RESUMO
Aberrant NOTCH3 signaling and overexpression is oncogenic, associated with cancer stem cells and drug resistance, yet therapeutic targeting remains elusive. Here, we develop NOTCH3-targeted antibody drug conjugates (NOTCH3-ADCs) by bioconjugation of an auristatin microtubule inhibitor through a protease cleavable linker to two antibodies with differential abilities to inhibit signaling. The signaling inhibitory antibody rapidly induces ligand-independent receptor clustering and internalization through both caveolin and clathrin-mediated pathways. The non-inhibitory antibody also efficiently endocytoses via clathrin without inducing receptor clustering but with slower lysosomal co-localization kinetics. In addition, DLL4 ligand binding to the NOTCH3 receptor mediates transendocytosis of NOTCH3-ADCs into ligand-expressing cells. NOTCH3-ADCs internalize into receptor and ligand cells independent of signaling and induce cell death in both cell types representing an atypical mechanism of ADC cytotoxicity. Treatment of xenografts with NOTCH3-ADCs leads to sustained tumor regressions, outperforms standard-of-care chemotherapy, and allows targeting of tumors that overexpress NOTCH3 independent of signaling inhibition.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Imunoconjugados/farmacologia , Receptor Notch3/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Humanos , Imunoconjugados/metabolismo , Oncogenes/efeitos dos fármacos , Receptor Notch3/imunologia , Receptores Notch/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
hSMG-1 kinase plays a dual role in a highly conserved RNA surveillance pathway termed nonsense-mediated RNA decay (NMD) and in cellular genotoxic stress response. Since deregulation of cellular responses to stress contributes to tumor growth and resistance to chemotherapy, hSMG-1 is a potential target for cancer treatment. From our screening efforts, we have identified pyrimidine derivatives as hSMG-1 kinase inhibitors. We report structure-based optimization of this pan-kinase scaffold to improve its biochemical profile and overall kinome selectivity, including mTOR and CDK, to generate the first reported selective hSMG-1 tool compound.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Pirimidinas/química , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Proteínas Serina-Treonina Quinases , Pirimidinas/síntese química , Pirimidinas/farmacologiaRESUMO
Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease associated with irreversible progressive airflow limitation. Matrix metalloproteinase-12 (MMP-12) has been characterized to be one of the major proteolytic enzymes to induce airway remodeling, destruction of elastin and the aberrant remodeling of damaged alveoli in COPD and asthma. The goal of this project is to develop and identify an orally potent and selective small molecule inhibitor of MMP-12 for treatment of COPD and asthma. Syntheses and structure-activity relationship (SAR) studies of a series of dibenzofuran (DBF) sulfonamides as MMP-12 inhibitors are described. Potent inhibitors of MMP-12 with excellent selectivity against other MMPs were identified. Compound 26 (MMP118), which exhibits excellent oral efficacy in the MMP-12 induced ear-swelling inflammation and lung inflammation mouse models, had been successfully advanced into Development Track status.
Assuntos
Desenho de Fármacos , Metaloproteinase 12 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz , Doença Pulmonar Obstrutiva Crônica/enzimologia , Animais , Asma/tratamento farmacológico , Asma/enzimologia , Química Farmacêutica/métodos , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Humanos , Inflamação , Concentração Inibidora 50 , Camundongos , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Relação Estrutura-Atividade , Sulfonamidas/química , Raios XRESUMO
We discovered 2-(4-substituted-pyrrolo[2,3-b]pyridin-3-yl)methylene-4-hydroxybenzofuran-3(2H)-ones as potent and selective ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR). Since phenolic OH groups pose metabolic liability, one of the two hydroxyl groups was selectively removed. The SAR data showed the structural features necessary for subnanomolar inhibitory activity against mTOR kinase as well as selectivity over PI3Kalpha. An X-ray co-crystal structure of one inhibitor with the mTOR-related PI3Kgamma revealed the key hydrogen bonding interactions.
Assuntos
Benzofuranos/química , Benzofuranos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Cristalografia por Raios X , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Nus , Microssomos/metabolismo , Modelos Moleculares , Neoplasias/tratamento farmacológico , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Relação Estrutura-Atividade , Serina-Treonina Quinases TORRESUMO
The mammalian target of rapamycin (mTOR), a central regulator of growth, survival, and metabolism, is a validated target for cancer therapy. Rapamycin and its analogues, allosteric inhibitors of mTOR, only partially inhibit one mTOR protein complex. ATP-competitive, global inhibitors of mTOR that have the potential for enhanced anticancer efficacy are described. Structural features leading to potency and selectivity were identified and refined leading to compounds with in vivo efficacy in tumor xenograft models.
Assuntos
Trifosfato de Adenosina/fisiologia , Antineoplásicos/síntese química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/síntese química , Pirimidinas/síntese química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Carbamatos/síntese química , Carbamatos/química , Carbamatos/farmacologia , Linhagem Celular Tumoral , Classe Ib de Fosfatidilinositol 3-Quinase , Cristalografia por Raios X , Desenho de Fármacos , Peptídeos e Proteínas de Sinalização Intracelular/química , Isoenzimas/química , Camundongos , Camundongos Nus , Microssomos/metabolismo , Modelos Moleculares , Fosfatidilinositol 3-Quinases/química , Proteínas Serina-Treonina Quinases/química , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR , Ureia/análogos & derivados , Ureia/síntese química , Ureia/química , Ureia/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
HCV-796 selectively inhibits hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In hepatoma cells containing a genotype 1b HCV replicon, HCV-796 reduced HCV RNA levels by 3 to 4 log(10) HCV copies/mug total RNA (the concentration of the compound that inhibited 50% of the HCV RNA level was 9 nM). Cells bearing replicon variants with reduced susceptibility to HCV-796 were generated in the presence of HCV-796, followed by G418 selection. Sequence analysis of the NS5B gene derived from the replicon variants revealed several amino acid changes within 5 A of the drug-binding pocket. Specifically, mutations were observed at Leu314, Cys316, Ile363, Ser365, and Met414 of NS5B, which directly interact with HCV-796. The impacts of the amino acid substitutions on viral fitness and drug susceptibility were examined in recombinant replicons and NS5B enzymes with the single-amino-acid mutations. The replicon variants were 10- to 1,000-fold less efficient in forming colonies in cells than the wild-type replicon; the S365L variant failed to establish a stable cell line. Other variants (L314F, I363V, and M414V) had four- to ninefold-lower steady-state HCV RNA levels. Reduced binding affinity with HCV-796 was demonstrated in an enzyme harboring the C316Y mutation. The effects of these resistance mutations were structurally rationalized using X-ray crystallography data. While different levels of resistance to HCV-796 were observed in the replicon and enzyme variants, these variants retained their susceptibilities to pegylated interferon, ribavirin, and other HCV-specific inhibitors. The combined virological, biochemical, biophysical, and structural approaches revealed the mechanism of resistance in the variants selected by the potent polymerase inhibitor HCV-796.