PO.ET09.04 · 实验与分子治疗
新型分子胶的理性发现
Rational discovery of novel molecular glues
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
目的：我们开发并应用了一个流程化的工作流来发现和优化cereblon（CRBN）介导的分子胶。
方法：我们结合计算和结构方法来定义CRBN靶点空间，并采用以CRBN为重点的小分子文库和DNA编码文库（DEL）来对新型分子胶进行高通量筛选。应用基于邻近的蛋白-蛋白相互作用检测来筛选新型胶降解剂靶点的诱导性CRBN邻近。随后基于细胞的降解方法可推动先导物优化，并采用先进的生物物理和结构方法进行结构和功能辅助设计。
结果与结论：我们已从理性筛选方法中鉴定出多个先导物，能够在TR-FRET和NanoBRET检测中诱导浓度依赖性的靶点-CRBN邻近。命中通过生物物理方法（包括表面等离子共振（SPR）和尺寸排阻色谱（SEC））经正交验证可诱导三元复合物形成。细胞读出通过HiBiT和基于免疫荧光的方法证明了依赖于CRBN E3连接酶表达或活性以及蛋白酶体降解的降解活性。这一整合的工作流连同用于靶上和脱靶选择性的蛋白质组学技术，构成了一个快速、以机制为中心的CRBN分子胶发现平台，可跨越多样化的靶点类别，充分利用先进但易于获取的方法学。
查看英文原文 English abstract
Purpose: We developed and applied a streamlined workflow to discover and optimize cereblon (CRBN)-mediated molecular glues.
Methods: We combine computational and structural approaches to define the CRBN target space with both CRBN-focused small molecule and DNA-encoded (DEL) libraries for high-throughput screening of novel molecular glues. Proximity based protein-protein interaction assays are applied to screen for induced CRBN proximity of novel glue degrader targets. Cell-based degradation methods can then drive lead optimization, with advanced biophysical and structural methodologies for structural and functional aided design.
Results and Conclusions: We have identified multiple leads from rational screening approaches inducing concentration-dependent target-CRBN proximity in TR-FRET and NanoBRET assays. Hits were orthogonally confirmed to induce ternary complex formation by biophysical methods including surface plasmon resonance (SPR) and size exclusion chromatography (SEC). Cellular readouts demonstrated degradation activity using HiBiT and immunofluorescence-based methods dependent on CRBN E3 ligase expression or activity and proteasomal degradation. This integrated workflow together with proteomics technologies for on-target and off-target selectivity, is a rapid, mechanism-centric discovery platform for CRBN molecular glues across diverse target classes taking advantage of advanced but readily available methodologies.
利益披露 Disclosure
S. Woehrle,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.
A. Blum,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.
B. Blume,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.
J. Bomke,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.
H. Buchstaller,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.
V. Dragone,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.
I. Ferrara,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.
A. Gambardella,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.
J. Gunera,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.
U. Grädler,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.
I. Kober,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.
J. Krieger,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.
B. Leuthner,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.
A. Unzue Lopez,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.
O. Schadt,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.
C. Schindler,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.
F. J. Sorrell,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.
A. M. Esteves,
Instituto de Biologia Experimental e Tecnológica (iBET) Employment.
S. P. Santos,
Instituto de Biologia Experimental e Tecnológica (iBET) Employment.
R. L. Sousa,
Instituto de Biologia Experimental e Tecnológica (iBET) Employment.
M. M. S. Silva,
Instituto de Biologia Experimental e Tecnológica (iBET) Employment.
A. R. Lemos,
Instituto de Biologia Experimental e Tecnológica (iBET) Employment.
P. M. F. Sousa,
Instituto de Biologia Experimental e Tecnológica (iBET) Employment.
T. M. Bandeiras,
Instituto de Biologia Experimental e Tecnológica (iBET) Employment.
M. Cuomo,
EMD Serono, Inc., The healthcare business of Merck KGaA, Darmstadt, Germany Employment.