RESUMEN
Pyrazolopyrimidines are well-established as covalent inhibitors of protein kinases such as the epidermal growth factor receptor or Bruton's tyrosine kinase, and we recently described their potential in targeting mitogen-activated protein kinase kinase 7 (MKK7). Herein, we report the structure-activity relationship of pyrazolopyrimidine-based MKK7 inhibitors and solved several complex crystal structures to gain insights into their binding mode. In addition, we present two structures of apo-MKK7, exhibiting a DFG-out and an unprecedented DFG-in/Leu-in conformation.
Asunto(s)
MAP Quinasa Quinasa 7/química , MAP Quinasa Quinasa 7/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Pirimidinas/química , Pirimidinas/metabolismo , Secuencias de Aminoácidos , MAP Quinasa Quinasa 7/antagonistas & inhibidores , Modelos Moleculares , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacologíaRESUMEN
The protein kinase MKK7 is linked to neuronal development and the onset of cancer. The field, however, lacks high-quality functional probes that would allow for the dissection of its detailed functions. Against this background, we describe an effective covalent inhibitor of MKK7 based on the pyrazolopyrimidine scaffold.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , MAP Quinasa Quinasa 7/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Cristalografía por Rayos X , Diseño de Fármacos , Fosforilación , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Pirazoles/química , Pirazoles/farmacología , Pirimidinas/química , Pirimidinas/farmacologíaRESUMEN
The examination of three-dimensional structural models in scientific publications allows the reader to validate or invalidate conclusions drawn by the authors. However, either due to a (temporary) lack of access to proper visualization software or a lack of proficiency, this information is not necessarily available to every reader. As the digital revolution is quickly progressing, technologies have become widely available that overcome the limitations and offer to all the opportunity to appreciate models not only in 2D, but also in 3D. Additionally, mobile devices such as smartphones and tablets allow access to this information almost anywhere, at any time. Since access to such information has only recently become standard practice, we want to outline straightforward ways to incorporate 3D models in augmented reality into scientific publications, books, posters, and presentations and suggest that this should become general practice.
Asunto(s)
Conformación Molecular , Publicaciones/normas , Humanos , Tecnología de la Información/normas , Tecnología de la Información/tendenciasRESUMEN
The identification of compounds for dissecting biological functions and the development of novel drug molecules are central tasks that often require screening campaigns. However, the required architecture is cost- and time-intensive. Herein we describe the devices and technologies that comprise a Robotics-Assisted Screening Platform for Efficient Ligand Discovery (RASPELD), which we set up in an academic laboratory. RASPELD provides semi-automated high-end screening, and it can be maintained by graduate students. We demonstrate its successful application in biochemical and cellular screens for the identification and validation of bioactive chemical entities as candidate cancer-relevant inhibitors. Specifically, we examined the interaction between a transcription factor, Nrf2, and its key regulator, Keap1. We also examined drug-resistant mutants of the epidermal growth factor receptor (EGFR). Screening campaigns with more than 30 000 compounds were performed in a reasonable period of time. We identified the molecule RSL6586 as a starting point for hit optimization, which is currently ongoing.
Asunto(s)
Antineoplásicos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Robótica/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Bioensayo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/instrumentación , Educación de Postgrado , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Ligandos , Mutación , Factor 2 Relacionado con NF-E2/metabolismo , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Robótica/instrumentaciónRESUMEN
In addition to the catalytically active kinase domain, most kinases feature regulatory domains that govern their activity. Modulating and interfering with these interdomain interactions presents a major opportunity for understanding biological systems and developing novel therapeutics. Therefore, small molecule inhibitors that target these interactions through an allosteric mode of action have high intrinsic selectivity, as these interactions are often unique to a single kinase or kinase family. Here we report the development of iFLiK (interface-Fluorescent Labels in Kinases), a fluorescence-based assay that can monitor such interdomain interactions. Using iFLiK, we have demonstrated selective detection of allosteric Akt inhibitors that induce an inactive closed conformation unique to Akt. This methodology easily distinguished small molecule allosteric inhibitors from classic ATP-competitive inhibitors. Screening an in-house compound library with iFLiK, we were able to identify novel compounds with a scaffold that has not been previously described for allosteric Akt inhibitors.