Unexpected Formation and Potent Antioxidant Activity of Macrocyclic Dimers Containing Disulfide and Selenide Groups.

During attempts to prepare spirodithiaselenuranes as GPx mimetics, a series of unexpected dimeric macrocycles was obtained, each containing two selenide and two disulfide moieties in rings ranging from 18- to 26-membered. The products showed potent GPx-like activity in an NMR assay based on their ability to catalyze the reduction of hydrogen peroxide with benzyl thiol. The high catalytic activity was attributed to transannular effects during selenide to selenoxide oxidation. This redox process was also characterized by an induction period that indicated autocatalysis in the formation of an intermediate selenoxide from the oxidation of the corresponding selenide.

Autoantibodies to Dense Fine Speckles in Pediatric Diseases and Controls.

OBJECTIVE: Autoantibodies to the dense fine speckled 70 kDa antigen (DFS70) are reported to be more common in individuals who do not have an antinuclear antibody (ANA)-associated rheumatic disease (AARD) than in patients with AARD. The frequency of anti-DFS70 antibodies has been thoroughly studied in adult but not in pediatric populations. The primary objective of this observational study was to determine the frequency of anti-DFS70 in pediatric AARD and reference cohorts. METHODS: Sera from 743 children with AARD and related conditions, and 345 samples from reference cohorts (healthy children and those being investigated for AARD) were studied for anti-DFS70 autoantibodies as measured by a chemiluminescence immunoassay. A de-identified administrative database was used to retrieve demographic, serologic, and clinical data. RESULTS: Anti-DFS70 antibodies were seen in 2.1% of healthy children and in 4.5% of sera from pediatric individuals referred for ANA testing. The frequency of anti-DFS70 was highest in juvenile localized scleroderma (LS; 4/29, 13.8%), juvenile dermatomyositis (JDM; 2/11, 18.2%), childhood systemic lupus erythematosus (cSLE; 19/331, 5.7%), diffuse cutaneous systemic sclerosis (1/22, 4.5%), celiac disease (2/49, 4.1%), and juvenile idiopathic arthritis (JIA; 5/202, 2.5%). Of note, anti-DFS70 antibodies were observed in 3/26 children (11.5%) with uveitis and JIA-associated uveitis. CONCLUSION: The frequency of anti-DFS70 autoantibodies in healthy pediatric subjects is within the lower range of that reported in adults. Anti-DFS70 antibodies can be found in childhood SSc and cSLE, but has a remarkably high frequency in children with LS, JDM, and uveitis.

Clinical and serological analysis of patients with positive anticyclic citrullinated Peptide antibodies referred through a Rheumatology Central Triage System.

OBJECTIVE: Anticitrullinated protein antibodies (ACPA) are a highly specific and sensitive biomarker for the diagnosis of rheumatoid arthritis (RA). Some patients who were found to have a positive ACPA test were referred to our Rheumatology Central Triage (CT; Calgary, Alberta, Canada) for assessment by a rheumatologist. The objectives of our study were to determine the clinical accuracy of ACPA in establishing a diagnosis of RA in a real-time clinical setting. METHODS: Cases that met 3 criteria were included in the study: (1) referred to the CT over 3 calendar years (n = 20,389), (2) reason for referral was a positive ACPA test (n = 568), and (3) evaluated by a certified rheumatologist (n = 314). An administrative serological database was used to retrieve specific ACPA results. RESULTS: Of patients referred through our CT for evaluation of a positive ACPA test, 57.6% received a diagnosis of RA; the remainder had a variety of other diagnoses, some of which might be considered early RA (9%). The predictive values of ACPA for the diagnosis of RA were increased when rheumatoid factor (RF) results were included in the analysis. When definite and possible RA were combined and the prevalence of moderate/high ACPA was compared to all other individuals, the positive and negative predictive values for moderate/high ACPA for RA were 74.3% and 68.4%, respectively. CONCLUSION: About 58% of patients with a positive ACPA referred through a triage system for a rheumatologist opinion received a diagnosis of RA at their first visit. RF provides additional useful information to guide the diagnosis and urgency of referral.

Multi-generational pharmacophore modeling for ligands to the cholane steroid-recognition site in the ß1 modulatory subunit of the BKCa channel.

Large conductance, voltage- and Ca(2+)-gated K(+) (BKCa) channels play a critical role in smooth muscle contractility and thus represent an emerging therapeutic target for drug development to treat vascular disease, gastrointestinal, bladder and uterine disorders. Several compounds are known to target the ubiquitously expressed BKCa channel-forming α subunit. In contrast, just a few are known to target the BKCa modulatory ß1 subunit, which is highly expressed in smooth muscle and scarce in most other tissues. Lack of available high-resolution structural data makes structure-based pharmacophore modeling of ß1 subunit-dependent BKCa channel activators a major challenge. Following recent discoveries of novel BKCa channel activators that act via ß1 subunit recognition, we performed ligand-based pharmacophore modeling that led to the successful creation and fine-tuning of a pharmacophore over several generations. Initial models were developed using physiologically active cholane steroids (bile acids) as template. However, as more compounds that act on BKCa ß1 have been discovered, our model has been refined to improve accuracy. Database searching with our best-performing model has uncovered several novel compounds as candidate BKCa ß1 subunit ligands. Eight of the identified compounds were experimentally screened and two proved to be activators of recombinant BKCa ß1 complexes. One of these activators, sobetirome, differs substantially in structure from any previously reported activator.

Activation of calcium- and voltage-gated potassium channels of large conductance by leukotriene B4.

Calcium/voltage-gated, large conductance potassium (BK) channels control numerous physiological processes, including myogenic tone. BK channel regulation by direct interaction between lipid and channel protein sites has received increasing attention. Leukotrienes (LTA4, LTB4, LTC4, LTD4, and LTE4) are inflammatory lipid mediators. We performed patch clamp studies in Xenopus oocytes that co-expressed BK channel-forming (cbv1) and accessory ß1 subunits cloned from rat cerebral artery myocytes. Leukotrienes were applied at 0.1 nm-10 µm to either leaflet of cell-free membranes at a wide range of [Ca(2+)]i and voltages. Only LTB4 reversibly increased BK steady-state activity (EC50 = 1 nm; Emax reached at 10 nm), with physiological [Ca(2+)]i and voltages favoring this activation. Homomeric cbv1 or cbv1-ß2 channels were LTB4-resistant. Computational modeling predicted that LTB4 docked onto the cholane steroid-sensing site in the BK ß1 transmembrane domain 2 (TM2). Co-application of LTB4 and cholane steroid did not further increase LTB4-induced activation. LTB4 failed to activate ß1 subunit-containing channels when ß1 carried T169A, A176S, or K179I within the docking site. Co-application of LTB4 with LTA4, LTC4, LTD4, or LTE4 suppressed LTB4-induced activation. Inactive leukotrienes docked onto a portion of the site, probably preventing tight docking of LTB4. In summary, we document the ability of two endogenous lipids from different chemical families to share their site of action on a channel accessory subunit. Thus, cross-talk between leukotrienes and cholane steroids might converge on regulation of smooth muscle contractility via BK ß1. Moreover, the identification of LTB4 as a highly potent ligand for BK channels is critical for the future development of ß1-specific BK channel activators.

Cerebrovascular dilation via selective targeting of the cholane steroid-recognition site in the BK channel ß1-subunit by a novel nonsteroidal agent.

The Ca(2+)/voltage-gated K(+) large conductance (BK) channel ß1 subunit is particularly abundant in vascular smooth muscle. By determining their phenotype, BK ß1 allows the BK channels to reduce myogenic tone, facilitating vasodilation. The endogenous steroid lithocholic acid (LCA) dilates cerebral arteries via BK channel activation, which requires recognition by a BK ß1 site that includes Thr169. Whether exogenous nonsteroidal agents can access this site to selectively activate ß1-containing BK channels and evoke vasodilation remain unknown. We performed a chemical structure database similarity search using LCA as a template, along with a two-step reaction to generate sodium 3-hydroxyolean-12-en-30-oate (HENA). HENA activated the BK (cbv1 + ß1) channels cloned from rat cerebral artery myocytes with a potency (EC50 = 53 µM) similar to and an efficacy (×2.5 potentiation) significantly greater than that of LCA. This HENA action was replicated on native channels in rat cerebral artery myocytes. HENA failed to activate the channels made of cbv1 + ß2, ß3, ß4, or ß1T169A, indicating that this drug selectively targets ß1-containing BK channels via the BK ß1 steroid-sensing site. HENA (3-45 µM) dilated the rat and C57BL/6 mouse pressurized cerebral arteries. Consistent with the electrophysiologic results, this effect was larger than that of LCA. HENA failed to dilate the arteries from the KCNMB1 knockout mouse, underscoring BK ß1's role in HENA action. Finally, carotid artery-infusion of HENA (45 µM) dilated the pial cerebral arterioles via selective BK-channel targeting. In conclusion, we have identified for the first time a nonsteroidal agent that selectively activates ß1-containing BK channels by targeting the steroid-sensing site in BK ß1, rendering vasodilation.

Multiple cholesterol recognition/interaction amino acid consensus (CRAC) motifs in cytosolic C tail of Slo1 subunit determine cholesterol sensitivity of Ca2+- and voltage-gated K+ (BK) channels.

Large conductance, Ca(2+)- and voltage-gated K(+) (BK) channel proteins are ubiquitously expressed in cell membranes and control a wide variety of biological processes. Membrane cholesterol regulates the activity of membrane-associated proteins, including BK channels. Cholesterol modulation of BK channels alters action potential firing, colonic ion transport, smooth muscle contractility, endothelial function, and the channel alcohol response. The structural bases underlying cholesterol-BK channel interaction are unknown. Such interaction is determined by strict chemical requirements for the sterol molecule, suggesting cholesterol recognition by a protein surface. Here, we demonstrate that cholesterol action on BK channel-forming Cbv1 proteins is mediated by their cytosolic C tail domain, where we identified seven cholesterol recognition/interaction amino acid consensus motifs (CRAC4 to 10), a distinct feature of BK proteins. Cholesterol sensitivity is provided by the membrane-adjacent CRAC4, where Val-444, Tyr-450, and Lys-453 are required for cholesterol sensing, with hydrogen bonding and hydrophobic interactions participating in cholesterol location and recognition. However, cumulative truncations or Tyr-to-Phe substitutions in CRAC5 to 10 progressively blunt cholesterol sensitivity, documenting involvement of multiple CRACs in cholesterol-BK channel interaction. In conclusion, our study provides for the first time the structural bases of BK channel cholesterol sensitivity; the presence of membrane-adjacent CRAC4 and the long cytosolic C tail domain with several other CRAC motifs, which are not found in other members of the TM6 superfamily of ion channels, very likely explains the unique cholesterol sensitivity of BK channels.

Structural determinants of monohydroxylated bile acids to activate beta 1 subunit-containing BK channels.

Lithocholate (LC) (10-300 microM) in physiological solution is sensed by vascular myocyte large conductance, calcium- and voltage-gated potassium (BK) channel beta(1) accessory subunits, leading to channel activation and arterial dilation. However, the structural features in steroid and target that determine LC action are unknown. We tested LC and close analogs on BK channel (pore-forming cbv1+beta(1) subunits) activity using the product of the number of functional ion channels in the membrane patch (N) and the open channel probability (Po). LC (5beta-cholanic acid-3alpha-ol), 5alpha-cholanic acid-3alpha-ol, and 5beta-cholanic acid-3beta-ol increased NPo (EC(50) approximately 45 microM). At maximal increase in NPo, LC increased NPo by 180%, whereas 5alpha-cholanic acid-3alpha-ol and 5beta-cholanic acid-3beta-ol raised NPo by 40%. Thus, the alpha-hydroxyl and the cis A-B ring junction are both required for robust channel potentiation. Lacking both features, 5alpha-cholanic acid-3beta-ol and 5-cholenic acid-3beta-ol were inactive. Three-dimensional structures show that only LC displays a bean shape with clear-cut convex and concave hemispheres; 5alpha-cholanic acid-3alpha-ol and 5beta-cholanic acid-3beta-ol partially matched LC shape, and 5alpha-cholanic acid-3beta-ol and 5-cholenic acid-3beta-ol did not. Increasing polarity in steroid rings (5beta-cholanic acid-3alpha-sulfate) or reducing polarity in lateral chain (5beta-cholanic acid 3alpha-ol methyl ester) rendered poorly active compounds, consistent with steroid insertion between beta(1) and bilayer lipids, with the steroid-charged tail near the aqueous phase. Molecular dynamics identified two regions in beta(1) transmembrane domain 2 that meet unique requirements for bonding with the LC concave hemisphere, where the steroid functional groups are located.