Ding Tianyi, Guseinov Abdul-Akim, Milligan Graeme, Plouffe Bianca, Tikhonova Irina G
School of Pharmacy, Queen's University Belfast, Belfast Bt9 7BL, Northern Ireland, U.K.
Centre for Translational Pharmacology, School of Molecular Biosciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland G12 8QQ, U.K.
ACS Pharmacol Transl Sci. 2024 Jul 16;7(8):2516-2526. doi: 10.1021/acsptsci.4c00330. eCollection 2024 Aug 9.
We applied our previously developed probe confined dynamic mapping protocol, which combines enhanced sampling molecular dynamics (MD) simulations and fragment-based approaches, to identify the binding site of GSK2239633A (-[[3-[[3-[(5-chlorothiophen-2-yl)sulfonylamino]-4-methoxyindazol-1-yl]methyl]phenyl]methyl]-2-hydroxy-2-methylpropanamide), a selective CC-chemokine receptor type 4 (CCR4) negative allosteric modulator, using CCR4 homology and AlphaFold models. By comparing the performance across five computational models, we identified conserved (K310 and Y304) and non-conserved (M243) residue hotspots for GSK2239633A binding, which were validated by mutagenesis and bioluminescence resonance energy transfer assay. Further analysis of 3D models and MD simulations highlighted the pair of residues 6.36 and 7.56 that might account for antagonist selectivity among chemokine receptors. Our protocol provides a promising approach for characterizing ligand binding sites in membrane proteins, considering receptor dynamics and adaptability and guiding protein template selection for ligand design.
我们应用了我们之前开发的探针受限动态映射协议，该协议结合了增强采样分子动力学（MD）模拟和基于片段的方法，使用CCR4同源模型和AlphaFold模型来识别选择性CC趋化因子受体4（CCR4）负变构调节剂GSK2239633A（-[[3-[[3-[(5-氯噻吩-2-基)磺酰氨基]-4-甲氧基吲唑-1-基]甲基]苯基]甲基]-2-羟基-2-甲基丙酰胺）的结合位点。通过比较五个计算模型的性能，我们确定了GSK2239633A结合的保守（K310和Y304）和非保守（M243）残基热点，这些热点通过诱变和生物发光共振能量转移测定得到了验证。对三维模型和MD模拟的进一步分析突出了可能解释趋化因子受体之间拮抗剂选择性的6.36和7.56这一对残基。考虑到受体动力学和适应性，并指导配体设计的蛋白质模板选择，我们的协议为表征膜蛋白中的配体结合位点提供了一种有前景的方法。
