RESUMO
Phosphopantetheine transferases (PPTases) can be used to efficiently prepare site-specific antibody-drug conjugates (ADCs) by enzymatically coupling coenzyme A (CoA)-linker payloads to 11-12 amino acid peptide substrates inserted into antibodies. Here, a two-step strategy is established wherein in a first step, CoA analogs with various bioorthogonal reactivities are enzymatically installed on the antibody for chemical conjugation with a cytotoxic payload in a second step. Because of the high structural similarity of these CoA analogs to the natural PPTase substrate CoA-SH, the first step proceeds very efficiently and enables the use of peptide tags as short as 6 amino acids compared to the 11-12 amino acids required for efficient one-step coupling of the payload molecule. Furthermore, two-step conjugation provides access to diverse linker chemistries and spacers of varying lengths. The potency of the ADCs was largely independent of linker architecture. In mice, proteolytic cleavage was observed for some C-terminally linked auristatin payloads. The in vivo stability of these ADCs was significantly improved by reduction of the linker length. In addition, linker stability was found to be modulated by attachment site, and this, together with linker length, provides an opportunity for maximizing ADC stability without sacrificing potency.
Assuntos
Anticorpos Monoclonais/química , Coenzima A/química , Citotoxinas/química , Imunoconjugados/química , Aminobenzoatos/administração & dosagem , Aminobenzoatos/química , Animais , Citotoxinas/administração & dosagem , Estabilidade de Medicamentos , Camundongos , Oligopeptídeos/administração & dosagem , Oligopeptídeos/química , Relação Estrutura-AtividadeRESUMO
The synthesis, preclinical profile, and in vivo efficacy in rat xenograft models of the novel and selective anaplastic lymphoma kinase inhibitor 15b (LDK378) are described. In this initial report, preliminary structure-activity relationships (SARs) are described as well as the rational design strategy employed to overcome the development deficiencies of the first generation ALK inhibitor 4 (TAE684). Compound 15b is currently in phase 1 and phase 2 clinical trials with substantial antitumor activity being observed in ALK-positive cancer patients.
Assuntos
Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/síntese química , Pirimidinas/síntese química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Sulfonas/síntese química , Quinase do Linfoma Anaplásico , Animais , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Cães , Humanos , Macaca fascicularis , Masculino , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/farmacocinética , Pirimidinas/uso terapêutico , Ratos , Relação Estrutura-Atividade , Sulfonas/farmacocinética , Sulfonas/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Through scaffold morphing of a known Smoothened antagonist Antag691, a series of novel phenyl imidazole derivatives were developed. Structure-activity-relationship studies and lead optimization led to the discovery of potent, selective and orally bioavailable Smoothened antagonist 19 that is suitable for in vivo studies.
Assuntos
Desenho de Fármacos , Imidazóis/síntese química , Imidazóis/farmacocinética , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Área Sob a Curva , Humanos , Imidazóis/química , Imidazóis/farmacologia , Concentração Inibidora 50 , Camundongos , Ligação Proteica/efeitos dos fármacos , Ratos , Receptor Smoothened , Relação Estrutura-AtividadeRESUMO
The blockade of aberrant hedgehog (Hh) signaling has shown promise for therapeutic intervention in cancer. A cell-based phenotypic high-throughput screen was performed, and the lead structure (1) was identified as an inhibitor of the Hh pathway via antagonism of the Smoothened receptor (Smo). Structure-activity relationship studies led to the discovery of a potent and specific Smoothened antagonist N-(6-((2S,6R)-2,6-dimethylmorpholino)pyridin-3-yl)-2-methyl-4'-(trifluoromethoxy)biphenyl-3-carboxamide (5m, NVP-LDE225), which is currently in clinical development.
RESUMO
Aurora family kinases regulate important events during mitosis including centrosome maturation and separation, mitotic spindle assembly, and chromosome segregation. Misregulation of Aurora kinases due to genetic amplification and protein overexpression results in aneuploidy and may contribute to tumorigenesis. Here we report the discovery of new small molecule aminothiazole inhibitors of Aurora kinases with exceptional kinase selectivity and report a 1.7 A cocrystal structure with the Aurora B:INCENP complex from Xenopus laevis. The compounds recapitulate the hallmarks of Aurora kinase inhibition, including decreased histone H3 serine 10 phosphorylation, failure to complete cytokinesis, and endoreduplication.
Assuntos
Aminas/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Tiazóis/química , Tiazóis/farmacologia , Animais , Aurora Quinases , Cianatos/química , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Sensibilidade e Especificidade , Relação Estrutura-Atividade , Xenopus laevisRESUMO
Constitutive overexpression and activation of NPM-ALK fusion protein [t(2:5)(p23;q35)] is a key oncogenic event that drives the survival and proliferation of anaplastic large-cell lymphomas (ALCLs). We have identified a highly potent and selective small-molecule ALK inhibitor, NVP-TAE684, which blocked the growth of ALCL-derived and ALK-dependent cell lines with IC(50) values between 2 and 10 nM. NVP-TAE684 treatment resulted in a rapid and sustained inhibition of phosphorylation of NPM-ALK and its downstream effectors and subsequent induction of apoptosis and cell cycle arrest. In vivo, NVP-TAE684 suppressed lymphomagenesis in two independent models of ALK-positive ALCL and induced regression of established Karpas-299 lymphomas. NVP-TAE684 also induced down-regulation of CD30 expression, suggesting that CD30 may be used as a biomarker of therapeutic NPM-ALK kinase activity inhibition.
Assuntos
Linfoma Difuso de Grandes Células B/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Antígeno Ki-1/análise , Camundongos , Camundongos SCID , Fosforilação , Pirimidinas/uso terapêutico , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Pyridoxal kinase (PDXK) catalyzes the phosphorylation of pyridoxal, pyridoxamine, and pyridoxine in the presence of ATP and Zn2+. This constitutes an essential step in the synthesis of pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, a cofactor for over 140 enzymes. (R)-Roscovitine (CYC202, Seliciclib) is a relatively selective inhibitor of cyclin-dependent kinases (CDKs), currently evaluated for the treatment of cancers, neurodegenerative disorders, renal diseases, and several viral infections. Affinity chromatography investigations have shown that (R)-roscovitine also interacts with PDXK. To understand this interaction, we determined the crystal structure of PDXK in complex with (R)-roscovitine, N6-methyl-(R)-roscovitine, and O6-(R)-roscovitine, the two latter derivatives being designed to bind to PDXK but not to CDKs. Structural analysis revealed that these three roscovitines bind similarly in the pyridoxal-binding site of PDXK rather than in the anticipated ATP-binding site. The pyridoxal pocket has thus an unexpected ability to accommodate molecules different from and larger than pyridoxal. This work provides detailed structural information on the interactions between PDXK and roscovitine and analogs. It could also aid in the design of roscovitine derivatives displaying strict selectivity for either PDXK or CDKs.
Assuntos
Inibidores de Proteínas Quinases/química , Estrutura Terciária de Proteína , Purinas/química , Piridoxal Quinase/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Ligantes , Substâncias Macromoleculares , Modelos Moleculares , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Inibidores de Proteínas Quinases/metabolismo , Purinas/metabolismo , Piridoxal Quinase/metabolismo , Roscovitina , SuínosRESUMO
(R)-Roscovitine (CYC202) is often referred to as a "selective inhibitor of cyclin-dependent kinases." Besides its use as a biological tool in cell cycle, neuronal functions, and apoptosis studies, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, viral infections, and glomerulonephritis. We have investigated the selectivity of (R)-roscovitine using three different methods: 1) testing on a wide panel of purified kinases that, along with previously published data, now reaches 151 kinases; 2) identifying roscovitine-binding proteins from various tissue and cell types following their affinity chromatography purification on immobilized roscovitine; 3) investigating the effects of roscovitine on cells deprived of one of its targets, CDK2. Altogether, the results show that (R)-roscovitine is rather selective for CDKs, in fact most kinases are not affected. However, it binds an unexpected, non-protein kinase target, pyridoxal kinase, the enzyme responsible for phosphorylation and activation of vitamin B6. These results could help in interpreting the cellular actions of (R)-roscovitine but also in guiding the synthesis of more selective roscovitine analogs.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Quinases/metabolismo , Purinas/química , Purinas/metabolismo , Piridoxal Quinase/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Ciclo Celular/fisiologia , Sobrevivência Celular , Células Cultivadas , Cromatografia de Afinidade , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Estrutura Molecular , Estrutura Terciária de Proteína , Piridoxal/metabolismo , Piridoxal Quinase/antagonistas & inibidores , Piridoxal Quinase/genética , Fosfato de Piridoxal/metabolismo , Ratos , Roscovitina , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Distribuição TecidualRESUMO
Hymenialdisine (HMD) is a sponge-derived natural product kinase inhibitor with nanomolar activity against CDKs, Mek1, GSK3beta, and CK1 and micromolar activity against Chk1. In order to explore the broader application of the pyrrolo[2,3-c]azepine skeleton of HMD as a general kinase inhibitory scaffold, we searched for additional protein targets using affinity chromatography in conjunction with the synthesis of diverse HMD analogs and profiled HMD against a panel of 60 recombinant enzymes. This effort has led to nanomolar to micromolar inhibitors of 11 new targets including p90RSK, KDR, c-Kit, Fes, MAPK1, PAK2, PDK1, PKCtheta, PKD2, Rsk1, and SGK. The synthesis of HMD analogs has resulted in the identification of compounds with enhanced and/or dramatically altered selectivities relative to HMD (28n) and in molecules with antiproliferative activities 30-fold higher than HMD (28p).
Assuntos
Azepinas/química , Inibidores Enzimáticos/química , Fosfotransferases/antagonistas & inibidores , Pirróis/química , Sequência de Aminoácidos , Azepinas/síntese química , Azepinas/farmacologia , Cromatografia de Afinidade , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Dados de Sequência Molecular , Fosfotransferases/metabolismo , Pirróis/síntese química , Pirróis/farmacologia , Relação Estrutura-AtividadeRESUMO
A molecular dithiane-based approach to synthesis of novel photolabile phospholipids is developed. These lipids are used in formulations with egg-POPC and cholesterol to prepare light-sensitive liposomes. Irradiation of such liposomes in PBS buffer (medium pressure mercury lamp, Pyrex filter, lambda > 300 nm) significantly increases the bilayer permeability and accelerates the release of entrapped small organic molecules by an order of magnitude. A simple assay, based on (1)H or (19)F PFG NMR measurements of diffusion coefficients, is developed to monitor light-induced unloading of the probe molecules.