Abstract by Yuting Qin

Reactive oxygen species (ROS) play diverse roles in both physiological processes and pathological conditions. Regulation of ROS levels rely on cytoprotective systems regulated by transcription factors, such as Nrf2. Targeting the Keap1-Nrf2 protein-protein interaction (PPI) is a promising strategy for scavenging ROS through upregulation of antioxidant enzymes and has emerged as a potential method ameliorating ROS-related disorders. Fragment-based drug discovery (FBDD), which exploits that screening fragments give a high hit rate due to less clash from smaller-sized fragments, is considered as an efficacious strategy for the development of inhibitors targeting PPIs.

Beginning in chapter 4, commercially available library of 2500 fragments was screened by thermal shift assay (TSA), fluorescence polarization (FP), and surface plasmon resonance (SPR). Hits were next validated by saturation transfer difference (STD) NMR, and prioritized by X-ray crystallography. A fragment 1/F#669 exhibited a K_i value of 490 µM, was selected as the starting point. Through systematic fragment growing and linking strategies, a lead compound 5/UCAB#813 with micromolar affinity (K_i = 3.19 µM) was obtained. Comprehensive SAR studies among 20 analogues generated a compound (20/UCAB#977-P1) with a K_i of 280 nM. This compound up-regulated Nrf2 downstream genes at both protein level and RNA level in HaCaT cells in an Nrf2-dependent manner without showing signs of cytotoxicity. To further improve the affinity and cellular activity, a conformational pre-organization strategy was implemented in chapter 5. A pair of X-ray crystallography for linear and cyclized compounds was obtained. 49 analogues were synthesized to explore the SAR. Overall, this led to a 245-fold improvement in binding affinity relative to linear compound UCAB#813, and compound 68/UCAB#1092 (K_i = 13 nM) was shown to upregulate Nrf2-regulated genes at 10 µM without off-target toxicity. A panel of 16 Keap1 Kelch homologues was tested for binding to our compounds by TSA demonstrating a high selectivity towards Keap1. An analysis of the physicochemical properties indicates that cellular activity and reasonable metabolic stability are only achieved within a narrow range of total polar surface area (tPSA) and cLogD.