Molecular cloning, expression, and characterization of a novel endo-alpha-N-acetylgalactosaminidase from Enterococcus faecalis

We report here the molecular cloning, expression and characterization of a novel endo-alpha-N-acetylgalactosaminidase, classified into the GH101 family, from Enterococcus faecalis (endo-EF). The recombinant endo-EF was found to catalyze the liberation of core1-disaccharides (Galbeta1-3GalNAc) from core1-pNP (Galbeta1-3GalNAcalpha-pNP) like other GH101 family enzymes. However, endo-EF seems to differ in specificity from the GH101 enzymes reported to date, because it was able to release trisaccharides from core2-pNP (Galbeta1-3[GlcNAcbeta1-6]GalNAcalpha-pNP) and tetrasaccharides from Gal-core2-pNP (Galbeta1-3[Galbeta1-3GlcNAcbeta1-6]GalNAcalpha-pNP). Interestingly, the enzyme could transfer not only core1-disaccharides but also core2-trisaccharides to alkanols generating alkyl-oligosaccharides. Endo-EF should facilitate O-glycoprotein research.     

Kinetochore stretching-mediated rapid silencing of the spindle-assembly checkpoint required for failsafe chromosome segregation

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Optimization and preclinical evaluation of novel histamine H3 receptor ligands: Acetyl and propionyl phenoxyalkyl piperazine derivatives

As a continuation of our search for novel histamine H₃ receptor ligands, a series of new acetyl and propionyl phenoxyalkylamine derivatives (2–25) was synthesized. Compounds with three to four carbon atoms alkyl chain spacer, composed of six various 4N-substituted piperazine moieties were evaluated for their binding properties at human histamine H₃ receptors (hH₃R). In vitro test results proved the 4-pyridylpiperazine moiety as crucial element for high hH₃R affinity (hH₃R K_i?=?5.2–115?nM). Moreover introduction of carbonyl group containing residues in the lipophilic part of molecules instead of branched alkyl substituents resulted in increased affinity in correlation to previously described series, whereas propionyl derivatives showed slightly higher affinities than those of acetyl (16 and 22vs.4 and 10; hH₃R K_i?=?5.2 and 15.4?nM vs. 10.2 and 115?nM, respectively). These findings were confirmed by molecular modelling studies, demonstrating multiple ligand-receptor interactions. Furthermore, pharmacological in vivo test results of compound 4 clearly indicate that it may affect the amount of calories consumed, thus act as an anorectic compound. Likewise, its protective action against hyperglycemia and the development of overweight has been shown. In order to estimate drug-likeness of compound 4, in silico and experimental evaluation of metabolic stability in human liver microsomes was performed.     

Synthesis and biological evaluation of N-arylpiperazine derivatives of 4,4-dimethylisoquinoline-1,3(2H,4H)-dione as potential antiplatelet agents

Despite the substantial clinical success of aspirin and clopidogrel in secondary prevention of ischemic stroke, up to 40% of patients remain resistant to the available antiplatelet treatment. Therefore, there is an urgent clinical need to develop novel antiplatelet agents with a novel mechanism of action. Recent studies revealed that potent alpha 2B-adrenergic receptor (alpha 2B-ARs) antagonists could constitute alternative antiplatelet therapy. We have synthesized a series of N-arylpiperazine derivatives of 4,4-dimethylisoquinoline-1,3(2H,4H)-dione as potential alpha 2B receptor antagonists. The most potent compound 3, effectively inhibited the platelet-aggregation induced both by collagen and ADP/adrenaline with IC₅₀ of 26.9?μM and 20.5?μM respectively. Our study confirmed that the alpha 2B-AR antagonists remain an interesting target for the development of novel antiplatelet agents with an alternative mechanism of action.     

Increase in the thermostability of Bacillus sp. strain TAR-1 xylanase using a site saturation mutagenesis library

Site saturation mutagenesis library is a recently developed technique, in which any one out of all amino acid residues in a target region is substituted into other 19 amino acid residues. In this study, we used this technique to increase the thermostability of a GH10 xylanase, XynR, from Bacillus sp. strain TAR-1. We hypothesized that the substrate binding region of XynR is flexible, and that the thermostability of XynR will increase if the flexibility of the substrate binding region is decreased without impairing the substrate binding ability. Site saturation mutagenesis libraries of amino acid residues Tyr43–Lys115 and Ala300–Asn325 of XynR were constructed. By screening 480 clones, S92E was selected as the most thermostable one, exhibiting the residual activity of 80% after heat treatment at 80°C for 15 min in the hydrolysis of Remazol Brilliant Blue-xylan. Our results suggest that this strategy is effective for stabilization of GH10 xylanase.

Abbreviations: DNS: 3,5-dinitrosalicylic acid; RBB-xylan: Remazol Brilliant Blue-xylan     

Radiosynthesis and evaluation of a fluorine-18 labeled radioligand targeting vesicular acetylcholine transporter

Vesicular acetylcholine transporter (VAChT) is a reliable biomarker for assessing the loss of cholinergic neurons in the brain that is associated with cognitive impairment of patients. 5-Hydrotetralin compound (±)-5-OH-VAT is potent (K_i?=?4.64?±?0.32?nM) and selective for VAChT (>1800-fold and 398-fold for σ₁ and σ₂ receptor, respectively) with favorable hydrophilicity (LogD?=?1.78), while (?)-5-OH-VAT originally serves as the radiolabeling precursor of (?)-[¹⁸F]VAT, a promising VAChT radiotracer with a logD value of 2.56. To evaluate (?)-5-OH-[¹⁸F]VAT as a radiotracer for VAChT, we performed in vitro binding assay to determine the potency of the minus enantiomer (?)-5-OH-VAT and plus enantiomer (+)-5-OH-VAT, indicating that (?)-5-OH-VAT is a more potent VAChT enantiomer. Radiosynthesis of (?)-5-OH-[¹⁸F]VAT was explored using three strategies. (?)-5-OH-[¹⁸F]VAT was achieved with a good yield (24?±?6%) and high molar activity (～37?GBq/µmol, at the end of synthesis) using a microwave assisted two-step one-pot procedure that started with di-MOM protected nitro-containing precursor (?)-6. MicroPET studies in the brain of nonhuman primate (NHP) suggest that (?)-5-OH-[¹⁸F]VAT readily penetrated the blood brain barrier and specifically accumulated in the VAChT-enriched striatum with improved washout kinetics from striatum compared to [¹⁸F]VAT. Nevertheless, the lower target to non-target ratio may limit its use for in vivo measurement of the VAChT level in the brain.     

Landscape of protein–small ligand binding modes

Elucidating the mechanisms of specific small‐molecule (ligand) recognition by proteins is a long‐standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein–ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all‐against‐all comparison of 20,040 protein–ligand complexes provided the landscape of the protein–ligand binding modes and its relationships with protein‐ and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R² = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein–ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them.     

Focus: Rare Disease: Desmoplastic Small Round Cell Tumor in a Pregnant Woman: A Case Report and Literature Review

Background: Desmoplastic small round cell tumor (DSRCT) is an aggressive malignant tumor commonly found in young men; most occurs in the abdominal cavity. Here we conducted an in-depth analysis of a pregnant patient in our hospital and explored all the case information in the literature on small round cell carcinoma of women. Case presentation: A 27-year-old pregnant woman underwent tumor resection in our hospital at 29 weeks gestational age for a large progressive shoulder lump. The postoperative pathology showed that the mass was a DSRCT. Genetic testing found no fusion gene. At 36 weeks gestation, a painful mass was found in the breast and proved to be a metastatic focus of the desmoplastic small round cell tumor. Twenty days after a successful cesarean section at 40 weeks gestation, she received the VAC-IE chemotherapy regimen, successfully completed the first course, but when awaiting the next chemotherapy, unfortunately, the patient died during follow-up due to tumor recurrence and metastasis. Conclusion: The treatment of DSRCT in pregnant women requires a multidisciplinary consultation, and the treatment and examination during pregnancy are subject to many constraints, which may have a negative impact on the patient’s prognosis. Also, a review of the literature found that there is still no standard treatment protocol for DSRCT, and its prognosis in female patients is independent of age and tissue origin.