RESUMO
There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small-molecule inhibitor of the WNT pathway discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex-associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a type 1 binding mode involving insertion of the CDK8 C terminus into the ligand binding site. In contrast to type II inhibitors of CDK8 and CDK19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogs alter WNT pathway-regulated gene expression and other on-target effects of modulating CDK8 and CDK19, including expression of genes regulated by STAT1. Consistent with this, we find that phosphorylation of STAT1(SER727) is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Quinase 8 Dependente de Ciclina/antagonistas & inibidores , Quinase 8 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Sondas Moleculares/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Compostos de Espiro/farmacologia , Linhagem Celular Tumoral , Quinase 8 Dependente de Ciclina/genética , Quinases Ciclina-Dependentes/genética , Humanos , Modelos Moleculares , Sondas Moleculares/química , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Piridinas/química , Compostos de Espiro/químicaRESUMO
An efficient total synthesis of integric acid is described starting from the Wieland-Miescher ketone. Key steps involve a one-step orthogonal deprotection/protection strategy of a thioacetal/aldehyde and the selective oxidative cleavage of a prenyl group in the presence of two other unsaturated moieties. The synthesis of both C4' diastereoisomers of integric acid delivered unambiguous evidence for (S)-stereochemistry at the C4' position.
Assuntos
Ácidos Carboxílicos/síntese química , Naftalenos/síntese química , Ácidos Carboxílicos/química , Conformação Molecular , Naftalenos/química , EstereoisomerismoRESUMO
Greater control is desirable in the stochastic conjugation technology used to synthesize antibody-drug conjugates (ADC). We have shown recently that a fluorescent dye can be stably conjugated to a mAb using a bifunctional platinum(II) linker. Here, we describe the general applicability of this novel linker technology for the preparation of stable and efficacious ADCs. The ethylenediamine platinum(II) moiety, herein called Lx, was coordinated to Desferal (DFO) or auristatin F (AF) to provide storable "semifinal" products, which were directly conjugated to unmodified mAbs. Conjugation resulted in ADCs with unimpaired mAb-binding characteristics, DAR in the range of 2.5 to 2.7 and approximately 85% payload bound to the Fc region, presumably to histidine residues. To evaluate the in vivo stability of Lx and its effect on pharmacokinetics and tumor targeting of an ADC, Lx-DFO was conjugated to the HER2 mAb trastuzumab, followed by radiolabeling with 89Zr. Trastuzumab-Lx-DFO-89Zr was stable in vivo and exhibited pharmacokinetic and tumor-targeting properties similar to parental trastuzumab. In a xenograft mouse model of gastric cancer (NCI-N87) or an ado-trastuzumab emtansine-resistant breast cancer (JIMT-1), a single dose of trastuzumab-Lx-AF outperformed its maleimide benchmark trastuzumab-Mal-AF and FDA-approved ado-trastuzumab emtansine. Overall, our findings show the potential of the Lx technology as a robust conjugation platform for the preparation of anticancer ADCs. Cancer Res; 77(2); 257-67. ©2016 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Imunoconjugados/farmacologia , Trastuzumab/farmacologia , Aminobenzoatos , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Desferroxamina , Desenho de Fármacos , Humanos , Imunoconjugados/química , Camundongos , Oligopeptídeos , Compostos de Platina , Traçadores Radioativos , Neoplasias Gástricas/patologia , Trastuzumab/química , Ensaios Antitumorais Modelo de Xenoenxerto , ZircônioAssuntos
Aminofenóis/síntese química , Aminofenóis/farmacologia , Antibacterianos/síntese química , Antibacterianos/farmacologia , Compostos Policíclicos/síntese química , Compostos Policíclicos/farmacologia , Aminofenóis/química , Antibacterianos/química , Cristalografia por Raios X , Enterococcus faecalis/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Conformação Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Compostos Policíclicos/química , Estereoisomerismo , Streptococcus pneumoniae/efeitos dos fármacosRESUMO
The potential of platinum(II) as a bifunctional linker in the coordination of small molecules, such as imaging agents or (cytotoxic) drugs, to monoclonal antibodies (mAbs) was investigated with a 4-nitrobenzo-2-oxa-1,3-diazole (NBD) fluorophore and trastuzumab (Herceptin™) as a model antibody. The effect of ligand and reaction conditions on conjugation efficiency was explored for [Pt(en)(L-NBD)Cl](NO3 ) (en=ethylenediamine), with L=N-heteroaromatic, N-alkyl amine, or thioether. Conjugation proceeded most efficiently at pHâ 8.0 in the presence of NaClO4 or Na2 SO4 in tricine or HEPES buffer. Reaction of N-coordinated complexes (20â equiv) with trastuzumab at 37 °C for 2â h, followed by removal of weakly bound complexes with excess thiourea, afforded conjugates with an NBD/mAb ratio of 1.5-2.9 that were stable in phosphate-buffered saline at room temperature for at least 48â h. In contrast, thioether-coordinated complexes afforded unstable conjugates. Finally, surface plasmon resonance analysis showed no loss in binding affinity of trastuzumab after conjugation.
Assuntos
Anticorpos Monoclonais/química , Corantes Fluorescentes/química , Imunoconjugados/química , Compostos Organoplatínicos/química , Oxidiazóis/química , Trastuzumab/química , Modelos Moleculares , Estrutura Molecular , Compostos Organoplatínicos/síntese químicaRESUMO
WNT signaling is frequently deregulated in malignancy, particularly in colon cancer, and plays a key role in the generation and maintenance of cancer stem cells. We report the discovery and optimization of a 3,4,5-trisubstituted pyridine 9 using a high-throughput cell-based reporter assay of WNT pathway activity. We demonstrate a twisted conformation about the pyridine-piperidine bond of 9 by small-molecule X-ray crystallography. Medicinal chemistry optimization to maintain this twisted conformation, cognisant of physicochemical properties likely to maintain good cell permeability, led to 74 (CCT251545), a potent small-molecule inhibitor of WNT signaling with good oral pharmacokinetics. We demonstrate inhibition of WNT pathway activity in a solid human tumor xenograft model with evidence for tumor growth inhibition following oral dosing. This work provides a successful example of hypothesis-driven medicinal chemistry optimization from a singleton hit against a cell-based pathway assay without knowledge of the biochemical target.