RESUMO
Stapled peptides have great potential as modulators of protein-protein interactions (PPIs). However, there is a vast landscape of chemical features that can be varied for any given peptide, and identifying a set of features that maximizes cellular uptake and subsequent target engagement remains a key challenge. Herein, we present a systematic analysis of staple functionality on the peptide bioactivity landscape in cellular assays. Through application of a "toolbox" of diversified dialkynyl linkers to the stapling of MDM2-binding peptides via a double-click approach, we conducted a study of cellular uptake and p53 activation as a function of the linker. Minor changes in the linker motif and the specific pairing of linker with peptide sequence can lead to substantial differences in bioactivity, a finding which may have important design implications for peptide-based inhibitors of other PPIs. Given the complexity of the structure-activity relationships involved, the toolbox approach represents a generalizable strategy for optimization when progressing from in vitro binding assays to cellular efficacy studies.
Assuntos
Peptídeos/química , Alcinos/química , Sequência de Aminoácidos , Linhagem Celular , Permeabilidade da Membrana Celular , Humanos , Estrutura Molecular , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/metabolismoRESUMO
Free energy perturbation theory, in combination with enhanced sampling of protein-ligand binding modes, is evaluated in the context of fragment-based drug design, and used to design two new small-molecule inhibitors of the Aurora A kinase-TPX2 protein-protein interaction.
Assuntos
Aurora Quinase A/antagonistas & inibidores , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Simulação de Dinâmica Molecular , Proteínas Nucleares/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Aurora Quinase A/química , Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Estrutura Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the 'Y pocket') that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases.