Globulin-specific Proteolytic Activity in Germinating Pumpkin Seeds as Detected by a Fluorescence Assay Method

The proteolytic activities of α-chymotrypsin, trypsin, pepsin, bromelain, and an extract from germinating pumpkin seeds (Cucurbita moschata) were determined by their ability to effect the release of 1-anilino-8-naphthalenesulfonate bound to internal hydrophobic sites in intact protein substrates. Casein, glyceraldehyde-3-P dehydrogenase, urease, catalase, pumpkin seed globulin, and bovine serum albumin enhanced the fluorescence of 1-anilino-8-naphthalenesulfonate sufficiently to be used as proteolytic substrates. Chymotrypsin, trypsin, pepsin, and bromelain exhibited activity against all or almost all of the protein substrates. The activity of 1 μg of α-chymotrypsin or trypsin and 100 ng of pepsin could be easily detected by this method of assay within 4 to 5 minutes depending upon the substrate. The enzyme extracted from 3-day germinated pumpkin seeds exhibited strong activity only against pumpkin seed globulin, weak activity against the globulins of squash and cucumber and casein, and no activity against the other protein substrates. Activity against pumpkin globulin was maximal at pH 7.4. When assayed by an increase in ninhydrin-positive products, the enzyme extract from pumpkin seeds also showed strong activity against pumpkin globulin and weak activity against casein. The 1-anilino-8-naphthalenesulfonate-fluorescence method was at least 20 times more sensitive than the ninhydrin method and was 10 to 20 times more rapid.     

Synthesis,biological evaluation and SAR analysis of O-alkylated analogs of quercetin for anticancer

O-Alkylated quercetin analogs were synthesized and their anticancer activities were assessed by a high-throughout screening (HTS) method. The structure–activity relationships (SAR) showed that introduction of long alkyl chain such as propyl group at the C-3 OH position or short alkyl chain such as ethyl group at the C-4′ OH position were very important for keeping inhibitory activities against the 16 cancer cell lines. Furthermore, when the two n-butyl groups were introduced into the C-3, C-7 or C-4′, C-7 positions, the anticancer activity was enhanced.     

Optimization of aqueous-assisted extraction of polysaccharides from pumpkin (Cucurbita moschata Duch) and their biological activities

The pumpkin pulp contains a greater composition of edible polysaccharides and has reported with excellent biological applications. This research pertains to optimize the extraction of polysaccharides from the fleshy portion of the pumpkin using aqueous assisted extraction (AAE). The result showed that the optimal extraction condition of pumpkin polysaccharide was as follows: extraction temperature at 55 °C, pH 4.5, and enzyme concentration of 4000 µ/g for 80 min. Under the optimal extraction condition, the yield of pumpkin polysaccharide via AAE (15.4) was significantly higher. The biological activities of extracted polysaccharide including α-amylase inhibition (57.41% at 1000 µg/mL) and anti-inflammatory (50.41% at 25 µg/mL) activity increased significantly. Additionally, the antioxidant activities of extracted pumpkin polysaccharides including IC₅₀ values of DPPH and ABTS were 59.87% and 58.74%, respectively. The pumpkin polysaccharide has maximum inhibitory effects against bacterial strains especially for Escherichia coli than that of fungal strains. It is suggested that the aqueous assisted extraction of is a cost-effective promising method to decrease the processing time as well as enhancing extracted polysaccharide yield – times.     

In vitro anticancer and antibacterial activates of the yttrium(III) complex and its nano-carriers toward DNA cleavage and biological interactions with DNA and BSA; An experimental and computational studie

ObjectivesIn this research, the biological properties of the yttrium (III) (Y) complex, with 2,9-dimethyl- 1,10-phenanthroline (Me₂Phen) ligand, were examined for in vitro fish DNA (FS-DNA)/ bovine serum albumin (BSA) interactions, DNA-cleavage, anticancer and antibacterial activities.MethodsMulti-spectrophotometric techniques and computational calculations were used for the interaction studies of the BSA and FS-DNA with the Y-complex. Absorption and fluorescence spectroscopy methods were used to define thermodynamic parameters, the binding constants (K_b), and the probable binding mechanism. Also, the DFT (density functional theory) study and molecular docking calculation of the Y-complex were done. Besides, the nanocarriers of Y-complex (lipid nanoencapsulation (LNEP) and the starch nanoencapsulation (SNEP)), as active anticancer candidates, were prepared. Finally, DNA-cleavage, anticancer, and antibacterial activities of this complex were investigated.ResultsThe absorption and fluorescence measurements were exhibited that the Y-complex has a high binding affinity to FS-DNA and BSA through a static mechanism. The negative thermodynamic parameter values for both DNA/BSA binding were confirmed that the hydrogen bonds and van der Waals forces played an essential role in the spontaneous bonding procedure. The site marker competitive studies for BSA confirmed that the Y-complex bonds to the sub-domain IB of protein (site III) on BSA, which was entirely agreement by docking calculation. The complex has displayed efficient DNA cleavage, antifungal and antibacterial activities. The anticancer activity of the Y-complex and its starch/lipid nano-encapsulated was carried out in cancer cell lines, which exposed considerably high activity.ConclusionsThus, Y-complex can be transported professionally through BSA in the blood and bonds in the groove of DNA. Base on biological applications of the Y-complex, it can be concluded that this complex and its nanocarriers can suggest as novel anticancer and antibacterial candidates.     

Structural basis of the antifungal activity of wheat PR4 proteins

PR4 proteins possess antifungal activity against several pathogenic fungi suggesting a pivotal role in defence reactions against plant pathogen attack. We already showed that wheatwin1, a wheat PR protein of class 4, is endowed with ribonuclease activity. In this study we produced three mutants altering the active site and performed comparative analysis with the native protein also in the presence of the ribonuclease inhibitor 5′-ADP. We characterized the RNA binding site and its interaction with 5′-ADP by 3D modelling and docking studies. Moreover, in vitro antifungal assays have been carried out in order to study the relationship between antifungal and ribonuclease activities. Finally, localization of wheatwin1 in Fusarium culmorum spores was evaluated using fluorescence light microscope.     

Ribosome-inactivating proteins in edible plants and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata

The basic protein fraction of tissue extracts from 40 edible plants inhibited cell-free protein synthesis and released adenine from herring sperm DNA, thus having adenine glycosylase activity. This suggested the presence of ribosome-inactivating proteins (RIPs) in the plant extracts. This indication was further strengthened by the presence of the two activities after a partial chromatographic purification of three extracts, including that from Lycopersicon esculentum (tomato), which had very low activity. From the extract of Cucurbita moschata (pumpkin), the most active one, a glycoprotein of 30,665 Da was purified which had the properties of a RIP, in that (i) it inhibited protein synthesis by a rabbit reticulocyte lysate with IC₅₀ (concentration giving 50% inhibition) 0.035 nM (1.08 ng ml⁻¹) and by HeLa, HT29 and JM cells with IC₅₀ in the 100 nM range, (ii) deadenylated hsDNA and other polynucleotidic substrates, and (iii) depurinated yeast rRNA at a concentration of 0.1 ng ml⁻¹, all values being comparable to those of other RIPs. The C. moschata RIP gave a weak cross-reaction only with an antiserum against dianthin 32, but not with antisera against other RIPs, and had superoxide dismutase, antifungal and antibacterial activities.     

Indolylmethylene benzo[h]thiazolo[2,3-b]quinazolinones: Synthesis,characterization and evaluation of anticancer and antimicrobial activities

A series of novel 10-((1H-indol-3-yl)methylene)-7-aryl-7,10-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-9(6H)-ones (8a–t) have been synthesized in good yields by the reaction of benzo[h]quinazoline-2(1H)-thiones (4a–f) with 2-chloro-N-phenylacetamide (5) followed by Knoevenagel condensation with various indole-3-carbaldehydes (7a–d) under conventional method. All the synthesized compounds were characterized by spectral studies and screened for their in vitro anticancer and antimicrobial activities. Compound 8c has exhibited excellent activity against MCF-7 (breast cancer cell line) than the standard drug Doxorubicin. Compound 8d against both the cancer cell lines, 8q against MCF-7 and 8c, 8h against HepG2 have also shown good activity. Remaining compounds have shown moderate activity against both the cell lines. Antimicrobial activity revealed that, the compound 8q and 8t against Staphylococcus aureus and 8i, 8k, 8l, 8q & 8t against Klebsiella pneumoniae have shown equipotent activity on comparing with the standard drug Streptomycin. Remaining compounds have shown significant antibacterial and comparable antifungal activities against all the tested microorganisms.     

Synthesis and bioactivities evaluation of l-pyroglutamic acid analogues from natural product lead

A series of l-pyroglutamic acid analogues from natural product lead were designed and synthesized, as well as their antifungal activities against Phytophthora infestans, neuritogenic activities, antibacterial activities and anti-inflammatory activities are described. The bioassays and SAR study showed that the majority of l-pyroglutamic acid esters have a significant antifungal activity against P. infestans, especially 2d and 2j demonstrated the best activities with EC₅₀ values of 1.44 and 1.21?μg?mL^?1, which were about seven times that of commercial azoxystrobin (7.85?μg?mL^?1). Moreover, compounds 2e, 2g and 4d displayed anti-inflammatory activity against LPS-induced NO production in BV-2 microglial cells; neuritogenic activity in NGF-induced PC-12 cells is the same activity. This study demonstrates that compounds 2d and 2j are potential drugs to control P. infestans.     

Synthesis,characterization, molecular modeling,and potential antimicrobial and anticancer activities of novel 2-aminoisoindoline-1,3-dione derivatives

In an effort to establish new drug candidates with improved antimicrobial and anticancer activities, we report here synthesis, molecular modeling, and in vitro biological evaluation of novel substituted N-amino phthalamide derivatives (3a-b, 4a-b, 5a-j, and 6). Structures of the newly synthesized compounds were described by IR, ¹H & ¹³CNMR and LC-MS spectral data. The novel compounds were evaluated for their antibacterial activity against four types of Gm+ve and two for Gm−ve types, and antifungal activity against three fungi microorganisms by well diffusion method. Of these novel compounds, Schiff bases showed mostly promising antibacterial activity compared to reference drugs. A successful step was done for explanation of their mode of action through molecular docking of most active molecules at DNA gyrase B enzyme and further were biologically tested. Moreover, the antiproliferative activity was tested against two human carcinoma cell lines (Human colon carcinoma (HCT-116) and human breast adenocarcinoma (MCF-7)) showing promising anticancer activity compared to doxorubicin drug. The data from structure-activity relationship (SAR) analysis revealed that the lypophilic properties of these compounds might be essential parameter for their activity and suggest that 2-amino phthalamide scaffold derivatives 5g and 5h exhibited good antimicrobial and anticancer activities and might used as leads for further optimization.     

Computational study,synthesis and evaluation of active peptides derived from Parasporin-2 and spike protein from Alphacoronavirus against colorectal cancer cells

Parasporin-2Aa1 (PS2Aa1) is a toxic protein of 37 KDa (30 kDa, activated form produced by proteolysis) that was shown to be cytotoxic against specific human cancer cells, although its mechanism of action has not been elucidated yet. In order to study the role of some native peptide fragments of proteins on anticancer activity, here we investigated the cytotoxic effect of peptide fragments from domain-1 of PS2Aa1 and one of the loops present in the binding region of the virus spike protein from Alphacoronavirus (HCoV-229E), the latter according to scientific reports, who showed interaction with the human APN (h-APN) receptor, evidence corroborated through computational simulations, and thus being possible active against colon cancer cells. Peptides namely P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa were synthesized using the Fmoc solid-phase synthesis and characterized by mass spectrometry (MS). Additionally, one region from loop 1 of HCoV-229E, Loop1-HCoV-229E, was also synthesized and characterized. The A4W-GGN5 anticancer peptide and 5-fluorouracil (5-FU) were taken as a control in all experiments. Circular dichroism revealed an α-helix structure for the peptides derived from PS2Aa1 (P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa) and β-laminar structure for the peptide derived from Alphacoronavirus spike protein Loop1-HCoV-229E. Peptides showed a hemolysis percentage of less than 20% at 100 µM concentration. Besides, peptides exhibited stronger anticancer activity against SW480 and SW620 cells after exposure for 48 h. Likewise, these compounds showed significantly lower toxicity against normal cells CHO-K1. The results suggest that native peptide fragments from Ps2Aa1 may be optimized as a novel potential cancer-therapeutic agents.     

Structure–activity relationship of cytochrome bc1 reductase inhibitor broad spectrum antifungal ilicicolin H

Ilicicolin H is a broad spectrum antifungal agent showing sub micro g/mL MICs against Candida spp., Aspergillus fumigatus and Cryptococcus spp. It is a potent inhibitor (C₅₀ 2–3 ng/mL) of the mitochondrial cytochrome bc1 reductase with over 1000-fold selectivity against rat liver cytochrome bc1 reductase. Structure–activity relationship of semisynthetic derivatives by chemical modification of ilicicolin H and its 19-hydroxy derivative produced by biotransformation have been described. Basic 4′-esters and moderately polar N- and O-alkyl derivatives retained antifungal and the cytochrome bc1 reductase activities. 4′,19-Diacetate and 19-cyclopropyl acetate retained antifungal and enzyme activity and selectivity with over 20-fold improvement of plasma protein binding.     

Design,synthesis, fungicidal activity and molecular docking studies of novel 2-((2-hydroxyphenyl)methylamino)acetamide derivatives

A series of novel 2-hydroxyphenyl substituted aminoacetamides was designed by molecular hybridization of the aminoacetamide scaffold and 2-hydroxyphenyl motif. The target compounds were synthesized and their fungicidal activities were evaluated. Some of the target compounds showed excellent antifungal activities against S. sclerotiorum and P. capsici. Significantly, compounds 5e displayed the most potent activity against S. sclerotiorum with EC₅₀ = 2.89 µg/mL, which was lower than that of commercial chlorothalonil. The systematic studies provided strong confidence that the hydroxyl group and the carbonyl group are crucial for the fungicidal activity. Molecular docking studies suggest that SDH enzyme could be one of the potential action targets of our compounds.     

Design,synthesis and molecular modeling of biquinoline–pyridine hybrids as a new class of potential EGFR and HER-2 kinase inhibitors

A new series of biquinoline–pyridine hybrids were designed and synthesized by a base-catalyzed cyclocondensation through one-pot multicomponent reaction. All compounds were tested for in vitro anticancer activities against two cancer cell lines A549 (adenocarcinomic human alveolar basal epithelial) and Hep G2 (liver cancer). Enzyme inhibitory activities of all compounds were carried out against EGFR and HER-2 kinase. Of the compounds studied, majority of the compounds showed effective anticancer activity against used cancer cell lines. Compound 9i (IC₅₀ = 0.09 μM) against EGFR and (IC₅₀ = 0.2 μM) against HER-2 kinase displayed the most potent inhibitory activity as compared to other member of the series. In the molecular modelling study, compound 9i was bound in to the active pocket of EGFR with four hydrogen bonds and two π–cation interactions having minimum binding energy ΔG_b = −54.4 kcal/mol.     

Antibacterial and antifungal activities of teucrium royleanum (Labiatea)

The crude methanolic extract and subsequent fractions of Teucrium royleanum (Labiatea) were screened for antibacterial and antifungal activities. Against tested pathogens, crude extract and subsequent fractions demonstrated moderate to excellent antibacterial activities. Highest antibacterial activity was displayed by the ethyl acetate fraction against S. typhi (100%), against E.coli (76.7%) and against P. aerugenosa (70.8%) followed by the chloroform fraction against S. typhi (85.7%). Similarly, the crude extract and its subsequent fractions showed mild to excellent activities in the antifungal bioassay with maximum antifungal activity against M. canis (87%) by the chloroform fraction followed by the ethyl acetate (71%) and n-butanol (70%) fractions.     

Design,synthesis and evaluation of novel sophoridinic imine derivatives containing conjugated planar structure as potent anticancer agents

Based on our previous study and the binding mode of camptothecin with Topo I, a series of novel sophoridine imine derivatives containing conjugated planar structure were designed, synthesized and tested for their in vitro anticancer activity. The results showed that most of the derivatives displayed potent activity. In particular, compounds 10b exhibited excellent anti-proliferative activities with IC₅₀ 5.7?µM and 8.5?µM against HepG-2 and HeLa cell lines, respectively. Molecular docking studies revealed that the introduction of conjugated planar structure could form π-π stacking interaction with DNA, leading to the improvement of biological activity. Its mode of action was to inhibit the activity of DNA Topo I, followed by the G0/G1 phase arrest. This work provides a theoretical basis for structural optimizations and exploring anticancer pathways of this kind of compound and 10b could emerge as promising lead compounds for the development of novel Topo I inhibitors.     

Scalable Production of Recombinant Membrane Active Peptides and Its Potential as a Complementary Adjunct to Conventional Chemotherapeutics

The production of short anticancer peptides in recombinant form is an alternative method for costly chemical manufacturing. However, the limitations of host toxicity, bioactivity and column purification have impaired production in mass quantities. In this study, short cationic peptides were produced in aggregated inclusion bodies by double fusion with a central protein that has anti-cancer activity. The anticancer peptides Tachiplicin I (TACH) and Latarcin 1 (LATA) were fused with the N- and C-terminus of the MAP30 protein, respectively. We successfully produced the recombinant TACH-MAP30-LATA protein and MAP30 alone in E. coli that represented 59% and 68% of the inclusion bodies. The purified form of the inclusion bodies was prepared by eliminating host cell proteins through multiple washing steps and semi-solubilization in alkaline buffer. The purified active protein was recovered by inclusive solubilization at pH 12.5 in the presence of 2 M urea and refolded in alkaline buffer containing oxides and reduced glutathione. The peptide-fusion protein showed lower CC₅₀ values against cancer cells (HepG2, 0.35±0.1 μM and MCF-7, 0.58±0.1 μM) compared with normal cells (WRL68, 1.83±0.2 μM and ARPE19, 2.5±0.1 μM) with outstanding activity compared with its individual components. The presence of the short peptides facilitated the entry of the peptide fusion protein into cancer cells (1.8 to 2.2-fold) compared with MAP30 alone through direct interaction with the cell membrane. The cancer chemotherapy agent doxorubicin showed higher efficiency and selectivity against cancer cells in combination with the peptide- fusion protein. This study provides new data on the mass production of short anticancer peptides as inclusion bodies in E. coli by fusion with a central protein that has similar activity. The product was biologically active against cancer cells compared with normal cells and enhanced the activity and selective delivery of an anticancer chemotherapy agent.     

In vitro cytotoxic potential of Solanum nigrum against human cancer cell lines

Plants have natural products which use to possess antiproliferative potential against many cancers. In the present study, six isolated fractions (ethyl acetate, petroleum ether, chloroform, n-butanol, ethanol and aqueous) from Solanum nigrum were evaluated for their cytotoxic effect on different cell lines. Hepatic carcinoma cell line (HepG2), cervical cancer cell line (HeLa) and baby hamster kidney (BHK) used as normal non-cancerous cells were evaluated for cytotoxicity against isolated fractions. Cell viability assay was performed to evaluate the cytotoxicity of all fractions on different cell lines followed by the lactate dehydrogenase and vascular endothelial growth factor assays of most active fraction among all screened for cytotoxic analysis. HPLC analysis of most active fractions against cytotoxicity was performed to check the biological activity of compounds. Results displayed the potent cytotoxic activity of ethyl acetate fraction of S. nigrum against HepG2 cells with IC₅₀ value of 7.89 μg/ml. Other fractions exhibited potent anticancer activity against HepG2 cells followed by HeLa cells. Fractions in our study showed no cytotoxicity in BHK cells. Cytotoxic activity observed in our current study exposed high antiproliferative potential and activity of ethyl acetate fraction against HepG2 cells. The results demonstrated that S. nigrum fractions exhibited anticancer activity against hepatic and cervical cancer cell lines with non-toxic effect in normal cells. These results reveal significant potential of S. nigrum for the therapeutic of cancers across the globe in future.     

Synthesis,biological evaluation,and in silico studies of new CDK2 inhibitors based on pyrazolo[3,4-d]pyrimidine and pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine scaffold with apoptotic activity

Cyclin-dependent kinase inhibition is considered a promising target for cancer treatment for its crucial role in cell cycle regulation. Pyrazolo pyrimidine derivatives were well established for their antitumor activity via CDK2 inhibition. In this research, new series of pyrazolopyrimidine derivatives (4–15) was designed and synthesised as novel CDK2 inhibitors. The anti-proliferative activities against MCF-7, HCT-116, and HepG-2 were used to evaluate their anticancer activity as novel CDK2 inhibitors. Most of the compounds showed superior cytotoxic activity against MCF-7 and HCT-116 compared to Sorafenib. Only compounds 8, 14, and 15 showed potent activity against HepG-2. The CDK2/cyclin A2 enzyme inhibitory activity was tested for all synthesised compounds. Compound 15 showed the most significant inhibitory activity with IC₅₀ 0.061 ± 0.003 µM. It exerted remarkable alteration in Pre G1 and S phase cell cycle progression and caused apoptosis in HCT cells. In addition, the normal cell line cytotoxicity for compound 15 was assigned revealing low cytotoxic results in normal cells rather than cancer cells. Molecular docking was achieved on the designed compounds and confirmed the two essential hydrogen binding with Leu83 in CDK2 active site. In silico ADMET studies and drug-likeness showed proper pharmacokinetic properties which helped in structure requirements prediction for the observed antitumor activity.     

Scaffold hopping of sampangine: Discovery of potent antifungal lead compound against Aspergillus fumigatus and Cryptococcus neoformans

Discovery of novel antifungal agents against Aspergillus fumigatus and Cryptococcus neoformans remains a significant challenge in current antifungal therapy. Herein the antifungal natural product sampangine was used as the lead compound for novel antifungal drug discovery. A series of D-ring scaffold hopping derivatives were designed and synthesized to improve antifungal activity and water solubility. Among them, the thiophene derivative S2 showed broad-spectrum antifungal activity, particularly for Aspergillus fumigatus and Cryptococcus neoformans. Moreover, compound S2 also revealed better water solubility than sampangine, which represents a promising antifungal lead compound for further structural optimization.     

Artemisinin-indole and artemisinin-imidazole hybrids: Synthesis,cytotoxic evaluation and reversal effects on multidrug resistance in MCF-7/ADR cells

A series of artemisinin derivatives with MDR reversal activity were designed and synthesized. All hybrids were screened to anticancer activities against four human cancer cell lines (A549, MCF-7, HepG-2, MDA-MB-231) and normal human hepatic cell (L02) in vitro. Most of the new compounds showed higher anticancer activities than artemisinin, among which compounds 11a and 11c displayed superior potency with IC₅₀ 6.78?μM and 5.25?μM against MCF-7, respectively. The further research indicated that the most potent 11c induced cell cycle arrest at G2 phase in MCF-7. Additionally, compound 11c showed remarkable MDR reversal activity which reversed adriamycin against MCF-7/ADR cells with IC₅₀ 0.76?μM.