RESUMO
Targeted covalent inhibitors have re-emerged as validated drugs to overcome acquired resistance in cancer treatment. Herein, by using a carbonyl boronic acid (CBA) warhead, we report the structure-based design of BCR-ABL inhibitors via reversible covalent targeting of the catalytic lysine with improved potency against both wild-type and mutant ABL kinases, especially ABLT315I bearing the gatekeeper residue mutation. We show the evolutionarily conserved lysine can be targeted selectively, and the selectivity depends largely on molecular recognition of the non-covalent pharmacophore in this class of inhibitors, probably due to the moderate reactivity of the warhead. We report the first co-crystal structures of covalent inhibitor-ABL kinase domain complexes, providing insights into the interaction of this warhead with the catalytic lysine. We also employed label-free mass spectrometry to evaluate off-targets of our compounds at proteome-wide level in different mammalian cells.
Assuntos
Desenho de Fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Lisina/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Lisina/síntese química , Lisina/química , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/químicaRESUMO
Cell growth and differentiation require precise coordination of cell cycle and differentiation proteins. This can be achieved by direct interactions between proteins, by indirect interaction in multiprotein complexes, or by modulation of gene expression levels of partner proteins. Contradictory data abound in the literature regarding the binding between some central cell cycle proteins, pRb, and CDK6, with myogenic differentiation promoting, MyoD, and inhibiting, Id-2, factors. We have tested these interactions using pure proteins and in vitro biophysical and biochemical methods, which included mass spectrometry, nuclear magnetic resonance (NMR), the affinity chromatography pull-down assays, and gel filtration chromatography. Using this multimethod approach, we were able to document interactions between pRb and HPV-E7, pRb and SV40 large T antigen, CDK6 and p19, and MyoD and DNA. Using the same methods, we could unambiguously show that there is no direct protein-protein interaction in vitro between the small pocket domain of pRb and the bHLH domain of MyoD, the small pocket domain of pRb and Id-2, and CDK6 and a 15-amino-acid peptide from the C-terminal domain of MyoD. Indirect interactions, through additional binding partners in multiprotein complexes or modulation of gene expression levels of these proteins, are therefore their probable mode of action.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína MyoD/metabolismo , Proteínas Repressoras/metabolismo , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Animais , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Proteínas de Ciclo Celular/genética , Diferenciação Celular/fisiologia , Galinhas , Quinase 6 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p19 , Quinases Ciclina-Dependentes/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Sequências Hélice-Alça-Hélice , Humanos , Proteína 2 Inibidora de Diferenciação , Espectroscopia de Ressonância Magnética , Espectrometria de Massas/métodos , Proteína MyoD/genética , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus , Mapeamento de Interação de Proteínas/métodos , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Proteína do Retinoblastoma/genética , Fatores de Transcrição/genéticaRESUMO
Seven different, but highly conserved 14-3-3 proteins are involved in diverse signaling pathways in human cells. It is unclear how the 14-3-3sigma isoform, a transcriptional target of p53, exerts its inhibitory effect on the cell cycle in the presence of other 14-3-3 isoforms, which are constitutively expressed at high levels. In order to identify structural differences between the 14-3-3 isoforms, we solved the crystal structure of the human 14-3-3sigma protein at a resolution of 2.8 Angstroms and compared it to the known structures of 14-3-3zeta and 14-3-3tau. The global architecture of the 14-3-3sigma fold is similar to the previously determined structures of 14-3-3zeta and 14-3-3t: two 14-3-3sigma molecules form a cup-shaped dimer. Significant differences between these 14-3-3 isoforms were detected adjacent to the amphipathic groove, which mediates the binding to phosphorylated consensus motifs in 14-3-3-ligands. Another specificity determining region is localized between amino-acids 203 to 215. These differences presumably select for the interaction with specific ligands, which may explain the different biological functions of the respective 14-3-3 isoforms. Furthermore, the two 14-3-3sigma molecules forming a dimer differ by the spatial position of the ninth helix, which is shifted to the inside of the ligand interaction surface, thus indicating adaptability of this part of the molecule. In addition, 5 non-conserved residues are located at the interface between two 14-3-3sigma proteins forming a dimer and represent candidate determinants of homo- and hetero-dimerization specificity. The structural differences among the 14-3-3 isoforms described here presumably contribute to isoform-specific interactions and functions.