Previously Uncharacterized Vacuolar-type ATPase Binding Site Discovered from Structurally Similar Compounds with Distinct Mechanisms of Action.

Using a comprehensive chemical genetics approach, we identified a member of the lignan natural product family, HTP-013, which exhibited significant cytotoxicity across various cancer cell lines. Correlation of compound activity across a panel of reporter gene assays suggested the vacuolar-type ATPase (v-ATPase) as a potential target for this compound. Additional cellular studies and a yeast haploinsufficiency screen strongly supported this finding. Competitive photoaffinity labeling experiments demonstrated that the ATP6V0A2 subunit of the v-ATPase complex binds directly to HTP-013, and further mutagenesis library screening identified resistance-conferring mutations in ATP6V0A2. The positions of these mutations suggest the molecule binds a novel pocket within the domain of the v-ATPase complex responsible for proton translocation. While other mechanisms of v-ATPase regulation have been described, such as dissociation of the complex or inhibition by natural products including bafilomycin A1 and concanamycin, this work provides detailed insight into a distinct binding pocket within the v-ATPase complex.

CCL20 and IL22 Messenger RNA Expression After Adalimumab vs Methotrexate Treatment of Psoriasis: A Randomized Clinical Trial.

IMPORTANCE: Methotrexate is a first-line systemic agent for treating of psoriasis, although its onset of effects is slower and overall it is less effective than tumor necrosis factor blockers. OBJECTIVE: To differentiate the response of psoriatic disease to adalimumab and methotrexate sodium. DESIGN, SETTING, AND PARTICIPANTS: Single-center, randomized, assessor-blind, 2-arm clinical trial of 30 patients from the outpatient dermatology center of Tufts Medical Center, enrolled from August 18, 2009, to October 11, 2011. Patients aged 18 to 85 years with chronic plaque-type psoriasis, a minimum Physician Global Assessment score of 3 (higher scores indicate more severe disease), and a psoriatic plaque of at least 2 cm were randomized in a 1:1 fashion to receive subcutaneous adalimumab or oral methotrexate. Skin biopsy specimens obtained at baseline and weeks 1, 2, 4, and 16 were given a histologic grade by blinded assessors to evaluate treatment response. Analyses were conducted from April 16, 2013, to January 5, 2015. INTERVENTIONS: A 16-week course of subcutaneous adalimumab (40 mg every 2 weeks after a loading dose) or low-dosage oral methotrexate sodium (7.5-25 mg/wk). MAIN OUTCOMES AND MEASURES: Changes in genomic, immunohistochemical, and messenger RNA (mRNA) profiles. RESULTS: Methotrexate responders experienced significant downregulation of helper T-cell-related (T(H)1, T(H)17, and T(H)22) mRNA expression compared with methotrexate nonresponders. Comparisons among adalimumab-treated patients were limited by the number of nonresponders (n = 1). Between adalimumab and methotrexate responders, we found no significant differences in gene expression at any study point or in the expression of T-cell-related mRNA at week 16. Adalimumab responders demonstrated early downregulation of chemokine (C-C motif) ligand 20 (CCL20) mRNA (mean [SE] at week 2, -1.83 [0.52], P < .001; week 16, -3.55 [0.54], P < .001) compared with late downregulation for methotrexate responders (week 2, 0.02 [0.51], P = .96; week 16, -2.96 [0.51], P < .001). Similar differences were observed with interleukin 22 (IL22) mRNA showing early downregulation for adalimumab responders (week 2, -3.17 [1.00], P < .001; week 16, -3.58 [1.00], P < .001) compared with late downregulation for methotrexate responders (week 2, -0.44 [0.68], P = .64; week 16, -5.14 [0.68], P < .001). Analysis of variance findings for key mRNA and immunohistochemical marker expression over the study course were significant only for CCL20 (P = .03) and IL22 (P = .006) mRNA comparing adalimumab and methotrexate responders. CONCLUSIONS AND RELEVANCE: Methotrexate is an immunomodulator with effects on helper T-cell signaling in psoriasis. Similar genomic and immunohistochemical response signatures and levels of mRNA downregulation at study completion among adalimumab and methotrexate responders suggest a disease-driven instead of therapeutic-driven pathway regulation. Adalimumab and methotrexate responses are differentiated by patterns of normalization of CCL20 and IL22 mRNA expression and may explain the varied onset and degree of clinical responses by each treatment. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00932113.

Prevalence of the metabolic syndrome in children with psoriatic disease.

Adults with psoriasis have a greater risk of developing metabolic syndrome (MetS) and cardiovascular disease (CVD), but few studies have investigated the prevalence of MetS and other risk factors for CVD in children with psoriasis. In an assessor-blinded study, 20 children ages 9-17 years with a current or previously documented history of psoriasis involving 5% or more of their body surface area or psoriatic arthritis were compared with a cohort of age- and sex-matched controls with benign nevi, warts, or acne. MetS, our primary endpoint, was defined by the presence of abnormal values in at least three of the following measures: triglycerides, high-density lipoprotein cholesterol (HDL-C), fasting blood glucose (FBG), waist circumference, and blood pressure. Secondary endpoints included high-sensitivity C-reactive protein (hs-CRP), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C). Thirty percent (6/20) of children with psoriasis met the criteria for MetS, compared with 5% (1/20) of the control group (p < 0.05). Subjects with psoriasis had higher mean FBG (91.1 mg/dL) than the control group (82.9 mg/dL) (p = 0.01). There were no statistically significant differences in the other four components of MetS, BMI, BMI percentile, hs-CRP, TC, or LDL-C. The results of this trial demonstrate that children with psoriasis have higher rates of MetS than age- and sex-matched controls. It may therefore be important to evaluate children with psoriasis for components of MetS to prevent future CVD morbidity and mortality.

Structural mechanism of GAF-regulated σ(54) activators from Aquifex aeolicus.

The σ subunits of bacterial RNA polymerase occur in many variant forms and confer promoter specificity to the holopolymerase. Members of the σ(54) family of σ subunits require the action of a 'transcriptional activator' protein to open the promoter and initiate transcription. The activator proteins undergo regulated assembly from inactive dimers to hexamers that are active ATPases. These contact σ(54) directly and, through ATP hydrolysis, drive a conformational change that enables promoter opening. σ(54) activators use several different kinds of regulatory domains to respond to a wide variety of intracellular signals. One common regulatory module, the GAF domain, is used by σ(54) activators to sense small-molecule ligands. The structural basis for GAF domain regulation in σ(54) activators has not previously been reported. Here, we present crystal structures of GAF regulatory domains for Aquifex aeolicus σ(54) activators NifA-like homolog (Nlh)2 and Nlh1 in three functional states-an 'open', ATPase-inactive state; a 'closed', ATPase-inactive state; and a 'closed', ligand-bound, ATPase-active state. We also present small-angle X-ray scattering data for Nlh2-linked GAF-ATPase domains in the inactive state. These GAF domain dimers regulate σ(54) activator proteins by holding the ATPase domains in an inactive dimer conformation. Ligand binding of Nlh1 dramatically remodels the GAF domain dimer interface, disrupting the contacts with the ATPase domains. This mechanism has strong parallels to the response to phosphorylation in some two-component regulated σ(54) activators. We describe a structural mechanism of GAF-mediated enzyme regulation that appears to be conserved among humans, plants, and bacteria.