Inhibition of NFATx activation by an oligopeptide: disrupting the interaction of NFATx with calcineurin

Calcium-dependent phosphatase calcineurin (CN) regulates the activation and nuclear translocation of NFAT. We identify here a novel CN-binding motif in one member of the NFAT family, NFATx, and a peptide based on this motif, Pep3. Pep3 binds CN and competes with wild-type NFATx for CN interaction. Amino acid mutations within Pep3 show that multiple amino acid residues are required for the effective functions of Pep3. Ectopic expression of Pep3 in a Th clone via a retrovirus-mediated gene transfer could selectively block the nuclear translocation of endogenous NFATx, whereas it had little effect on the nuclear translocation of another member of the NFAT family, NFATp. Furthermore, in transfection experiments, Pep3 also blocked the nuclear translocation of transfected NFATx, but not NFATp, in the B cell line M12, demonstrating specific inhibition of Pep3 for NFATx. Importantly, several cytokines produced by the T cell clone were severely repressed by ectopic Pep3, and indeed, the production of these cytokines was enhanced by the expression of wild-type NFATx. Our results show selective inhibition of NFATx activation and cytokine expression by Pep3 and suggest a new approach for studying the biology of each NFAT family member. This approach may provide an opportunity for pharmacological targeting of Ca(2+)-dependent signaling events.     

Discovery of novel paeonol-based derivatives against skin inflammation in vitro and in vivo

T-LAK-cell-originated protein kinase (TOPK), a novel member of the mitogen-activated protein kinase family, is considered an effective therapeutic target for skin inflammation. In this study, a series (A − D) of paeonol derivatives was designed and synthesised using a fragment growing approach, and their anti-inflammatory activities against lipopolysaccharide (LPS)-induced nitric oxide production in RAW264.7 cells were tested. Among them, compound B12 yielded the best results (IC₅₀ = 2.14 μM) with low toxicity (IC₅₀ > 50 µM). Preliminary mechanistic studies indicated that this compound could inhibit the TOPK-p38/JNK signalling pathway and phosphorylate downstream related proteins. A murine psoriasis-like skin inflammation model was used to determine its therapeutic effect.     

Design,synthesis and in vitro and in vivo biological evaluation of flurbiprofen amides as new fatty acid amide hydrolase/cyclooxygenase-2 dual inhibitory potential analgesic agents

Compounds combining dual inhibitory action against FAAH and cyclooxygenase (COX) may be potentially useful analgesics. Here, we describe a novel flurbiprofen analogue, N-(3-bromopyridin-2-yl)-2-(2-fluoro-(1,1''-biphenyl)-4-yl)propanamide (Flu-AM4). The compound is a competitive, reversible inhibitor of FAAH with a K_i value of 13 nM and which inhibits COX activity in a substrate-selective manner. Molecular modelling suggested that Flu-AM4 optimally fits a hydrophobic pocket in the ACB region of FAAH, and binds to COX-2 similarly to flurbiprofen. In vivo studies indicated that at a dose of 10 mg/kg, Flu-AM4 was active in models of prolonged (formalin) and neuropathic (chronic constriction injury) pain and reduced the spinal expression of iNOS, COX-2, and NFκB in the neuropathic model. Thus, the present study identifies Flu-AM4 as a dual-action FAAH/substrate-selective COX inhibitor with anti-inflammatory and analgesic activity in animal pain models. These findings underscore the potential usefulness of such dual-action compounds.     

Preclinical in vitro screening of newly synthesised amidino substituted benzimidazoles and benzothiazoles

Newly synthesised benzimidazole/benzotiazole derivatives bearing amidino, namely 3,4,5,6-tetrahydropyrimidin-1-ium chloride, substituents have been evaluated for their potential antitumor activity in vitro. Compounds and standard drugs (doxorubicin, staurosporine and vandetanib) were tested on three human lung cancer cell lines A549, HCC827 and NCI-H358. We tested compounds in MTS citotoxicity assay and in BrdU proliferative assay performed on 2 D and 3 D assay format. Because benzmidazole scaffold is similar to natural purines, we tested the most active compounds for ability to induce cell apoptosis of A549 by binding to DNA in comparison with doxorubicin and saturosporine. Additionally, the ADME properties of the most active benzothiazole/benzimidazole and non-active compounds were determined to see if the different ADME properties are the cause of different activity in 2 D and 3 D assays, as well as to see if the tested active compounds have drug like properties and potency for further profilation. ADME characterisation included solubility, lipophilicity, permeability, metabolic stability and binding to plasma proteins. In general, the benzothiazole derivatives were more active in comparison to their benzimidazole analogues. The exception was 2-phenyl substituted benzimidazole 6a being active with very pronounced activity especially towards HCC827 cells. All active compounds have similar mode of action on A549 cell line as standard compound doxorubicin, which binds to nucleic acids with the DNA double helix. Tested active benzothiazole compounds were characterised by moderate to good solubility, good metabolic stability, low permeability and high binding to plasma proteins. One tested active benzimidazole derivative showed ADME properties, but lower lipophilicity resulted in low PPB and higher metabolic instability. In addition, no significant difference was observed in ADME profile between active and non-active compounds.     

Sulfation of glycosaminoglycans depends on the catalytic activity of lithium-inhibited phosphatase BPNT2 in vitro

Golgi-resident bisphosphate nucleotidase 2 (BPNT2) is a member of a family of magnesium-dependent, lithium-inhibited phosphatases that share a three-dimensional structural motif that directly coordinates metal binding to effect phosphate hydrolysis. BPNT2 catalyzes the breakdown of 3′-phosphoadenosine-5′-phosphate, a by-product of glycosaminoglycan (GAG) sulfation. KO of BPNT2 in mice leads to skeletal abnormalities because of impaired GAG sulfation, especially chondroitin-4-sulfation, which is critical for proper extracellular matrix development. Mutations in BPNT2 have also been found to underlie a chondrodysplastic disorder in humans. The precise mechanism by which the loss of BPNT2 impairs sulfation remains unclear. Here, we used mouse embryonic fibroblasts (MEFs) to test the hypothesis that the catalytic activity of BPNT2 is required for GAG sulfation in vitro. We show that a catalytic-dead Bpnt2 construct (D108A) does not rescue impairments in intracellular or secreted sulfated GAGs, including decreased chondroitin-4-sulfate, present in Bpnt2-KO MEFs. We also demonstrate that missense mutations in Bpnt2 adjacent to the catalytic site, which are known to cause chondrodysplasia in humans, recapitulate defects in overall GAG sulfation and chondroitin-4-sulfation in MEF cultures. We further show that treatment of MEFs with lithium (a common psychotropic medication) inhibits GAG sulfation and that this effect depends on the presence of BPNT2. Taken together, this work demonstrates that the catalytic activity of an enzyme potently inhibited by lithium can modulate GAG sulfation and therefore extracellular matrix composition, revealing new insights into lithium pharmacology.     

Recognition mechanism of p63 by the E3 ligase Itch: Novel strategy in the study and inhibition of this interaction

The HECT-containing E3 ubiquitin ligase Itch mediates the degradation of several proteins, including p63 and p73, involved in cell specification and fate. Itch contains four WW domains, which are essential for recognition on the target substrate, which contains a short proline-rich sequence. Several signaling complexes containing these domains have been associated with human diseases such as muscular dystrophy, Alzheimer’s or Huntington’s diseases. To gain further insight into the structural determinants of the Itch-WW2 domain, we investigated its interaction with p63. We assigned, by 3D heteronuclear NMR experiments, the backbone and side chains of the uniformly ¹³C-¹⁵N-labeled Itch-WW2. In vitro interaction of Itch-WW2 domain with p63 was studied using its interactive p63 peptide, pep63. Pep63 is an 18-mer peptide corresponding to the region from 534–551 residue of p63, encompassing the PPxY motif that interacts with the Itch-WW domains, and we identified the residues involved in this molecular recognition. Moreover, here, a strategy of stabilization of the conformation of the PPxY peptide has been adopted, increasing the WW-ligand binding. We demonstrated that cyclization of pep63 leads to an increase of both the biological stability of the peptide and of the WW-ligand complex. Stable metal-binding complexes of the pep63 have been also obtained, and localized oxidative damage on Itch-WW2 domain has been induced, demonstrating the possibility of use of metal-pep63 complexes as models for the design of metal drugs to inhibit the Itch-WW-p63 recognition in vivo. Thus, our data suggest a novel strategy to study and inhibit the recognition mechanism of Itch E3-ligase.     

Conservative initial postoperative anticoagulation strategy after HeartMate 3 left ventricular assist device implantation

IntroductionAlthough anticoagulation therapy is mandated after implantation of a left ventricular assist device (LVAD), postoperative bleedings and reoperations occur relatively frequently and are associated with worse outcomes. We evaluated the use of a conservative postoperative anticoagulation protocol in patients implanted with a HeartMate 3 (HM3) LVAD.MethodsIn a single-centre retrospective analysis of postoperative outcomes after HM3 LVAD implantation, a standard (old) anticoagulation protocol (i.e. early, full-dose anticoagulation with low-molecular weight heparin and overlapping vitamin K antagonist) was compared with a new conservative anticoagulation protocol (i.e. slow initiation of vitamin K antagonists without overlapping heparin). Main outcomes were changes in international normalised ratio (INR), lactate dehydrogenase (LDH), bleeding and/or tamponade events requiring reoperation, length of stay and adverse events.ResultsIn total, 73 patients (48 in old vs 25 in new protocol group) were evaluated. Mean age was 56 years (standard deviation 13) and most patients (78%) were males. Changes in INR and LDH in the first 14 days were similar in both groups (p = 0.50 and p = 0.997 for interaction, respectively). Number of bleeding/tamponade events requiring reoperation was lower in the new than in the old protocol group (4% vs 33%, p = 0.005). Postoperative 30-day mortality was similar, and we observed no thromboembolic events. Median (25th–75th percentiles) total length of postoperative hospital stay (27 (25–41) vs 21 (19–27) days, p < 0.001) and length of intensive care unit stay (5 (2–9) vs 2 (2–5) days, p = 0.022) were significantly shorter in the new protocol group.ConclusionThese retrospective data suggest that conservative slow initiation of anticoagulation therapy after HM3 LVAD implantation is associated with less bleeding/tamponade events requiring reoperation, a similar safety profile and a shorter duration of stay than the currently advised standard anticoagulation protocol.Supplementary InformationThe online version of this article (10.1007/s12471-022-01671-1) contains supplementary material, which is available to authorized users.     

Discovery of new acetylcholinesterase inhibitors for Alzheimer’s disease: virtual screening and in vitro characterisation

For more than two decades, the development of potent acetylcholinesterase (AChE) inhibitors has been an ongoing task to treat dementia associated with Alzheimer’s disease and improve the pharmacokinetic properties of existing drugs. In the present study, we used three docking-based virtual screening approaches to screen both ZINC15 and MolPort databases for synthetic analogs of physostigmine and donepezil, two highly potent AChE inhibitors. We characterised the in vitro inhibitory concentration of 11 compounds, ranging from 14 to 985 μM. The most potent of these compounds, S-I 26, showed a fivefold improved inhibitory concentration in comparison to rivastigmine. Moderate inhibitors carrying novel scaffolds were identified and could be improved for the development of new classes of AChE inhibitors.     

Inhibitory evaluation of Curculigo latifolia on α-glucosidase,DPP (IV) and in vitro studies in antidiabetic with molecular docking relevance to type 2 diabetes mellitus

The inhibition of α-glucosidase and DPP enzymes capable of effectively reducing blood glucose level in the management of type 2 diabetes. The purpose of the present study is to evaluate the inhibitory potential of α-glucosidase and DPP (IV) activity including with the 2-NBDG uptake assay and insulin secretion activities through in vitro studies. The selected of active compounds obtained from the screening of compounds by LC-MS were docked with the targeted enzyme that involved in the mechanism of T2DM. From the results, root extracts displayed a better promising outcome in α-glucosidase (IC₅₀ 2.72 ± 0.32) as compared with the fruit extracts (IC₅₀ 3.87 ± 0.32). Besides, root extracts also displayed a better activity in the inhibition of DPP (IV), enhance insulin secretion and glucose uptake activity. Molecular docking results revealing that phlorizin binds strongly with α-glucosidase, DPP (IV) and Insulin receptor (IR) enzymes with achieving the lowest binding energy value. The present work suggests several of the compounds have the potential that contribute towards inhibiting α-glucosidase and DPP (IV) and thus effective in lowering post-prandial hyperglycaemia.     

Outcome of transcatheter aortic valve replacement in patients over 85 years of age versus patients aged 85 and younger

IntroductionThe Dutch general population is aging rapidly. Many of these patient are fit and eligible for TAVR. However, studies on outcome in older versus younger patients are scant.Material and methodsA single-centre retrospective study comparing patients older and younger than age 85 on outcome.Results190 patients underwent TAVR: 136 were aged 85 or younger (U85), 54 were older than 85 (O85). The U85 group had more men (U85: 71 [52.2%] vs O85: 19 [35.2%]; p = 0.034), a higher incidence of diabetes (U85: 36 [26.5%] vs O85: 3 [5.6%]; p = 0.001) and atrial fibrillation (U85: 35 [25.7%] vs O85: 5 [9.3%]; p = 0.03) and a higher body mass index (U85: 27.5 [± 5.24] vs O85: 26 [± 3.78]; p = 0.027). In the O85 group there was a lower estimated glomerular filtration rate (O85: 50.28 [± 15.32] ml/min vs U85: 65.25 [± 29.97] ml/min; p = 0.012). There was no difference in 30-day mortality (U85: 6 [4.4%] vs O85: 3 [5.6%]) and 1‑year mortality (U85 9 [6.6%] vs O85 3 [5.6%]) (p = 0.521). There was an equal amount of new onset permanent left bundle branch block (U85: 38 [27.9%] vs O85: 14 [25.9%]; p = 0.896) and permanent pacemaker implantation (U85: 28 [20.6%] vs O85: 28 [20.6%]; p = 0.748). There was no difference in bleeding events (p = 0.469), vascular complications (p = 0.195) or moderate/severe regurgitation (p = 0.972). The U85 group had a slightly longer admission duration (U85 6.29 [± 5.289] days vs O85 5.98 [± 3.328] days (p = 0.037)).ConclusionTAVR in patients over 85 years of age has excellent outcome, comparable to those aged 85 and younger.     

Epistatic interactions promote persistence of NS3-Q80K in HCV infection by compensating for protein folding instability

The Q80K polymorphism in the NS3-4A protease of the hepatitis C virus is associated with treatment failure of direct-acting antiviral agents. This polymorphism is highly prevalent in genotype 1a infections and stably transmitted between hosts. Here, we investigated the underlying molecular mechanisms of evolutionarily conserved coevolving amino acids in NS3-Q80K and revealed potential implications of epistatic interactions in immune escape and variants persistence. Using purified protein, we characterized the impact of epistatic amino acid substitutions on the physicochemical properties and peptide cleavage kinetics of the NS3-Q80K protease. We found that Q80K destabilized the protease protein fold (p < 0.0001). Although NS3-Q80K showed reduced peptide substrate turnover (p < 0.0002), replicative fitness in an H77S.3 cell culture model of infection was not significantly inferior to the WT virus. Epistatic substitutions at residues 91 and 174 in NS3-Q80K stabilized the protein fold (p < 0.0001) and leveraged the WT protease stability. However, changes in protease stability inversely correlated with enzymatic activity. In infectious cell culture, these secondary substitutions were not associated with a gain of replicative fitness in NS3-Q80K variants. Using molecular dynamics, we observed that the total number of residue contacts in NS3-Q80K mutants correlated with protein folding stability. Changes in the number of contacts reflected the compensatory effect on protein folding instability by epistatic substitutions. In summary, epistatic substitutions in NS3-Q80K contribute to viral fitness by mechanisms not directly related to RNA replication. By compensating for protein-folding instability, epistatic interactions likely protect NS3-Q80K variants from immune cell recognition.     

Multi-stage structure-based virtual screening approach towards identification of potential SARS-CoV-2 NSP13 helicase inhibitors

On account of its crucial role in the virus life cycle, SARS-COV-2 NSP13 helicase enzyme was exploited as a promising target to identify a novel potential inhibitor using multi-stage structure-based drug discovery approaches. Firstly, a 3D pharmacophore was generated based on the collected data from a protein-ligand interaction fingerprint (PLIF) study using key interactions between co-crystallised fragments and the NSP13 helicase active site. The ZINC database was screened through the generated 3D-pharmacophore retrieving 13 potential hits. All the retrieved hits exceeded the benchmark score of the co-crystallised fragments at the molecular docking step and the best five-hit compounds were selected for further analysis. Finally, a combination between molecular dynamics simulations and MM-PBSA based binding free energy calculations was conducted on the best hit (compound FWM-1) bound to NSP13 helicase enzyme, which identified FWM-1 as a potential potent NSP13 helicase inhibitor with binding free energy equals −328.6 ± 9.2 kcal/mol.     

Genetic dissection of days to flowering via genome-wide association studies in Turkish common bean germplasm

Common bean is a nutrient‐dense legume crop serving as a source of food for millions of people. Characterization of unexplored common bean germplasm to unlock the phenotypic and genetic variations is still needed to explore the breeding potential of this crop. The current study aimed to dissect the genetic basis having association for days to flowering (DF). A total of 188 common bean accessions collected from 19 provinces of Turkey were used as plant material under five environments and two locations. Analysis of variance (ANOVA) revealed that genotypes and genotype by environment interaction have significant effects on DF. A total of 10 most stable accessions were evaluated from stability analysis. Overall maximum (75) and minimum (54) DF were observed for Hakkari-51 and Mus-46 accessions, respectively. The implemented constellation plot divided studied germplasm according to their DF and growth habit. A total of 7900 DArTseq markers were used for association analysis. Mixed linear model using the Q + K Model resulted a total of 18 DArTseq markers from five environments. DArT-8668385 marker identified in Bolu during 2016 was also associated with DF in Sivas during 2017. Combined data of five years resulted a total of four markers (DArT-22346534, DArT-3369768, DArT-3374613, and DArT-3370801) having significant association ( p  <  0.01 ) for DF. DArT-22346534 present on Pv 08 accounted a maximum of 9.89% variation to the studied trait. A total of four putative candidate genes were predicted from sequences reflecting homology to identified four DArTseq markers. We envisage that exploitation of identified DArTseq markers will hopefully beneficial for the development of new common bean varieties having better adaptation ability to changing climatic conditions.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01029-8.     

A single-centre prospective evaluation of left bundle branch area pacemaker implantation characteristics

BackgroundLeft bundle branch area pacing (LBBAP) has recently been introduced as a physiological pacing technique with synchronous left ventricular activation. It was our aim to evaluate the feasibility and learning curve of the technique, as well as the electrical characteristics of LBBAP.Methods and resultsLBBAP was attempted in 80 consecutive patients and electrocardiographic characteristics were evaluated during intrinsic rhythm, right ventricular septum pacing (RVSP) and LBBAP. Permanent lead implantation was successful in 77 of 80 patients (96%). LBBAP lead implantation time and fluoroscopy time shortened significantly from 33 ± 16 and 21 ± 13 min to 17 ± 5 and 12 ± 7 min, respectively, from the first 20 to the last 20 patients. Left bundle branch (LBB) capture was achieved in 54 of 80 patients (68%). In 36 of 45 patients (80%) with intact atrioventricular conduction and narrow QRS, an LBB potential (LBB_pot) was present with an LBB_pot to onset of QRS interval of 22 ± 6 ms. QRS duration increased significantly more during RVSP (141 ± 20 ms) than during LBBAP (125 ± 19 ms), compared to 130 ± 30 ms without pacing. An even clearer difference was observed for QRS area, which increased significantly more during RVSP (from 32 ± 16 µVs to 73 ± 20 µVs) than during LBBAP (41 ± 15 µVs). QRS area was significantly smaller in patients with LBB capture compared to patients without LBB capture (43 ± 18 µVs vs 54 ± 21 µVs, respectively). In patients with LBB capture (n = 54), the interval from the pacing stimulus to R‑wave peak time in lead V6 was significantly shorter than in patients without LBB capture (75 ± 14 vs 88 ± 9 ms, respectively).ConclusionLBBAP is a safe and feasible technique, with a clear learning curve that seems to flatten after 40–60 implantations. LBB capture is achieved in two-thirds of patients. Compared to RVSP, LBBAP largely maintains ventricular electrical synchrony at a level close to intrinsic (narrow QRS) rhythm.Supplementary InformationThe online version of this article (10.1007/s12471-022-01679-7) contains supplementary material, which is available to authorized users.