Design,synthesis, docking,ADMET studies,and anticancer evaluation of new 3-methylquinoxaline derivatives as VEGFR-2 inhibitors and apoptosis inducers

Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a critical role in cancer angiogenesis. Inhibition of VEGFR-2 activity proved effective suppression of tumour propagation. Accordingly, two series of new 3-methylquinoxaline derivatives have been designed and synthesised as VEGFR-2 inhibitors. The synthesised derivatives were evaluated in vitro for their cytotoxic activities against MCF-7and HepG2 cell lines. In addition, the VEGFR-2 inhibitory activities of the target compounds were estimated to indicate the potential mechanism of their cytotoxicity. To a great extent, the results of VEGFR-2 inhibition were highly correlated with that of cytotoxicity. Compound 27a was the most potent VEGFR-2 inhibitor with IC₅₀ of 3.2 nM very close to positive control sorafenib (IC₅₀ = 3.12 nM). Such compound exhibited a strong cytotoxic effect against MCF-7 and HepG2, respectively with IC₅₀ of 7.7 and 4.5 µM in comparison to sorafenib (IC₅₀ = 3.51 and 2.17 µM). In addition, compounds 28, 30f, 30i, and 31b exhibited excellent VEGFR-2 inhibition activities (IC₅₀ range from 4.2 to 6.1 nM) with promising cytotoxic activity. Cell cycle progression and apoptosis induction were investigated for the most active member 27a. Also, the effect of 27a on the level of caspase-3, caspase-9, and BAX/Bcl-2 ratio was determined. Molecular docking studies were implemented to interpret the binding mode of the target compounds with the VEGFR-2 pocket. Furthermore, toxicity and ADMET calculations were performed for the synthesised compounds to study their pharmacokinetic profiles     

Babesia gibsoni: Identification, expression, localization, and serological characterization of a Babesia gibsoni 22-kDa protein

Babesia gibsoni causes canine babesiosis. Here, we describe the identification and characterization of a novel gene, BgP22, containing an open reading frame of 621 bp and encoding a 22-kDa protein from B. gibsoni, as a serodiagnostic candidate. The recombinant BgP22 (rBgP22) was expressed and used as an antigen to produce anti-rBgP22 sera in mice. Using these anti-rBgP22 sera, a native 22-kDa protein was recognized by Western blot analysis and observed in the membrane of the parasites by immunofluorescent antibody tests (IFAT). The enzyme-linked immunosorbent assay (ELISA) using the rBgP22 detected specific antibodies to this protein in the sera of dogs experimentally and naturally infected with B. gibsoni in chronic stage. Furthermore, it did not show a cross reaction with the closely related apicomplexan parasites, indicating that the rBgP22 could be used as a diagnostic antigen for a detection of the chronic carrier stages of B. gibsoni infection.     

Development and validation of an EI-GC/MS method for the determination of sertraline and its major metabolite desmethyl-sertraline in blood

A sensitive and specific GC/MS method for the determination of sertraline and its main metabolite desmethyl-sertraline in whole blood has been developed, optimized and validated. Sample preparation included solid-phase extraction of both analytes and their derivatization with heptafluorobutyric anhydride (HFBA). Protriptyline was used as internal standard for the determination of both analytes. Limits of detection and quantification for both sertraline and desmethyl-sertraline were 0.30 and 1.00 μg/L, respectively. The calibration curves were linear within the dynamic range of each analyte (1.00-500.0 μg/L) with a correlation coefficient (R(2)) exceeding 0.991. Extraction efficiency ranged from 90.1(± 5.8)% to 95.4(± 3.0)% for sertraline, and from 84.9(± 8.2)% to 107.7(± 4.4)% for desmethyl-sertraline. The precision for sertraline and desmethyl-sertraline was between 3.6-5.5% and 4.7-7.2%, respectively, while the accuracy was in the range of -6.67% to 2.20% and -6.33% to 2.88% for sertraline and desmethyl-sertraline, respectively. The method was applied to real blood samples obtained from patients that follow sertraline treatment and also in cases of forensic interest. The developed method can be used in routine every day analysis by clinical and forensic laboratories, for pharmacokinetic studies, for therapeutic sertraline monitoring or for the investigation of forensic cases where sertraline is involved.     

A pilot study on nitration/dysfunction of NK1 segment of myogenic stem cell activator HGF

Protein tyrosine residue (Y) nitration, a post-translational chemical-modification mode, has been associated with changes in protein activity and function; hence the accumulation of specific nitrated proteins in tissues may be used to monitor the onset and progression of pathological disorders. To verify the possible impact of nitration on postnatal muscle growth and regeneration, a pilot study was designed to examine the nitration/dysfunction of hepatocyte growth factor (HGF), a key ligand that is released from the extracellular tethering and activates myogenic stem satellite cells to enter the cell cycle upon muscle stretch and injury. Exposure of recombinant HGF (a hetero-dimer of α- and β-chains) to peroxynitrite induces Y nitration in HGF α-chain under physiological conditions. Physiological significance of this finding was emphasized by Western blotting that showed the NK1 segment of HGF (including a K1 domain critical for signaling-receptor c-met binding) undergoes nitration with a primary target of Y198. Peroxynitrite treatment abolished HGF-agonistic activity of the NK1 segment, as revealed by in vitro c-met binding and bromodeoxyuridine-incorporation assays. Importantly, direct-immunofluorescence microscopy of rat lower hind-limb muscles from two aged-groups (2-month-old “young” and 12-month-old “retired/adult”) provided in vivo evidence for age-related nitration of extracellular HGF (Y198). Overall, findings provide the insight that HGF/NK1 nitration/dysfunction perturbs myogenic stem cell dynamics and homeostasis; hence NK1 nitration may stimulate progression of muscular disorders and diseases including sarcopenia.     

ErbB4-neuregulin signaling modulates synapse development and dendritic arborization through distinct mechanisms

Perturbations in neuregulin-1 (NRG1)/ErbB4 function have been associated with schizophrenia. Affected patients exhibit altered levels of these proteins and display hypofunction of glutamatergic synapses as well as altered neuronal circuitry. However, the role of NRG1/ErbB4 in regulating synapse maturation and neuronal process formation has not been extensively examined. Here we demonstrate that ErbB4 is expressed in inhibitory interneurons at both excitatory and inhibitory postsynaptic sites. Overexpression of ErbB4 postsynaptically enhances size but not number of presynaptic inputs. Conversely, knockdown of ErbB4 using shRNA decreases the size of presynaptic inputs, demonstrating a specific role for endogenous ErbB4 in synapse maturation. Using ErbB4 mutant constructs, we demonstrate that ErbB4-mediated synapse maturation requires its extracellular domain, whereas its tyrosine kinase activity is dispensable for this process. We also demonstrate that depletion of ErbB4 decreases the number of primary neurites and that stimulation of ErbB4 using a soluble form of NRG1 results in exuberant dendritic arborization through activation of the tyrosine kinase domain of ErbB4 and the phosphoinositide 3-kinase pathway. These findings demonstrate that NRG1/ErbB4 signaling differentially regulates synapse maturation and dendritic morphology via two distinct mechanisms involving trans-synaptic signaling and tyrosine kinase activity, respectively.     

Saprotrophic basidiomycete mycelia and their interspecific interactions affect the spatial distribution of extracellular enzymes in soil

Saprotrophic cord-forming basidiomycetes are important decomposers of lignocellulosic substrates in soil. The production of extracellular hydrolytic enzymes was studied during the growth of two saprotrophic basidiomycetes, Hypholoma fasciculare and Phanerochaete velutina, across the surface of nonsterile soil microcosms, along with the effects of these basidiomycetes on fungi and bacteria within the soil. Higher activities of α-glucosidase, β-glucosidase, cellobiohydrolase, β-xylosidase, phosphomonoesterase and phosphodiesterase, but not of arylsulphatase, were recorded beneath the mycelia. Despite the fact that H. fasciculare, with exploitative hyphal growth, produced much denser hyphal cover on the soil surface than P. velutina, with explorative growth, both fungi produced similar amounts of extracellular enzymes. In the areas where the mycelia of H. fasciculare and P. velutina interacted, the activities of N-acetylglucosaminidase, α-glucosidase and phosphomonoesterase, the enzymes potentially involved in hyphal cell wall damage, and the utilization of compounds released from damaged hyphae of interacting fungi, were particularly increased. No significant differences in fungal biomass were observed between basidiomycete-colonized and noncolonized soil, but bacterial biomass was reduced in soil with H. fasciculare. The increases in the activities of β-xylosidase, β-glucosidase, phosphomonoesterase and cellobiohydrolase with increasing fungal:bacterial biomass ratio indicate the positive effects of fungal enzymes on nutrient release and bacterial abundance, which is reflected in the positive correlation of bacterial and fungal biomass content.     

Increased MMPs expression and decreased contraction in the rat myometrium during pregnancy and in response to prolonged stretch and sex hormones

Normal pregnancy is associated with uterine relaxation to accommodate the stretch imposed by the growing fetus; however, the mechanisms underlying the relationship between pregnancy-associated uterine stretch and uterine relaxation are unclear. We hypothesized that increased uterine stretch during pregnancy is associated with upregulation of matrix metalloproteinases (MMPs), which in turn cause inhibition of myometrium contraction and promote uterine relaxation. Uteri from virgin, midpregnant (day 12), and late-pregnant rats (day 19) were isolated, and myometrium strips were prepared for measurement of isometric contraction and MMP expression and activity using RT-PCR, Western blot analysis, and gelatin zymography. Oxytocin caused concentration-dependent contraction of myometrium strips that was reduced in mid- and late-pregnant rats compared with virgin rats. Pretreatment with the MMP inhibitors SB-3CT (MMP-2/MMP-9 Inhibitor IV), BB-94 (batimastat), or Ro-28-2653 (cipemastat) enhanced contraction in myometrium of pregnant rats. RT-PCR, Western blot analysis, and gelatin zymography demonstrated increased mRNA expression, protein amount, and activity of MMP-2 and MMP-9 in myometrium of late-pregnant>midpregnant>virgin rats. Prolonged stretch of myometrium strips of virgin rats under 8 g basal tension for 18 h was associated with reduced contraction and enhanced expression and activity of MMP-2 and MMP-9, which were reversed by MMP inhibitors. Concomitant treatment of stretched myometrium of virgin rats with 17β-estradiol (E2), progesterone (P4), or E2+P4 was associated with further reduction in contraction and increased MMP expression and activity. MMP-2 and MMP-9 caused significant reduction of oxytocin-induced contraction of myometrium of virgin rat. Thus, normal pregnancy is associated with reduced myometrium contraction and increased MMPs expression and activity. The results are consistent with the possibility that myometrium stretch and concomitant increase in sex hormones during pregnancy are associated with increased expression/activity of specific MMPs, which in turn inhibit uterine contraction and promote uterine relaxation.     

Radiosensitizer EXO-miR-197-3p Inhibits Nasopharyngeal Carcinoma Progression and Radioresistance by Regulating the AKT/mTOR Axis and HSPA5-mediated Autophagy

The biological functions of exosomes and microRNAs (miRs) in nasopharyngeal carcinoma (NPC) remain largely unexplored. Here, miR-197-3p was screened and identified, and whose level was reduced in serum and exosomes of patients with NPC. MiR-197-3p might be a good diagnostic and prognostic indicator. Our data showed that miR-197-3p expression was closely related to radioresistance, apoptosis, proliferation, migration, and survival of NPC. Inhibition of miR-197-3p expression in vitro could promote the proliferation and migration of NPC cells, while promotion of miR-197-3p expression in vivo could significantly inhibit the growth and enhance the radiosensitivity of NPC cells. From the perspective of mechanism, miR-197-3p could inhibit AKT/mTOR phosphorylation activation, inhibit an activated pathway of AKT/mTOR, target Heat Shock 70-kDa Protein 5(HSPA5) related to endoplasmic reticulum homeostasis, inhibit HSPA5-mediated autophagy, and reverse the radioresistance of NPC. Interestingly, exosomal miR-197-3p (EXO-miR-197-3p) reduced the proliferation and migration potential of NPC cells in vitro, and tumor growth and radioresistance of NPC cells in vivo. EXO-miR-197-3p inhibited NPC progression and radioresistance by regulating AKT/mTOR phosphorylation activation and HSPA5-mediated autophagy. In conclusion, our results highlight the potential of EXO-miR-197-3p as an effective radiosensitizer and therapeutic agent for refractory NPC.     

Recognition of the gluconeogenic enzyme,Pck1, via the Gid4 E3 ligase: An in silico perspective

Gluconeogenesis, the reverse process of glycolysis, is a favorable mechanism at conditions of glucose deprivation. Pck1 is a rate‐limiting gluconeogenic enzyme, where its deficiency or mutation contributes to serious clinical situations as neonatal hypoglycemia and liver failure. A recent report confirms that Pck1 is a target for proteasomal degradation through its proline residue at the penultimate position, recognized by Gid4 E3 ligase, but with a lack of informative structural details. In this study, we delineate the localized sequence motif, degron, that specifically interact with Gid4 ligase and unravel the binding mode of Pck1 to the Gid4 ligase by using molecular docking and molecular dynamics. The peptide/protein docking HPEPDOCK web server along with molecular dynamic simulations are applied to demonstrate the binding mode and interactions of a Pck1 wild type (SPSK) and mutant (K4V) with the recently solved structure of Gid4 ligase. Results unveil a distinct binding mode of the mutated peptide compared with the wild type despite having comparable binding affinities to Gid4. Moreover, the four‐residue peptide is found insufficient for Gid4 binding, while the seven‐residue peptide suffices for binding to Gid4. The amino acids S134, K135, and N137 in the loop L1 (between β1 and β2) of the Gid4 are essential for the stabilization of the seven‐residue peptide in the binding site of the ligase. The presence of Val⁴ instead of Lys⁴ smashes the H‐bonds that are formed between Lys⁴ and Gid4 in the wild type peptide, making the peptide prone to bind with the other side of the binding pocket (L4 loop of Gid4). The dynamics of Gid4 L3 loop is affected dramatically once K4V mutant Pck1 peptide is introduced. This opens the door to explore the mutation effects on the binding mode and smooth the path to target protein degradation by design competitive and non‐competitive inhibitors.