RESUMEN
This laboratory and others have shown that agents that inhibit the in vitro catalytic activity of methionine aminopeptidase-2 (MetAP2) are effective in blocking angiogenesis and tumor growth in preclinical models. However, these prototype MetAP2 inhibitors are clearly not optimized for therapeutic use in the clinic. We have discovered an orally active class of MetAP2 inhibitors, the anthranilic acid sulfonamides exemplified by A-800141, which is highly specific for MetAP2. This orally bioavailable inhibitor exhibits an antiangiogenesis effect and a broad anticancer activity in a variety of tumor xenografts including B cell lymphoma, neuroblastoma, and prostate and colon carcinomas, either as a single agent or in combination with cytotoxic agents. We also have developed a biomarker assay to evaluate in vivo MetAP2 inhibition in circulating mononuclear cells and in tumors. This biomarker assay is based on the N-terminal methionine status of the MetAP2-specific substrate GAPDH in these cells. In cell cultures in vitro, the sulfonamide MetAP2 inhibitor A-800141 caused the formation of GAPDH variants with an unprocessed N-terminal methionine. A-800141 blocked tumor growth and MetAP2 activity in a similar dose-response in mouse models, demonstrating the antitumor effects seen for A-800141 are causally connected to MetAP2 inhibition in vivo. The sulfonamide MetAP2 inhibitor and GAPDH biomarker in circulating leukocytes may be used for the development of a cancer treatment.
Asunto(s)
Aminopeptidasas/antagonistas & inhibidores , División Celular/efectos de los fármacos , Metaloendopeptidasas/antagonistas & inhibidores , Neoplasias/patología , Inhibidores de Proteasas/farmacología , Administración Oral , Aminopeptidasas/metabolismo , Animales , Catálisis , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Masculino , Metaloendopeptidasas/metabolismo , Ratones , Ratones SCID , Neoplasias/enzimología , Inhibidores de Proteasas/administración & dosificación , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismoRESUMEN
This Letter describes the lead discovery, optimization, and biological characterization of a series of substituted 4-amino-1H-pyrazolo[3,4-d]pyrimidines as potent inhibitors of IGF1R, EGFR, and ErbB2. The leading compound 11 showed an IGF1R IC(50) of 12 nM, an EGFR (L858R) IC(50) of 31 nM, and an ErbB2 IC(50) of 11 nM, potent activity in cellular functional and anti-proliferation assays, as well as activity in an in vivo pharmacodynamic assay.
Asunto(s)
Adenina/análogos & derivados , Antineoplásicos/química , Antineoplásicos/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptor ErbB-2/antagonistas & inhibidores , Receptor IGF Tipo 1/antagonistas & inhibidores , Adenina/química , Adenina/farmacocinética , Adenina/farmacología , Animales , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Receptores ErbB/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias/tratamiento farmacológico , Ratas , Receptor ErbB-2/metabolismo , Receptor IGF Tipo 1/metabolismo , Relación Estructura-ActividadRESUMEN
The design and enzyme activities of a novel class of imidazo[2,1-b]thiazoles is presented.
Asunto(s)
Receptores ErbB/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Tiazoles/farmacología , Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Tiazoles/químicaRESUMEN
BACKGROUND: The insulin-like growth factor (IGF) axis is an important signaling pathway in the growth and survival of many cell and tissue types. This pathway has also been implicated in many aspects of cancer progression from tumorigenesis to metastasis. The multiple roles of IGF signaling in cancer suggest that inhibition of the pathway might yield clinically effective therapeutics. METHODS: We describe A-928605, a novel pyrazolo [3,4-d]pyrimidine small molecule inhibitor of the receptor tyrosine kinases (IGF1R and IR) responsible for IGF signal transduction. This compound was first tested for its activity and selectivity via conventional in vitro kinome profiling and cellular IGF1R autophosphorylation. Additionally, cellular selectivity and efficacy of A-928605 were analyzed in an IGF1R oncogene-addicted cell line by proliferation, signaling and microarray studies. Finally, in vivo efficacy of A-928605 was assessed in the oncogene-addicted cell line and in a neuroblastoma model as a single agent as well as in combination with clinically approved therapeutics targeting EGFR in models of pancreatic and non-small cell lung cancers. RESULTS: A-928605 is a selective IGF1R inhibitor that is able to abrogate activation of the pathway both in vitro and in vivo. This novel compound dosed as a single agent is able to produce significant growth inhibition of neuroblastoma xenografts in vivo. A-928605 is also able to provide additive effects when used in combination with clinically approved agents directed against EGFR in non-small cell lung and human pancreatic tumor models. CONCLUSION: These results suggest that a selective IGF1R inhibitor such as A-928605 may provide a useful clinical therapeutic for IGF pathway affected tumors and warrants further investigation.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Neoplasias/fisiopatología , Proteínas Oncogénicas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Receptores de Somatomedina/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Desnudos , Ratones SCID , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Oncogénicas/metabolismo , Fosforilación/efectos de los fármacos , Receptores de Somatomedina/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Emerging clinical and pre-clinical data indicate that both insulin-like growth factor receptor (IGF-IR) and members of the epidermal growth factor (EGF) family of receptor tyrosine kinases (RTKs) exhibit significant cross-talk in human cancers. Therefore, a small molecule that successfully inhibits the signaling of both classes of oncogenic kinases might provide an attractive agent for chemotherapeutic use. Herein, we disclose the structure activity relationships that led to the synthesis and biological characterization of 14, a novel small molecule inhibitor of both IGF-IR and members of the epidermal growth factor family of RTKs.
Asunto(s)
Antineoplásicos/síntesis química , Química Farmacéutica/métodos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptores de Somatomedina/antagonistas & inhibidores , Receptores de Somatomedina/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Dimerización , Diseño de Fármacos , Humanos , Pulmón/metabolismo , Modelos Químicos , Neoplasias/metabolismo , Fosforilación , Pirimidinas/química , Proteínas Tirosina Quinasas Receptoras/química , Transducción de SeñalRESUMEN
Methionine aminopeptidase-2 (MetAP2) is a novel target for cancer therapy. As part of an effort to discover orally active reversible inhibitors of MetAP2, a series of anthranilic acid sulfonamides with micromolar affinities for human MetAP2 were identified using affinity selection by mass spectrometry (ASMS) screening. These micromolar hits were rapidly improved to nanomolar leads on the basis of insights from protein crystallography; however, the compounds displayed extensive binding to human serum albumin and had limited activity in cellular assays. Modifications based on structural information on the binding of lead compounds to both MetAP2 and domain III of albumin allowed the identification of compounds with significant improvements in both parameters, which showed good cellular activity in both proliferation and methionine processing assays.
Asunto(s)
Aminopeptidasas/química , Antineoplásicos/síntesis química , Metaloendopeptidasas/química , Albúmina Sérica/química , Sulfonamidas/síntesis química , ortoaminobenzoatos/síntesis química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Técnicas In Vitro , Espectrometría de Masas , Metionina/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Ratas , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacología , ortoaminobenzoatos/química , ortoaminobenzoatos/farmacologíaRESUMEN
The heptapeptide 1, NAc-Gly-Val-DIle-Thr-Arg-Ile-ArgNHEt, a structurally modified fragment derived from the second type-1 repeat of thrombospondin-1 (TSP-1), is known to possess antiangiogenic activity. However, therapeutic utility could not be demonstrated because this peptide has a very short half-life in rodents. To optimize the PD/PK profile of 1, we initiated a systematic SAR study. The initial structural modifications were performed at positions 5 and 7 of peptide 1 and at the N- and C-termini. Out of several hundred peptides synthesized, the nonapeptide 5 (ABT-526) emerged as a promising lead. ABT-526 inhibited VEGF-induced HMVEC cell migration and tube formation in the nanomolar range and increased apoptosis of HUAEC cells. ABT-526 showed acceptable PK in rodents, dog, and monkey. ABT-526, when incorporated in an angiogenic pellet implanted in the rat cornea at 10 microM, reduced neovascularization by 92%. Substitution of DalloIle in place of DIle in ABT-526 provided nonapeptide 6 (ABT-510), which was 30-fold less active than ABT-526 in the EC migration but 20-fold more active in the tube formation assay. In comparison to ABT-526, ABT-510 has increased water solubility and slower clearance in dog and monkey. Radiolabeled ABT-510 demonstrated saturable binding to HMVEC cells at 0.02-20 nM concentrations and was displaceable by TSP-1. ABT-510 and ABT-526 were shown to significantly increase apoptosis of HUAEC cells. ABT-510 was effective in blocking neovascularization in the mouse Matrigel plug model and inhibited tumor growth in the mouse Lewis lung carcinoma model. Previous studies had shown that ABT-510 was effective in inhibiting the outgrowth of murine melanoma metastases in syngeneic mice and in blocking the growth of human bladder carcinoma implanted in nude mice. It had been also shown that ABT-510 could regress tumor lesions in pet dogs or cause unexpected stabilization of the disease in advanced canine cancer. ABT-526 and ABT-510 are the first compounds in the class of potent inhibitors of angiogenesis that mimic the antiangiogenic function of TSP-1. ABT-510 is currently in phase II clinical studies.
Asunto(s)
Inhibidores de la Angiogénesis/síntesis química , Oligopéptidos/síntesis química , Trombospondina 1/química , Inhibidores de la Angiogénesis/farmacocinética , Inhibidores de la Angiogénesis/farmacología , Animales , Apoptosis , Capilares/citología , Quimiotaxis/efectos de los fármacos , Córnea/irrigación sanguínea , Córnea/efectos de los fármacos , Perros , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Endotelio Vascular/citología , Femenino , Haplorrinos , Humanos , Técnicas In Vitro , Inyecciones Intravenosas , Ratones , Ratones Endogámicos C57BL , Imitación Molecular , Neovascularización Fisiológica/efectos de los fármacos , Oligopéptidos/farmacocinética , Oligopéptidos/farmacología , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Cordón Umbilical/citología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Eotaxin, an inducer of eosinophil migration and activation, exerts its activity by binding to CCR3, the C-C chemokine receptor 3. An inhibitor of the eotaxin-CCR3 binding interaction may have potential as an anti-inflammatory drug for treatment of asthma, parasitic infections, and allergic disorders. A radioligand binding assay was developed using HEK cells transfected with CCR3, with (125)I eotaxin as the ligand. Whole cells grown on polylysine-coated plates were used as the receptor source for the screen. Screening of more than 200,000 compounds with this assay yielded a number of screening hits, and of these, 2 active novel antagonists were identified. These compounds showed inhibitory effects on eosinophil chemotaxis in both in vitro and in vivo assays.
Asunto(s)
Bioquímica/métodos , Receptores de Quimiocina/química , Receptores de Quimiocina/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Membrana Celular/metabolismo , Movimiento Celular , Quimiocina CCL11 , Quimiocinas CC/química , Quimiocinas CC/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Eosinófilos/metabolismo , Humanos , Ligandos , Ratones , Ratones Endogámicos BALB C , Modelos Químicos , Polilisina/química , Unión Proteica , Ensayo de Unión Radioligante , Receptores CCR3 , TransfecciónRESUMEN
The catalytic activity of methionine aminopeptidase-2 (MetAP2) has been pharmacologically linked to cell growth, angiogenesis, and tumor progression, making this an attractive target for cancer therapy. An assay for monitoring specific protein changes in response to MetAP2 inhibition, allowing pharmacokinetic (PK)/pharmacodynamic (PD) models to be established, could dramatically improve clinical decision-making. Candidate MetAP2-specific protein substrates were discovered from undigested cell culture-derived proteomes by MALDI-/SELDI-MS profiling and a biochemical method using (35)S-Met labeled protein lysates. Substrates were identified either as intact proteins by FT-ICR-MS or applying in-gel protease digestions followed by LC-MS/MS. The combination of these approaches led to the discovery of novel MetAP2-specific substrates including thioredoxin-1 (Trx-1), SH3 binding glutamic acid rich-like protein (SH3BGRL), and eukaryotic elongation factor-2 (eEF2). These studies also confirmed glyceraldehye 3-phosphate dehydrogenase (GAPDH) and cyclophillin A (CypA) as MetAP2 substrates. Additional data in support of these proteins as MetAP2-specific substrates were provided by in vitro MetAP1/MetAP2 enzyme assays with the corresponding N-terminal derived peptides and 1D/2D Western analyses of cellular and tissue lysates. FT-ICR-MS characterization of all intact species of the 18 kDa substrate, CypA, enabled a SELDI-MS cell-based assay to be developed for correlating N-terminal processing and inhibition of proliferation. The MetAP2-specific protein substrates discovered in this study have diverse properties that should facilitate the development of reagents for testing in preclinical and clinical environments.
Asunto(s)
Aminopeptidasas/antagonistas & inhibidores , Biomarcadores de Tumor/metabolismo , Metaloendopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Animales , Biomarcadores de Tumor/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Células K562 , Leucemia Eritroblástica Aguda/patología , Ratones , Peso Molecular , Inhibidores de Proteasas/clasificación , Proteómica/métodos , Factores de TiempoRESUMEN
The insulin-like growth factor-1 receptor (IGF-1R) and ErbB family of receptors are receptor tyrosine kinases that play important roles in cancer. Lack of response and resistance to therapies targeting ErbB receptors occur and are often associated with activation of the IGF-1R pathway. Combinations of agents that inhibit IGF-1R and ErbB receptors have been shown to synergistically block cancer cell proliferation and xenograft tumor growth. To determine the mechanism by which targeting both IGF-1R and ErbB receptors causes synergistic effects on cell growth and survival, we investigated the effects of combinations of selective IGF-1R and ErbB kinase inhibitors on proliferative and apoptotic signaling. We identified A431 squamous cell carcinoma cells as most sensitive to combinations of ErbB and IGF-1R inhibitors. The inhibitor combinations resulted in not only blockade of A431 cell proliferation, but also induced apoptosis, which was not seen with either agent alone. Upon examining phosphorylation states and expression levels of proteins in the IGF-1R and ErbB signaling pathways, we found a correlation between the ability of combinations to inhibit proliferation and to decrease levels of phosphorylated Akt and cyclin D1. In addition, the massive cell death induced by combined IGF-1R/ErbB inhibition was associated with Mcl-1 reduction and Bax activation. Thus, targeting both IGF-1R and ErbB receptors simultaneously results in cell cycle arrest and apoptosis through combined effects on Akt, cyclin D1, and Bax activation.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Proteínas Oncogénicas v-erbB/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Animales , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ciclina D , Ciclinas/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Trasplante de Neoplasias , Neoplasias/enzimología , Proteínas Oncogénicas v-erbB/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptor IGF Tipo 1/metabolismo , Transducción de Señal/efectos de los fármacos , Trasplante Heterólogo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
A series of aryl sulfonamides of 5,6-disubstituted anthranilic acids were identified as potent inhibitors of methionine aminopeptidase-2 (MetAP2). Small alkyl groups and 3-furyl were tolerated at the 5-position of anthranilic acid, while -OCH(3), CH(3), and Cl were found optimal for the 6-position. Placement of 2-aminoethoxy group at the 6-position enabled interaction with the second Mn(2+) but did not result in enhancement in potency. Introduction of a tertiary amino moiety at the ortho-position of the sulfonyl phenyl ring gave reduced protein binding and improved cellular activity, but led to lower oral bioavailability.
Asunto(s)
Aminopeptidasas/antagonistas & inhibidores , Plomo/química , Metaloendopeptidasas/antagonistas & inhibidores , Sulfonamidas/química , ortoaminobenzoatos/farmacología , Aminopeptidasas/química , Metaloendopeptidasas/química , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Relación Estructura-Actividad , ortoaminobenzoatos/químicaRESUMEN
A high throughput screen of Abbott's compound repository revealed that the pyrazolo[3,4-d]pyrimidine class of kinase inhibitors possessed moderate potency for IGF-IR, a promising target for cancer chemotherapy. The synthesis and subsequent optimization of this class of compounds led to the discovery of 14, a compound that possesses in vivo IGF-IR inhibitory activity.
Asunto(s)
Pirazoles/síntesis química , Pirazoles/farmacología , Pirimidinas/síntesis química , Pirimidinas/farmacología , Receptores de Somatomedina/antagonistas & inhibidores , Administración Oral , Animales , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Inyecciones Intravenosas , Ratones , Fosforilación , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
We have screened molecules for inhibition of MetAP2 as a novel approach toward antiangiogenesis and anticancer therapy using affinity selection/mass spectrometry (ASMS) employing MetAP2 loaded with Mn(2+) as the active site metal. After a series of anthranilic acid sulfonamides with micromolar affinities was identified, chemistry efforts were initiated. The micromolar hits were quickly improved to potent nanomolar inhibitors by chemical modifications guided by insights from X-ray crystallography.
Asunto(s)
Aminopeptidasas/antagonistas & inhibidores , Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Glicoproteínas/antagonistas & inhibidores , Sulfonamidas/farmacología , Inhibidores de la Angiogénesis/síntesis química , Inhibidores de la Angiogénesis/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Sitios de Unión , Línea Celular , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Manganeso/química , Espectrometría de Masas/métodos , Metionil Aminopeptidasas , Modelos Moleculares , Estructura Molecular , Sensibilidad y Especificidad , Estereoisomerismo , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
Kringle 5, a proteolytic fragment of human plasminogen has been shown to potently inhibit angiogenesis. The tetrapeptide KLYD derived from kringle 5 has been shown to capture many activities of kringle 5 in vitro. Further simplification has been achieved by replacement of the two central amino acids with a 4-aminobenzoic acid spacer group. Molecules displaying the required recognition groups on this core show similar in vitro properties to kringle 5, and are able to displace radiolabeled protein from a high affinity binding site on endothelial cells.
Asunto(s)
Ácido 4-Aminobenzoico/química , Kringles , Imitación Molecular , Plasminógeno/farmacología , para-Aminobenzoatos , Inhibición de Migración Celular , Movimiento Celular/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Humanos , Estructura Molecular , Plasminógeno/química , Plasminógeno/genética , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Nonsteriodal anti-inflammatory drugs (NSAIDs) are efficacious for the treatment of pain associated with inflammatory disease. Clinical experience with marketed selective cyclooxygenase-2 (COX-2) inhibitors (celecoxib, rofecoxib, and valdecoxib) has confirmed the utility of these agents in the treatment of inflammatory pain with an improved gastrointestinal safety profile relative to NSAID comparators. These COX-2 inhibitors belong to the same structural class. Each contains a core heterocyclic ring with two appropriately substituted phenyl rings appended to adjacent atoms. Here, we report the identification of vicinally disubstituted pyridazinones as potent and selective COX-2 inhibitors. The lead compound in the series, ABT-963 [2-(3,4-difluoro-phenyl)-4-(3-hydroxy-3-methyl-butoxy)-5-(4-methanesulfonyl-phenyl)-2H-pyridazin-3-one], has excellent selectivity (ratio of 276, COX-2/COX-1) in human whole blood, improved aqueous solubility compared with celecoxib and rofecoxib, high oral anti-inflammatory potency in vivo, and gastric safety in the animal studies. After oral administration, ABT-963 reduced prostaglandin E2 production in the rat carrageenan air pouch model (ED50 of 0.4 mg/kg) and reduced the edema in the carrageenan induced paw edema model with an ED30 of 1.9 mg/kg. ABT-963 dose dependently reduced nociception in the carrageenan hyperalgesia model (ED50 of 3.1 mg/kg). After 14 days of dosing in the adjuvant arthritis model, ABT-963 had an ED(50) of 1.0 mg/kg in reducing the swelling of the hind paws. Magnetic resonance imaging examination of the diseased paws in the adjuvant model showed that ABT-963 significantly reduced bone loss and soft tissue destruction. ABT-963 is a highly selective COX-2 inhibitor that may have utility in the treatment of the pain and inflammation associated with arthritis.