Isolation, Identification and Assessment of Efficient Cellulase Producing Bacteria from the Termite Guts

The present study is concerned with the screening the cellulase-producing bacteria from termite gut, assessed potential cellulase-producing bacteria and partial characterization (optimum parameters) of cellulase from isolated bacteria. The result showed that 15 out of 48 isolated strains was positive for degrading the carboxy methyl cellulose (CMC) in agar by congo-red method. After screening by DNS assay, three selected bacteria exhibited high cellulase activity that were identified as Citrobacter amalonaticus CM 1-3, Bacillus cereus CM 5-1 and Streptococcus salivarius CE 5-1 using 16S rRNA sequence analysis. All bacterial strains utilized CMC and showed the highest cellulase activity. Cellulase characterization of C. amalonaticus CM 1-3 and S. salivarius CE 5-1 was revealed optimum activity at 35°C, pH 7.0 and for 48 h. Bacillus cereus CM 5-1 represented its potential use in industrial processes due to thermostable cellulase production. The crude cellulase of this strain was purified by (NH4)2SO4 precipitation with 1.58 purification fold and 74.38% overall recovery. The optimal temperature and pH for cellulase activity of B. cereus CM 5-1 were at 40°C and pH 7.0. Thus, this study provided additional information about the diversity and partial characteristic cellulase of cellulolytic bacteria from termite gut for future industrial applications

Evaluation of the anti-malarial activity and cytotoxicity of 2,4-diamino-pyrimidine-based kinase inhibitors

A series of 2,4 diamino-pyrimidines have been identified from an analysis of open access high throughput anti-malarial screening data reported by GlaxoSmithKline at the 3D7 and resistant Dd2 strains. SAR expansion has been performed using structural knowledge of the most plausible parasite target. Seventeen new analogs have been synthesized and tested against the resistant K1 strain of Plasmodium falciparum (Pf). The cytotoxicity of the compounds was assessed in Vero and A549 cells and their selectivity towards human kinases including JAK2 and EGFR were undertaken. We identified compound 5n and 5m as sub-micromolar inhibitors, with equivalent anti-malarial activity to Chloroquine (CQ). Compounds 5d and 5k, mM inhibitors of Pf, displayed improved cytotoxicity with weak inhibition of the human kinases

Anti-feline immunodeficiency virus reverse transcriptase properties of some medicinal and edible mushrooms

Background and Aim: Feline immunodeficiency virus (FIV) causes AIDS-like symptoms in domestic and wild cats. Treatment of infected cats has been performed using human anti-HIV drugs, which showed some limitations. This study aimed to determine the anti-FIV potential of some mushrooms. Materials and Methods: A total of 17 medicinal and edible mushrooms were screened to find their inhibitory effect against FIV reverse transcriptase (FIV-RT). Three solvents, water, ethanol, and hexane, were used to prepare crude mushroom extracts. Fluorescence spectroscopy was used to perform relative inhibition and 50% inhibitory concentrations (IC50) studies. Results: The ethanol extract from dried fruiting bodies of Inonotus obliquus showed the strongest inhibition with an IC50 value of 0.80±0.16 μg/mL. The hexane extract from dried mycelium of I. obliquus and ethanol and water extracts from fresh fruit bodies of Phellinus igniarius also exhibited strong activities with the IC50 values of 1.22±0.20, 4.33±0.39, and 6.24±1.42 μg/mL, respectively. The ethanol extract from fresh fruiting bodies of Cordyceps sinensis, hexane extracts from dried mycelium of I. obliquus, ethanol extracts of Ganoderma lucidum, hexane extracts of fresh fruiting bodies of Morchella esculenta, and fresh fruiting bodies of C. sinensis showed moderate anti-FIV-RT activities with IC50 values of 29.73±12.39, 49.97±11.86, 65.37±14.14, 77.59±8.31, and 81.41±17.10 μg/mL, respectively. These mushroom extracts show anti-FIV potential. Conclusion: The extracts from I. obliquus, P. igniarius, C. sinensis, and M. esculenta showed potential anti-FIV activity

Structural analysis of cannabinoids against EGFR-TK leads a novel target against EGFR-driven cell lines

Epidermal growth factor receptor (EGFR) is a member of the ErbB family of proteins and are involved in downstream signal transduction, plays prominent roles in cell growth regulation, proliferation, and the differentiation of many cell types. They are correlated with the stage and severity of cancer. Therefore, EGFRs are targeted proteins for the design of new drugs to treat cancers that overexpress these proteins. Currently, several bioactive natural extracts are being studied for therapeutic purposes. Cannabis has been reported in many studies to have beneficial medicinal effects, such as anti-inflammatory, analgesic, antibacterial, and anti-inflammatory effects, and antitumor activity. However, it is unclear whether cannabinoids reduce intracellular signaling by inhibiting tyrosine kinase phosphorylation. In this study, cannabinoids (CBD, CBG, and CBN) were simulated for binding to the EGFR-intracellular domain to evaluate the binding energy and binding mode based on molecular docking simulation. The results showed that the binding site was almost always located at the kinase active site. In addition, the compounds were tested for binding affinity and demonstrated their ability to inhibit kinase enzymes. Furthermore, the compounds potently inhibited cellular survival and apoptosis induction in either of the EGFR-overexpressing cell lines

Biological Evaluation and Molecular Dynamics Simulation of Chalcone Derivatives as Epidermal Growth Factor-Tyrosine Kinase Inhibitors

Targeted cancer therapy has become a high potential cancer treatment. Epidermal growth factor receptor (EGFR), which plays an important role in cell signaling, enhanced cell survival and proliferation, has been suggested as molecular target for the development of novel cancer therapeutics. In this study, a series of chalcone derivatives was screened by in vitro cytotoxicity against the wild type (A431 and A549) and mutant EGFR (H1975 and H1650) cancer cell lines, and, subsequently, tested for EGFR-tyrosine kinase (TK) inhibition. From the experimental screening, all chalcones seemed to be more active against the A431 than the A549 cell line, with chalcones 1c, 2a, 3e, 4e, and 4t showing a more than 50% inhibitory activity against the EGFR-TK activity and a high cytotoxicity with IC50 values of < 10 µM against A431 cells. Moreover, these five chalcones showed more potent on H1975 (T790M/L858R mutation) than H1650 (exon 19 deletion E746-A750) cell lines. Only three chalcones (1c, 2a and 3e) had an inhibitory activity against EGFR-TK with a relative inhibition percentage that was close to the approved drug, erlotinib. Molecular dynamics studies on their complexes with EGFR-TK domain in aqueous solution affirmed that they were well-occupied within the ATP binding site and strongly interacted with seven hydrophobic residues, including the important hinge region residue M793. From the above information, as well as ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties, all three chalcones could serve as lead compounds for the development of EGFR-TK inhibitors

Identification of Vinyl Sulfone Derivatives as EGFR Tyrosine Kinase Inhibitor: In Vitro and In Silico Studies

Epidermal growth factor receptor (EGFR), overexpressed in many types of cancer, has been proved as a high potential target for targeted cancer therapy due to its role in regulating proliferation and survival of cancer cells. In the present study, a series of designed vinyl sulfone derivatives was screened against EGFR tyrosine kinase (EGFR-TK) using in silico and in vitro studies. The molecular docking results suggested that, among 78 vinyl sulfones, there were eight compounds that could interact well with the EGFR-TK at the ATP-binding site. Afterwards, these screened compounds were tested for the inhibitory activity towards EGFR-TK using ADP-Glo™ kinase assay, and we found that only VF16 compound exhibited promising inhibitory activity against EGFR-TK with the IC50 value of 7.85 ± 0.88 nM. In addition, VF16 showed a high cytotoxicity with IC50 values of 33.52 ± 2.57, 54.63 ± 0.09, and 30.38 ± 1.37 µM against the A431, A549, and H1975 cancer cell lines, respectively. From 500-ns MD simulation, the structural stability of VF16 in complex with EGFR-TK was quite stable, suggesting that this compound could be a novel small molecule inhibitor targeting EGFR-TK

In Silico and In Vitro Study of Janus Kinases Inhibitors from Naphthoquinones

Janus kinases (JAKs) are involved in numerous cellular signaling processes related to immune cell functions. JAK2 and JAK3 are associated with the pathogenesis of leukemia and common lymphoid-derived illnesses. JAK2/3 inhibitors could reduce the risk of various diseases by targeting this pathway. Herein, the naphthoquinones were experimentally and theoretically investigated to identify novel JAK2/3 inhibitors. Napabucasin and 2′-methyl napabucasin exhibited potent cell growth inhibition in TF1 (IC50 = 9.57 and 18.10 μM) and HEL (IC50 = 3.31 and 6.65 μM) erythroleukemia cell lines, and they significantly inhibited JAK2/3 kinase activity (in a nanomolar range) better than the known JAK inhibitor, tofacitinib. Flow cytometric analysis revealed that these two compounds induced apoptosis in TF1 cells in a time and dose-dependent manner. From the molecular dynamics study, both compounds formed hydrogen bonds with Y931 and L932 residues and hydrophobically contacted with the conserved hinge region, G loop, and catalytic loop of the JAK2. Our obtained results suggested that napabucasin and its methylated analog were potential candidates for further development of novel anticancer drug targeting JAKs

Generation of a Single-Chain Variable Fragment Antibody against Feline Immunoglobulin G for Biosensor Applications

For many decades, feline infectious disease has been among the most common health problems and a leading cause of death in cats. These diseases include toxoplasmosis, feline leukemia virus (FeLV), and particularly feline immunodeficiency virus (FIV) disease. Early diagnosis is essential to increase the chance of successful treatment. Generally, measurement of the IgG level is considered to be indicative of an individual’s immune status for a particular pathogen. The antibodies specific to feline IgG are crucial components for the development of a detection kit. In this study, feline IgG-bound scFv was selected using phage display technology. Three rounds of biopanning were conducted against purified feline IgG. Through an indirect enzyme-linked immunosorbent assay (ELISA), two scFv clones demonstrating the best binding ability to feline IgG were chosen for biochemical characterization. In addition, the selected scFv (N14) was expressed and purified in a bacterial system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the size of the purified N14 was 29 kDa. A sandwich ELISA was used to evaluate the binding capacity of the purified scFv to feline IgG. As expected, the purified N14 had the capacity to bind feline IgG. Furthermore, N14 was modified to create a scFv-alkaline phosphatase (scFv-AP) fusion platform. The surface plasmon resonance (SPR) results revealed that N14-AP bound to feline IgG with an affinity binding value of 0.3 ± 0.496 μM. Additionally, the direct ELISA demonstrated the binding capacity of N14-AP to feline IgG in both cell lysate and purified protein. Moreover, N14-AP could be applied to detect feline IgG based on electrosensing with a detection limit of 10.42 nM. Overall, this study successfully selected a feline IgG-bound scFv and developed a scFv-AP platform that could be further engineered and applied in a feline infectious disease detection kit

Generation of a nanobody against HER2 tyrosine kinase using phage display library screening for HER2-positive breast cancer therapy development

Human epidermal growth factor receptor 2 (HER2) protein overexpression is found in ~30% of invasive breast carcinomas and in a high proportion of noninvasive ductal carcinomas in situ. Targeted cancer therapy is based on monoclonal antibodies and kinase inhibitors and reflects a new era of cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa) of conventional antibodies. Furthermore, there are many disadvantages with the current anti-HER2 drug, including drug resistance and adverse effects. Nanobodies (15 kDa), single-domain antibody (sdAb) fragments, can overcome these limitations. This study produced the recombinant sdAb against the HER2-tyrosine kinase (HER2-TK) domain using phage display technology. Three specific anti-HER2-TK sdAbs were selected for further characterization. Hallmark VHH residue identification and amino acid sequence analysis revealed that clone numbers 4 and 22 were VH antibodies, whereas clone number 17 was a VH H antibody (nanobody). The half-maximal inhibitory concentration of VHH17 exhibited significantly greater HER2 kinase-inhibition activity than the other clones. Consistent with these results, several charges and polar residues of the HER2-TK activation loop that were predicted based on mimotope analysis also appeared in the docking result and interacted via the CDR1, CDR2 and CDR3 loops of VHH17. Furthermore, the cell-penetrable VHH17 (R9 VHH17) showed cell-penetrability and significantly decreased HER2-positive cancer cell viability. Thus, the VH H17 could be developed as an effective therapeutic agent to treat HER2-positive breast cancer