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.

The role of T cells in chronic obstructive pulmonary disease (COPD) is not well understood. We have previously demonstrated that chronic cigarette smoke exposure can lead to the accumulation of CD4(+) and CD8(+) T cells in the alveolar airspaces in a mouse model of COPD, implicating these cells in disease pathogenesis. However, whether specific inhibition of T cell responses represents a therapeutic strategy has not been fully investigated. In this study inhibition of T cell responses through specific depleting antibodies, or the T cell immunosuppressant drug cyclosporin A, prevented airspace enlargement and neutrophil infiltration in a mouse model of chronic cigarette smoke exposure. Furthermore, individual inhibition of either CD4(+) T helper or CD8(+) T cytotoxic cells prevented airspace enlargement to a similar degree, implicating both T cell subsets as critical mediators of the adaptive immune response induced by cigarette smoke exposure. Importantly, T cell depletion resulted in significantly decreased levels of the Th17-associated cytokine IL-17A, and of caspase 3 and caspase 7 gene expression and activity, induced by cigarette smoke exposure. Finally, inhibition of T cell responses in a therapeutic manner also inhibited cigarette smoke-induced airspace enlargement, IL-17A expression, and neutrophil influx in mice. Together these data demonstrate for the first time that therapeutic inhibition of T cell responses may be efficacious in the treatment of COPD. Given that broad immunosuppression may be undesirable in COPD patients, this study provides proof-of-concept for more targeted approaches to inhibiting the role of T cells in emphysema development.

PL, Rizi RR. Regional function-structure relationships in lungs of an elastase murine model of emphysema. J Appl Physiol 112: 135-148, 2012. First published September 22, 2011 doi:10.1152/japplphysiol.01181.2010.-Changes in lung function and structure were studied using hyperpolarized 3 He MRI in an elastaseinduced murine model of emphysema. The combined analysis of the apparent diffusion coefficient (ADC) and fractional ventilation (R) were used to distinguish emphysematous changes and also to develop a model for classifying sections of the lung into diseased and normal. Twelve healthy male BALB/c mice (26 Ϯ 2 g) were randomized into healthy and elastase-induced mice and studied ϳ8 -11 wk after model induction. ADC and R were measured at a submillimeter planar resolution. Chord length (Lx) data were analyzed from histology samples from the corresponding imaged slices. Logistic regression was applied to estimate the probability that an imaged pixel came from a diseased animal, and bootstrap methods (1,000 samples) were used to compare the regression results for the morphological and imaging results. Multivariate ANOVA (MANOVA) was used to analyze transformed ADC (ADCBC), and R (RBC) data and also to control for the experiment-wide error rate. MANOVA and ANOVA showed that elastase induced a statistically measureable change in the average transformed Lx and ADCBC but not in the average RBC. Marginal mean analysis demonstrated that ADCBC was on average 0.19 [95% confidence interval (CI): 0.16, 0.22] higher in the emphysema group, whereas RBC was on average 0.05 (95% CI: 0.04, 0.06) lower. Logistic regression supported the hypothesis that ADCBC and R BC, together, were better at differentiating normal from diseased tissue than either measurement alone. The odds ratios for ADCBC and R BC were 7.73 (95% CI: 5.23, 11.42) and 9.14 ϫ 10 Ϫ5 (95% CI: 3.33 ϫ 10 Ϫ5 , 25.06 ϫ 10 Ϫ5 ), respectively. Using a 50% probability cutoff, this model classified 70.6% of pixels correctly. The sensitivity and specificity of this model at the 50% cutoff were 74.9% and 65.2%, respectively. The area under the receiver operating characteristic curve was 0.76 (95% CI: 0.74, 0.78). The regression model presented can be used to map MRI data to disease probability maps. These probability maps present a future possibility of using both measurements in a more clinically feasible method of diagnosing this disease. lung physiology; relationship of lung function and structure; regional fractional ventilation; apparent diffusion coefficient; hyperpolarized gas magnetic resonance imaging

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.