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Bupropion inhibits human α3β4 nicotinic acetylcholine receptors by interacting with luminal and non-luminal sites
No full textThe interaction of (±)-bupropion [(±)-BP] with the human (h) α3β4 nicotinic acetylcholine receptor (AChR) was determined by functional and structural approaches. The Ca2+ influx results indicated that (±)-BP inhibits hα3β4 AChRs with ~2-fold lower potency than that for its photoreactive analog (±)-2-(N-tert-butylamino)-3’-iodo-4’-azidopropiophenone [(±)-SADU-3-72], indicating that this compound can be used to further characterize the (±)-BP binding sites. The competition binding results showed that (±)-BP binds to the [3H]imipramine sites at desensitized hα3β4 AChRs with ~4-fold higher affinity compared to the resting state. Molecular docking results indicated that both enantiomers of BP and SADU-3-72, in the protonated state, interact with luminal and non-luminal sites. BP interacts with a luminal site which overlaps with that for imipramine, and with non-luminal sites located in the transmembrane domain (TMD) at interfacial (+α3/-β4) and α3 intrasubunit sites, in the extracellular domain (ECD), and in the TMD-ECD junction at the +β4/-α3 and +β4/-β4 interfaces and within each subunit. Our results are consistent with a hα3β4 model where BP and SADU-3-72 bind to overlapping non-luminal sites as well as to non-overlapping luminal sites
Sclerostin-deficiency modifies the development of CKD-MBD in mice
No full textSclerostin is a soluble antagonist of canonical Wnt signaling and a strong inhibitor of bone formation. We present experimental data on the role of sclerostin in chronic kidney disease - bone mineral disorder (CKD-MBD).
Methods
We performed 5/6 nephrectomies in 36-week-old sclerostin-deficient (SOST−/−) B6-mice and in C57BL/6J wildtype (WT) mice. Animals received a high phosphate diet for 11 weeks. The bones were analyzed by high-resolution micro-computed tomography (μCT) and quantitative bone histomorphometry. Aortic tissue was analyzed regarding the extent of vascular calcification.
Results
All nephrectomized mice had severe renal failure, and parathyroid hormone was highly increased compared to corresponding sham animals. All SOST−/− animals revealed the expected high bone mass phenotype. Overall, the bone compartment in WT and SOST−/− mice responded similarly to nephrectomy. In uremic WT animals, μCT data at both the distal femur and lumbar spine revealed significantly increased trabecular volume compared to non-uremic WTs. In SOST−/− mice, the differences between trabecular bone volume were less pronounced when comparing uremic with sham animals. Cortical thickness and cortical bone density at the distal femur decreased significantly and comparably in both genotypes after 5/6 nephrectomy compared to sham animals (cortical bone density − 18% and cortical thickness − 32%). Overall, 5/6 nephrectomy and concomitant hyperparathyroidism led to a genotype-independent loss of cortical bone volume and density. Overt vascular calcification was not detectable in either of the genotypes.
Conclusion
Renal osteodystrophy changes were more pronounced in WT mice than in SOST−/− mice. The high bone mass phenotype of sclerostin deficiency was detectable also in the setting of chronic renal failure with severe secondary hyperparathyroidism
Methyldopa blocks MHC class II binding to disease-specific antigens in autoimmune diabetes
No full textMajor histocompatibility (MHC) class II molecules are strongly associated with many autoimmune disorders. In type 1 diabetes, the DQ8 molecule is common, confers significant disease risk and is involved in disease pathogenesis. We hypothesized blocking DQ8 antigen presentation will provide a treatment by preventing recognition of self-peptides by pathogenic T-cells. We used the crystal structure of DQ8 to select drug-like small molecules predicted to bind structural pockets in the antigen binding cleft. A number of compounds inhibited DQ8 antigen presentation in vitro with one compound preventing insulin autoantibody production and delaying diabetes onset in an animal model of spontaneous autoimmune diabetes. We discovered an existing drug, methyldopa, blocked DQ8 and treated recent onset type 1 diabetes patients having the DQ8 allele. Methyldopa specifically inhibited DQ8 antigen presentation along with reducing inflammatory T-cell responses toward insulin, highlighting the relevance of blocking disease specific MHC II antigen presentation to treat autoimmunity
Comparison of 3He and 129Xe MRI for evaluation of lung microstructure and ventilation at 1.5 T
No full textPurpose: To evaluate the sensitivity and reproducibility of quantitative metrics of lung ventilation and microstructure from hyperpolarized 129Xe MRI alongside equivalent 3He MRI measurements at 1.5 T.
Materials & Methods: MR data from eleven healthy volunteers (HV), five ex-smokers (ES), five patients with COPD and fourteen patients with non-small-cell lung cancer (LC) were analyzed in this retrospective study. LC and COPD patients underwent 3D MRI of lung ventilation, and HV, ES and COPD patients underwent 2D multi-slice diffusion-weighted lung MRI using both 3He and 129Xe gases. Ventilated volume percentage (VV%) and mean apparent diffusion coefficient (ADC) were derived from ventilation and diffusion-weighted imaging, respectively. COPD patients performed the complete MR protocol in four separate scan sessions to assess reproducibility. MR measurements were compared to same-day pulmonary function tests.
Results: A positive correlation between 3He and 129Xe VV% was identified (r=0.798, P=0.001). VV% was larger for 3He than 129Xe (P=0.001), with an average bias of 11%. A strong correlation between mean 3He and 129Xe ADC was obtained (r=0.922, P<0.001), and ADCs of both nuclei exhibited good correlations with PFTs. In COPD patients, mean coefficient of variation (CV) values of 3He and 129Xe VV% over repeated scans were 5.78% and 13.01%, respectively, with intra-class correlation (ICC) coefficients (significances) of 0.576 (P=0.025); 0.458 (P=0.095). Mean 3He and 129Xe ADC values were both highly reproducible (CV<3%; ICC significance, P<0.001).
Conclusion: 129Xe lung MRI is approaching readiness as an alternative to 3He for quantitative clinical MRI of lung microstructure and ventilation at 1.5 T
Hypertonic saline has a prolonged effect on mucociliary clearance in adults with cystic fibrosis
No full textBackground: Inhaled hypertonic saline (HS) has been shown to increase mucociliary clearance (MCC) and improve clinical outcomes in adults and adolescents with cystic fibrosis (CF). However, in younger children with CF, a large study failed to demonstrate clinical benefits. This discrepancy could reflect pharmacodynamic differences in the MCC response to HS in different populations. We previously demonstrated the absence of a sustained effect of HS on MCC in healthy adults and in this study sought to characterize the durability of the MCC response to HS in adults with CF. Methods: At two study sites, MCC was measured in CF adults using gamma scintigraphy during three separate visits: at baseline, 15 min, and 4 h after a single dose of HS (7% NaCl, 4 mL). Particle clearance rates at these visits were used to assess the durability of the MCC response to HS. Results: The average 90-minute clearance rate measured 4 h after HS was significantly increased (21.81% ± 12.8) when compared to baseline (13.77% ± 8.7, p = .048) and showed no apparent slowing relative to the rate measured 15 min after HS. While not all subjects responded to HS, the acute response strongly predicted the sustained effect in these subjects (r = 0.896, p < .0001). Conclusions: These results suggest that, in contrast to healthy adults, a single dose of HS has a prolonged effect on MCC in adults with CF, which lasts at least 4 h. This may explain its clinical efficacy in this population
A CARD10-dependent tonic signalosome activates MALT1 paracaspase and regulates IL-17/TNF-a driven keratinocyte inflammation
No full textThe paracaspase MALT1 (Mucosa associated lymphoid tissue lymphoma translocation protein 1) controls signaling downstream of several cell surface receptors, such as C-type lectin receptors on myeloid cells and antigen receptors on lymphocytes. Upon receptor engagement, MALT1, BCL10 (B-cell lymphoma/leukemia 10) and a CARD (Caspase recruitment domain) family member assemble into a ‘CBM’ complex, which is required to trigger MALT1 paracaspase activity and downstream transcriptional activation mechanisms (Meininger and Krappmann 2016; Rosebeck et al. 2011). Here, we found that CARD10 is highly expressed in proliferating keratinocytes and is responsible for a tonic level of paracaspase activity, driven by MALT1 isoform A. Furthermore, using the potent and selective MALT1 inhibitor MLT-827 (Bardet et al. 2018; Unterreiner et al. 2017), we reveal that MALT1 activity regulates pro-inflammatory responses downstream of IL-17/TNF-α
Mouse models of cancer-induced cachexia: Hind limb muscle mass and evoked force as readouts
No full textThe majority of patients with advanced cancer suffer from cachexia, a systemic wasting syndrome, which subsequently impacts the tolerance to anti-cancer treatments, response to therapy, quality of life, and eventually, survival. Despite a high unmet medical need, there is currently no specific remedy available for an effective treatment of cachexia and its sequelae. A key feature of cachexia is the inexorable loss of skeletal muscle mass, which constitutes a main contributor to body weight loss and progressive functional impairments. Therefore, it's crucial to identify early readouts to detect and monitor the loss of muscle mass and function to initiate appropriate treatments timely.
Here, we describe experimental cancer models using mouse (syngeneic) or human (xenograft) cancer cell lines with a rapid onset of tumor growth and cachexia. These models are easier to establish, monitor and reproduce compared to the genetically engineered mouse models currently available. Moreover, we establish readouts such as hind limb muscle mass and volume, as well as evoked force and food intake measurements, to allow the evaluation of potential therapeutic agents for the early treatment of cachexia and associated impairments
Spontaneous and Drug-induced Arteritis/Polyarteritis in the Göttingen Minipig—Review
No full textArteritis/polyarteritis occurs spontaneously in many species used in preclinical toxicology studies. In Göttingen minipigs, arteritis/polyarteritis is an occasionally observed background change. In the minipig, this finding differs in frequency and nature from age-related polyarteritis nodosa in rats or monkeys, and Beagle pain syndrome in dogs. In minipigs, it can be present in a single small- or medium-sized artery of an organ or a few organs and is most commonly recorded in the cardiac and extracardiac blood vessels, vagina, oviduct, rectum, epididymis, spinal cord, pancreas, urinary bladder, kidneys, and stomach. The etiology is unknown although it has been considered in minipigs as well as in rats, dogs, and monkeys to be possibly immune mediated. This background change is important with respect to its nature and distribution in the minipig in order to distinguish it from drug-induced vascular changes, which might occur in similar locations and have similar morphologic features. This review summarizes the morphology, incidence, and predilection sites of arteritis as a spontaneously occurring background change and as a drug-induced vasculopathy in the minipig, and also describes the main aspects to consider when evaluating vascular changes in Göttingen minipig toxicity studies and their human relevance
Supplimentary information for: Microtiter Plate (MTP) Reaction Screening and Optimization of Surfactant Chemistry: Examples of Suzuki–Miyaura and Buchwald–Hartwig Cross-Couplings in Water
No full textA screening method to evaluate Suzuki–Miyaura and Buchwald–Hartwig coupling reactions under aqueous surfactant conditions has been established, leading to high yielding and highly selective Suzuki–Miyaura and Buchwald–Hartwig reactions under mild reaction temperatures and ecologically improved conditions. The screening method highlighted the utility of Buchwald–Hartwig third generation precatalysts for unprecedented high conversion Buchwald–Hartwig C–N coupling reactions in water
Fragment-Based Drug Discovery of Inhibitors of Phosphopantetheine Adenylyltransferase from Gram-Negative Bacteria
No full textThe discovery and development of new antibiotics capable of curing infections due to multidrug-resistant and pandrug-resistant Gram-negative bacteria are a major challenge with fundamental importance to our global healthcare system. Part of our broad program at Novartis to address this urgent, unmet need includes the search for new agents that inhibit novel bacterial targets. Here we report the discovery and hit-to-lead optimization of new inhibitors of phosphopantetheine adenylyltransferase (PPAT) from Gram-negative bacteria. Utilizing a fragment-based screening approach, we discovered a number of unique scaffolds capable of interacting with the pantetheine site of E. coli PPAT and inhibiting enzymatic activity, including triazolopyrimidinone 6. Structure-based optimization resulted in the identification of two lead compounds as selective, small molecule inhibitors of bacterial PPAT: triazolopyrimidinone 53 and azabenzimidazole 54 efficiently inhibited E. coli and P. aeruginosa PPAT and displayed modest cellular potency against the efflux-deficient E. coli ΔtolC mutant strain