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.

Clinical utility of phosphodiesterase 4 (PDE4) inhibitors as antiinflammatory agents has, to date, been limited by adverse effects including nausea and emesis, making accurate assessment of emetic versus anti-inflammatory potencies critical to the development of inhibitors with improved therapeutic indices. In the present study we determined the in vitro and in vivo anti-inflammatory potencies of the first-generation PDE4 inhibitor, rolipram, the second-generation inhibitors, roflumilast and cilomilast, and a novel third generation inhibitor, 1-ethyl-5-{5-[(4-methyl-1-piperazinyl)methyl]-1,3,4-oxadiazol-2-yl}-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo [3,4-b]pyridin-4-amine (EPPA-1). The rank-order potency against lipopolysaccharide (LPS)-induced tumor necrosis factor-␣ production by human peripheral blood mononuclear cells was roflumilast (IC 50 ϭ 5 nM) Ͼ EPPA-1 (38) Ͼ rolipram (269) Ͼ cilomilast (389), and against LPS-induced pulmonary neutrophilia in the rat was EPPA-1 (D 50 ϭ 0.042 mg/ kg) Ͼ roflumilast (0.24) Ͼ rolipram (3.34) Ͼ cilomilast (4.54). Pica, the consumption of non-nutritive substances in response to gastrointestinal stress, was used as a surrogate measure for emesis, giving a rank-order potency of rolipram (D 50 ϭ 0.495 mg/kg) Ͼ roflumilast (1.6) Ͼ cilomilast (6.4) Ͼ EPPA-1 (24.3). The low and high emetogenic activities of EPPA-1 and rolipram, respectively, detected in the pica model were confirmed in a second surrogate model of emesis, reversal of ␣ 2 -adrenoceptor-mediated anesthesia in the mouse. The rank order of therapeutic indices derived in the rat [(pica D 50 )/(neutrophilia D 50 )] was EPPA-1 (578) Ͼ roflumilast (6.4) Ͼ cilomilast (1.4) Ͼ rolipram (0.15), consistent with the rank order derived in the ferret [(emesis D 50 )/(neutrophilia D 50 )]. These data validate rat pica feeding as a surrogate for PDE4 inhibitor-induced emesis in higher species, and identify EPPA-1 as a novel PDE4 inhibitor with an improved therapeutic index.Phosphodiesterases are a superfamily of enzymes that hydrolyze cAMP and/or cGMP to their inactive nucleotides. Phosphodiesterase 4 (PDE4) is selective for cAMP, and consists of the four subtypes A, B, C, and D. PDE4 inhibitors have shown efficacy in various in vitro and in vivo inflammatory models by increasing the intracellular levels of cAMP in many immune cells (T lymphocytes, monocytes, neutrophils, and eosinophils). As such, PDE4 inhibitors have been pursued as therapeutics for pulmonary diseases with an inflammatory component, including chronic obstructive pulmo-

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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)-2-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 (-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-1-induced nuclear factor-B 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.