Identification of N,N-arylalkyl-picolinamide derivatives targeting the RNA-binding protein HuR, by combining biophysical fragment-screening and molecular hybridization

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.

Automated summary

In this study, a biophysical fragment screening approach was used to identify promising small molecule fragments that interact with HuR, providing insights for the development of new therapeutic drugs. By designing and synthesizing compounds with interfering effects, the formation of the HuR-mRNA complex was successfully disrupted, offering a new strategy and pathway for rational modulation of RNA-binding protein activity.