The NRF2-mediated cytoprotective response is central to cellular homoeostasis, and there is increasing interest in developing small-molecule activators of this pathway as therapeutics for diseases involving chronic oxidative stress. The protein KEAP1, which regulates NRF2, is a key point for pharmacological intervention, and we recently described the use of fragment-based drug discovery to develop a tool compound that directly disrupts the protein-protein interaction between NRF2 and KEAP1. We now present the identification of a second, chemically distinct series of KEAP1 inhibitors, which provided an alternative chemotype for lead optimization. Pharmacophoric information from our original fragment screen was used to identify new hit matter through database searching and to evolve this into a new lead with high target affinity and cell-based activity. We highlight how knowledge obtained from fragment-based approaches can be used to focus additional screening campaigns in order to de-risk projects through the rapid identification of novel chemical series.

Nuclear factor (erythroid-derived 2)–like 2 (Nrf2) is a key regulator of oxidative stress and cellular repair and can be activated through inhibition of its cytoplasmic repressor, Kelch-like ECH-associated protein 1 (Keap1). Several small molecule disrupters of the Nrf2-Keap1 complex have recently been tested and/or approved for human therapeutic use but lack either potency or selectivity. The main goal of our work was to develop a potent, selective activator of NRF2 as protection against oxidative stress. In human bronchial epithelial cells, our Nrf2 activator, 3-(pyridin-3-ylsulfonyl)-5-(trifluoromethyl)-2H-chromen-2-one (PSTC), induced Nrf2 nuclear translocation, Nrf2-regulated gene expression, and downstream signaling events, including induction of NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme activity and heme oxygenase-1 protein expression, in an Nrf2-dependent manner. As a marker of subsequent functional activity, PSTC restored oxidant (tert-butyl hydroperoxide)–induced glutathione depletion. The compound's engagement of the Nrf2 signaling pathway translated to an in vivo setting, with induction of Nrf2-regulated gene expression and NQO1 enzyme activity, as well as restoration of oxidant (ozone)–induced glutathione depletion, occurring in the lungs of PSTC-treated rodents. Under disease conditions, PSTC engaged its target, inducing the expression of Nrf2-regulated genes in human bronchial epithelial cells derived from patients with chronic obstructive pulmonary disease, as well as in the lungs of cigarette smoke–exposed mice. Subsequent to the latter, a dose-dependent inhibition of cigarette smoke–induced pulmonary inflammation was observed. Finally, in contrast with bardoxolone methyl and sulforaphane, PSTC did not inhibit interleukin-1β–induced nuclear factor-κB translocation or insulin-induced S6 phosphorylation in human cells, emphasizing the on-target activity of this compound. In summary, we characterize a potent, selective Nrf2 activator that offers protection against pulmonary oxidative stress in several cellular and in vivo models.

Word Count: 250 Introduction Word Count: 690 Discussion Word Count: 886 This article has not been copyedited and formatted. The final version may differ from this version. JPET Fast Forward. Published on August 8, 2017 as DOI: 10.1124/jpet.117.241794 at A PE T Jornals on A uust 0, 2017 jpet.asjournals.org D ow nladed from

KEAP1 is the key regulator of the NRF2-mediated cytoprotective response, and increasingly recognized as a target for diseases involving oxidative stress. Pharmacological intervention has focused on molecules that decrease NRF2-ubiquitination through covalent modification of KEAP1 cysteine residues, but such electrophilic compounds lack selectivity and may be associated with off-target toxicity. We report here the first use of a fragment-based approach to directly target the KEAP1 Kelch-NRF2 interaction. X-ray crystallographic screening identified three distinct hot-spots for fragment binding within the NRF2 binding pocket of KEAP1, allowing progression of a weak fragment hit to molecules with nanomolar affinity for KEAP1 while maintaining drug-like properties. This work resulted in a promising lead compound which exhibits tight and selective binding to KEAP1, and activates the NRF2 antioxidant response in cellular and in vivo models, thereby providing a high quality chemical probe to explore the therapeutic potential of disrupting the KEAP1-NRF2 interaction.

Endoplasmic reticulum stress plays a critical role to restore the homeostasis of protein production in eukaryotic cells. This vital process is hence involved in many types of diseases including COPD. PERK, one branch in the ER stress signaling pathways, has been reported to activate NRF2 signaling pathway, a known protective response to COPD. Based on this scientific rationale, we aimed to identify PERK activators as a mechanism to achieve NRF2 activation. In this report, we describe a phenotypic screening assay to identify PERK activators. This assay measures phosphorylation of GFP-tagged eIF2α upon PERK activation via a cell-based LanthaScreen technology. To obtain a robust assay with sufficient signal to background and low variation, multiple parameters were optimized including GFP-tagged eIF2α BacMam concentration, cell density and serum concentration. The assay was validated by a tool compound, Thapsigargin, which induces phosphorylation of eIF2α. In our assay, this compound showed maximal signal window of approximately 2.5-fold with a pEC50 of 8.0, consistent with literature reports. To identify novel PERK activators through phosphorylation of eIF2α, a focused set of 8,400 compounds was screened in this assay at 10 µM. A number of hits were identified and validated. The molecular mechanisms for several selected hits were further characterized in terms of PERK activation and effects on PERK downstream components. Specificity of these compounds in activating PERK was demonstrated with a PERK specific inhibitor and in PERK knockout mouse embryonic fibroblast (MEF) cells. In addition, these hits showed NRF2-dependent anti-oxidant gene induction. In summary, our phenotypic screening assay is demonstrated to be able to identify PERK specific activators. The identified PERK activators could potentially be used as chemical probes to further investigate this pathway as well as the link between PERK activation and NRF2 pathway activation.

With many safety and technical limitations partly mitigated through chemical modifications, antisense oligonucleotides (ASOs) are gaining recognition as therapeutic entities. The increase in potency realized by 'third generation chemistries' may, however, simultaneously increase affinity to unintended targets with partial sequence complementarity. However, putative hybridization-dependent off-target effects (OTEs), a risk historically regarded as low, are not being adequately investigated. Here we show an unexpectedly high OTEs confirmation rate during screening of fully phosphorothioated (PS)-LNA gapmer ASOs designed against the BACH1 transcript. We demonstrate in vitro mRNA and protein knockdown of off-targets with a wide range of mismatch (MM) and gap patterns. Furthermore, with RNase H1 activity residing within the nucleus, hybridization predicted against intronic regions of pre-mRNAs was tested and confirmed. This dramatically increased ASO-binding landscape together with relatively high potency of such interactions translates into a considerable safety concern. We show here that with base pairing-driven target recognition it is possible to predict the putative off-targets and address the liability during lead design and optimization phases. More-over, in silico analysis performed against both primary as well as spliced transcripts will be invaluable in elucidating the mechanism behind the hepatoxicity observed with some LNA-modified gapmers.

Chronic obstructive pulmonary disease (COPD) is an increasing health problem primarily associated with cigarette smoking, and one of the leading causes of morbidity and mortality worldwide. Despite recent advances in understanding the pathogenesis of the disease, overall patient outcome remains poor with limited therapeutic intervention. Chronic inflammation, an imbalance between proteolytic and anti-proteolytic activities (leading to lung parenchyma destruction) and excessive oxidative stress contribute to COPD pathophysiology. Oxidative stress-triggered apoptosis of alveolar structural cells, including epithelial cells, may be an important event in the development of COPD. In this study, we developed a cell-based oxidative stress-induced apoptosis assay and performed a high-throughput screen (HTS) using a human druggable genome siRNA library. Our results have identified potential novel pathways (e.g. unfolded protein response, proteosomal activity) and targets (e.g. MAP3K14, HMGB2) that regulate the response of lung epithelial cells to oxidative stress. This assay has proven to be a useful tool for the identification of potential new therapeutic targets for lung disease.