asunto(s)
Antiinflamatorios/síntesis química , Benzopiranos/síntesis química , Prednisolona/farmacología , Quinolinas/síntesis química , Receptores de Glucocorticoides/metabolismo , Animales , Antiinflamatorios/química , Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Benzopiranos/química , Benzopiranos/metabolismo , Benzopiranos/farmacología , Línea Celular , Selectina E/genética , Selectina E/metabolismo , Genes Reporteros , Humanos , Ligandos , Luciferasas/genética , Quinolinas/química , Quinolinas/metabolismo , Quinolinas/farmacología , Ratas , Estereoisomerismo , Activación Transcripcional , TransfecciónRESUMEN
In search of a uroselective agent that exhibits a high level of selectivity for the alpha(1A) receptor, a novel series of tricyclic hexahydrobenz[e]isoindoles was synthesized. A generic pharmacophoric model was developed requiring the presence of a basic amine core and a fused heterocyclic side chain separated by an alkyl chain. It was shown that the 6-OMe substitution with R, R stereochemistry of the ring junction of the benz[e]isoindole and a two-carbon spacer chain were optimal. In contrast to the highly specific requirements for the benz[e]isoindole portion and linker chain, a wide variety of tricyclic fused heterocyclic attachments were tolerated with retention of potency and selectivity. In vitro functional assays for the alpha(1) adrenoceptor subtypes were used to further characterize these compounds, and in vivo models of vascular vs prostatic tone were used to assess uroselectivity.