Hu proteins are members of the RNA-binding protein (RBP) family and play a pivotal role in the regulation of post-transcriptional processes. Through interaction with selected mRNAs, RBPs regulate their function and stability; as a consequence, RBP dysregulation can cause abnormal translation of key proteins involved in several pathologies. In the past few years, this observation has sparked interest to develop new treatments against these pathologies by using small molecules able to modulate RBP activity. Among the four Hu proteins, we have directed our efforts towards the isoform HuR, which is mainly involved in cancer, inflammation and retinopathy. Aimed at developing compounds able to modulate the stability of HuR–mRNA complexes, in the present work, we applied a biophysical fragment screening by assessing a library of halogen-enriched heterocyclic fragments (HEFLibs) via Surface Plasmon Resonance (SPR) and Saturation Transfer Difference (STD) NMR to select promising fragments able to interact with HuR. One selected fragment and a few commercially available congeners were exploited to design and synthesize focused analogues of compound N-(3-chlorobenzyl)-N-(3,5-dihydroxyphenethyl)-4-hydroxybenzamide (1), our previously reported hit. STD NMR spectroscopy, molecular modeling, and SPR offered further insight into the HuR-small molecule interaction and showed that fragment-based approaches represent a promising and yet underexplored strategy to tackle such unusual targets. Lastly, fluorescence polarization (FP) studies revealed the capability of the new compounds to interfere with the formation of the HuR-mRNA complex. This is, to our knowledge, the first fragment-based campaign performed on the Hu protein class, and one of the few examples in the larger RBP field and constitutes an important step in the quest for the rational modulation of RBPs and related RNA functions by small molecules.

Hu 蛋白是 RNA 结合蛋白 (RBP) 家族的成员，在转录后过程的调节中起关键作用。通过与选定的 mRNA 相互作用，RBP 调节其功能和稳定性；因此，RBP 失调会导致涉及多种病理的关键蛋白质的异常翻译。在过去几年中，这一观察引起了人们的兴趣，即通过使用能够调节 RBP 活性的小分子来开发针对这些病理的新疗法。在四种 Hu 蛋白中，我们已将我们的努力集中在 HuR 异构体上，它主要与癌症、炎症和视网膜病变有关。在目前的工作中，旨在开发能够调节 HuR-mRNA 复合物稳定性的化合物，我们通过表面等离子共振 (SPR) 和饱和转移差 (STD) NMR 评估富含卤素的杂环片段 (HEFLibs) 库来应用生物物理片段筛选，以选择能够与 HuR 相互作用的有希望的片段。一种选定的片段和一些市售的同类物被用来设计和合成化合物的聚焦类似物N- (3-氯苄基) -N- (3,5-二羟基苯乙基)-4-羟基苯甲酰胺( 1 )，我们之前报道的命中。STD  NMR 光谱、分子建模和 SPR 提供了对 HuR-小分子相互作用的进一步洞察，并表明基于片段的方法代表了一种有前途但尚未探索的策略来解决此类不寻常的目标。最后，荧光偏振 (FP) 研究揭示了新化合物干扰 HuR-mRNA 复合物形成的能力。据我们所知，这是对 Hu 蛋白类进行的第一次基于片段的运动，也是更大的 RBP 领域中为数不多的例子之一，并且构成了寻求合理调节 RBP 和相关 RNA 功能的重要一步。小分子。