Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom.
Proc Natl Acad Sci U S A. 2013 Aug 6;110(32):12984-9. doi: 10.1073/pnas.1304045110. Epub 2013 Jul 19.
In fragment-based drug discovery, the weak affinities exhibited by fragments pose significant challenges for screening. Biophysical techniques are used to address this challenge, but there is no clear consensus on which cascade of methods is best suited to identify fragment hits that ultimately translate into bound X-ray structures and provide bona fide starting points for synthesis. We have benchmarked an integrated biophysical approach for fragment screening and validation against Mycobacterium tuberculosis pantothenate synthetase. A primary screen of 1,250 fragments library was performed by thermal shift, followed by secondary screen using one-dimensional NMR spectroscopy (water ligand observed gradient spectroscopy and saturation transfer difference binding experiments) and ultimate hit validation by isothermal titration calorimetry and X-ray crystallography. Our multibiophysical approach identified three distinct binding sites for fragments and laid a solid foundation for successful structure-based elaboration into potent inhibitors.
在基于片段的药物发现中，片段表现出的弱亲和力给筛选带来了重大挑战。生物物理技术被用于解决这一挑战，但对于哪种方法序列最适合识别最终转化为结合 X 射线结构并为合成提供真实起点的片段命中，尚无明确共识。我们已经针对分枝杆菌泛酸合酶对基于片段的筛选和验证的综合生物物理方法进行了基准测试。通过热位移对 1250 个片段文库进行了初步筛选，然后使用一维 NMR 光谱学（水配体观察梯度光谱和饱和转移差结合实验）进行二次筛选，并通过等温滴定量热法和 X 射线晶体学进行最终命中验证。我们的多生物物理方法确定了三个不同的片段结合位点，为成功的基于结构的精心设计成有效的抑制剂奠定了坚实的基础。
