Optimization of fragment size D-amino acid oxidase (DAAO) inhibitors was investigated using a combination of computational and experimental methods. Retrospective free energy perturbation (FEP) calculations were performed for benzo[d]isoxazole derivatives, a series of known inhibitors with two potential binding modes derived from X-ray structures of other DAAO inhibitors. The good agreement between experimental and computed binding free energies in only one of the hypothesized binding modes strongly support this bioactive conformation. Then, a series of 1-H-indazol-3-ol derivatives formerly not described as DAAO inhibitors was investigated. Binding geometries could be reliably identified by structural similarity to benzo[d]isoxazole and other well characterized series and FEP calculations were performed for several tautomers of the deprotonated and protonated compounds since all these forms are potentially present owing to the experimental pKa values of representative compounds in the series. Deprotonated compounds are proposed to be the most important bound species owing to the significantly better agreement between their calculated and measured affinities compared to the protonated forms. FEP calculations were also used for the prediction of the affinities of compounds not previously tested as DAAO inhibitors and for a comparative structure-activity relationship study of the benzo[d]isoxazole and indazole series. Selected indazole derivatives were synthesized and their measured binding affinity towards DAAO was in good agreement with FEP predictions.

中文翻译：

---

通过自由能计算来验证互变异构和原型结合模式。基于结构的D-氨基酸氧化酶抑制剂优化研究。

结合计算和实验方法研究了片段大小D-氨基酸氧化酶（DAAO）抑制剂的优化。对苯并[d]异恶唑衍生物进行了回顾性自由能扰动（FEP）计算，苯并[d]异恶唑衍生物是一系列已知的抑制剂，具有从其他DAAO抑制剂的X射线结构得出的两种潜在结合模式。仅在一种假设的结合模式下，实验和计算的结合自由能之间的良好一致性强烈支持了这种生物活性构象。然后，研究了一系列以前未被描述为DAAO抑制剂的1-H-吲唑-3-醇衍生物。结合几何形状可以通过与苯并[d]异恶唑的结构相似性可靠地鉴定，并且可以进行其他特征明​​确的系列，并且对去质子化和质子化化合物的几种互变异构体进行了FEP计算，因为由于代表性化合物的实验pKa值，所有这些形式都可能存在系列中。提议去质子化的化合物是最重要的结合物种，因为与质子化形式相比，它们的计算亲和力和测量亲和力之间的一致性更好。FEP计算还用于预测以前未作为DAAO抑制剂测试过的化合物的亲和力，并用于对苯并[d]异恶唑和吲唑系列的结构-活性关系的比较研究。