The crystal structure of recombinant murine liver cytosolic epoxide hydrolase (EC 3.3.2.3) has been determined at 2.8-Å resolution. The binding of a nanomolar affinity inhibitor confirms the active site location in the C-terminal domain; this domain is similar to that of haloalkane dehalogenase and shares the ␣͞␤ hydrolase fold. A structure-based mechanism is proposed that illuminates the unique chemical strategy for the activation of endogenous and man-made epoxide substrates for hydrolysis and detoxification. Surprisingly, a vestigial active site is found in the N-terminal domain similar to that of another enzyme of halocarbon metabolism, haloacid dehalogenase. Although the vestigial active site does not participate in epoxide hydrolysis, the vestigial domain plays a critical structural role by stabilizing the dimer in a distinctive domain-swapped architecture. Given the genetic and structural relationships among these enzymes of xenobiotic metabolism, a structure-based evolutionary sequence is postulated.During the course of mammalian evolution, myriad catabolic pathways have evolved to defend against harmful environmental chemicals and their metabolites. For example, aromatic hydrocarbons such as styrene, stilbene, or benzo[a]pyrene can be oxidized in vivo to form mutagenic epoxides that readily alkylate nucleic acids. Olefinic terpenoid natural products can be similarly activated and oxidized to generate terpenoid epoxides with various reactivities and potencies (1). The first line of chemical defense against xenobiotic-derived epoxides is the liver enzyme epoxide hydrolase (EC 3.3.2.3) (1-3), which exists as microsomal and soluble enzymes designated mEH and sEH, respectively; sEH is found in both the cytosol and the peroxisomal matrix of liver cells (4-8). Both mEH and sEH hydrolyze epoxides to form vicinal 1,2-diols, which are typically less reactive, less mutagenic, and more rapidly excreted due to increased solubility (1, 9). Accordingly, sEH activity is found to decrease the mutagenicity of epoxides in the Ames Salmonella assay (10), and it decreases the induction of sister chromatid exchange by trans-␤-ethylstyrene (11).The catalytic mechanism of sEH proceeds through a covalent alkylenzyme ester intermediate with an active site aspartate nucleophile. Strikingly, hydrolysis of this intermediate requires that an oxygen atom of the enzyme is incorporated into the vicinal diol product with each turnover (12)(13)(14). Enzymological studies of the related mEH isozyme (21% sequence identity) indicate an identical mechanism proceeding through an alkylenzyme ester intermediate (15)(16)(17). Interestingly, GJ10 haloalkane dehalogenase (18-21) and haloacid dehalogenase (22-24) from Xanthobacter autotrophicus each hydrolyze carbon-halogen bonds through mechanisms similarly involving an aspartate nucleophile and an alkylenzyme ester intermediate. These are the first and last enzymes, respectively, in the catabolism of 1,2-dihalogenated alkanes such as 1,2-dichloroethane and 1,2-dibromoethane. Like the pare...

Background Anti-cytokine therapies such as adalimumab, tocilizumab, and the small molecule JAK inhibitor tofacitinib have proven that cytokines and their subsequent downstream signaling processes are important in the pathogenesis of rheumatoid arthritis. Tofacitinib, a pan-JAK inhibitor, is the first approved JAK inhibitor for the treatment of RA and has been shown to be effective in managing disease. However, in phase 2 dose-ranging studies tofacitinib was associated with dose-limiting tolerability and safety issues such as anemia. Upadacitinib (ABT-494) is a selective JAK1 inhibitor that was engineered to address the hypothesis that greater JAK1 selectivity over other JAK family members will translate into a more favorable benefit:risk profile. Upadacitinib selectively targets JAK1 dependent disease drivers such as IL-6 and IFNγ, while reducing effects on reticulocytes and natural killer (NK) cells, which potentially contributed to the tolerability issues of tofacitinib. Methods Structure-based hypotheses were used to design the JAK1 selective inhibitor upadacitinib. JAK family selectivity was defined with in vitro assays including biochemical assessments, engineered cell lines, and cytokine stimulation. In vivo selectivity was defined by the efficacy of upadacitinib and tofacitinib in a rat adjuvant induced arthritis model, activity on reticulocyte deployment, and effect on circulating NK cells. The translation of the preclinical JAK1 selectivity was assessed in healthy volunteers using ex vivo stimulation with JAK-dependent cytokines. Results Here, we show the structural basis for the JAK1 selectivity of upadacitinib, along with the in vitro JAK family selectivity profile and subsequent in vivo physiological consequences. Upadacitinib is ~ 60 fold selective for JAK1 over JAK2, and > 100 fold selective over JAK3 in cellular assays. While both upadacitinib and tofacitinib demonstrated efficacy in a rat model of arthritis, the increased selectivity of upadacitinib for JAK1 resulted in a reduced effect on reticulocyte deployment and NK cell depletion relative to efficacy. Ex vivo pharmacodynamic data obtained from Phase I healthy volunteers confirmed the JAK1 selectivity of upadactinib in a clinical setting. Conclusions The data presented here highlight the JAK1 selectivity of upadacinitinib and supports its use as an effective therapy for the treatment of RA with the potential for an improved benefit:risk profile. Electronic supplementary material The online version of this article (10.1186/s41927-018-0031-x) contains supplementary material, which is available to authorized users.

The structures of two alkylurea inhibitors complexed with murine soluble epoxide hydrolase have been determined by x-ray crystallographic methods.

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