Bioactive compounds from Morchella esculenta as potential inhibitors of RNA-binding protein La in ovarian cancer: a molecular modeling and quantum mechanics approach.

La protein is significantly expressed in various malignant tumors, including ovarian cancer (OC), which is related to the poor response to platinum-based chemotherapy. Thus, inhibiting La protein could control the expression of the potential downstream genes involved in promoting proliferation and chemotherapy resistance to OC, which could serve as a therapeutic intervention. Through a molecular docking approach, 12 compounds from Morchella esculenta were screened against the crystal structure of La protein and four hit compounds were identified, including beta-carotene, p-hydroxybenzoic acid, gamma-tocopherol, and alpha-tocopherol, with a binding affinity of - 10.7, - 8.1, - 7.9, and - 7.6 kcal/mol, respectively, higher than pyridine-2-carboxylate (control), with a binding affinity of - 5.2 kcal/mol. To explore the interaction of the hit compounds with the target receptor, they were selected for a molecular dynamic simulation and post-simulation analysis for 100 ns. The result showed promising reliability of the ligands due to a stable interaction with the La protein crystal structure. Furthermore, the drug-likeness and physicochemical chemical properties of the compounds were investigated using ADMET study and density functional theory analysis, respectively, and the result shows that the hit compounds could serve as a promising starting for the development of novel LA protein inhibitors for OC therapeutics. Finally, this study compared HOMO and LUMO values from global hybrids with long-range corrected DFAs, and the result from the two followed the same qualitative pattern while calculating HOMO values; however, MO62X/cc-pVTZ could better predict LUMO values when considering a global hybrid.

Improving Pharmacokinetics of Peptides Using Phage Display.

Phage display is a versatile method often used in the discovery of peptides that targets disease-related biomarkers. A major advantage of this technology is the ease and cost efficiency of affinity selection, also known as biopanning, to identify novel peptides. While it is relatively straightforward to identify peptides with optimal binding affinity, the pharmacokinetics of the selected peptides often prove to be suboptimal. Therefore, careful consideration of the experimental conditions, including the choice of using in vitro, in situ, or in vivo affinity selections, is essential in generating peptides with high affinity and specificity that also demonstrate desirable pharmacokinetics. Specifically, in vivo biopanning, or the combination of in vitro, in situ, and in vivo affinity selections, has been proven to influence the biodistribution and clearance of peptides and peptide-conjugated nanoparticles. Additionally, the marked difference in properties between peptides and nanoparticles must be considered. While peptide biodistribution depends primarily on physiochemical properties and can be modified by amino acid modifications, the size and shape of nanoparticles also affect both absorption and distribution. Thus, optimization of the desired pharmacokinetic properties should be an important consideration in biopanning strategies to enable the selection of peptides and peptide-conjugated nanoparticles that effectively target biomarkers in vivo.

Computational evaluation of bioactive compounds from Viscum album (mistletoe) as inhibitors of p63 for pancreatic cancer treatment.

Pancreatic ductal adenocarcinoma is an aggressive malignancy usually detectable at the advanced stage, with a 5-year survival rate of less than 8%. It has been reported that a gene called tumor-protein 63 (TP63) is expressed in an aggressive form of pancreatic cancer with a squamous signature. Thus, inhibiting the activity of p63 can be a means of treating and managing PDA. Different studies have shown that plant constituents are rich and can be a promising source for discovering drug candidates. The extract from mistletoe (Viscum album) is known to contain anticancer compounds; however, the specific molecular mechanism of the bioactive compounds is unknown. This study examines the pancreatic cancer therapeutic potential of the bioactive compounds in the flavonoid and phenolic acid constituents of mistletoe by adopting structural bioinformatics and advanced theoretical chemistry techniques via molecular docking, molecular dynamics simulation, molecular mechanics/generalized Born surface area (MM/GBSA) calculations, pharmacokinetic analysis, and density functional theory analysis. The six best compounds from the flavonoid constituent with the highest binding affinity ranging from -6.8 kcal/mol to -6.7 kcal/mol were selected with the control gemcitabine (-5.5 kcal/mol) for further computational analysis after molecular docking. Furthermore, MM/GBSA calculation showed the highest binding energy for the selected docked compounds, which validates their inhibitory potential. Hence, the molecular dynamics simulation, post-simulation analysis, pharmacokinetics model, and DFT results showed that mistletoe compounds are reliable due to their stable interaction with the target protein and drug-likeness properties.Communicated by Ramaswamy H. Sarma.

Effect of Grafting Rootstock on the Antioxidant Capacity and Content of Heirloom Tomatoes (Solanum lycopersicum L.) in Hydroponic Culture.

Heirloom tomato varieties are in demand by consumers due to high antioxidant levels. However, these varieties are difficult to produce and are prone to disease. To overcome these problems, heirloom tomatoes may be cultivated in hydroponic systems and grafted onto disease-resistant rootstocks. However, it is unknown if the antioxidant content and capacity are affected by grafting. In this study, heirloom (Black Krim and Green Zebra) and standard (Big Beef) varieties were grafted onto wild type (WT) or productive rootstocks (Arnold and Supernatural). The tomatoes were harvested at maturity, freeze-dried, and ground into a powder. Lycopene was extracted using hexane, and the content was determined spectrophotometrically at 503 nm. The antioxidant capacity of methanol extracts was evaluated by the 2,2'-azino-di[3-ethylbenzthiazoline sulfonsyr]sulphonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays, whereas the phenolic content was determined using the Folin-Ciocalteu assay. Interestingly, the grafting of Big Beef and Green Zebra onto Supernatural rootstock resulted in an increased antioxidant capacity, as determined by the DPPH assay. Moreover, the phenolic content was changed for Big Beef grafted onto Arnold, and Big Beef and Green Zebra grafted onto Supernatural. Taken together, these results indicate that certain combinations of standard and heirloom tomato varieties and productive rootstocks may influence the antioxidant capacity and phenolic content. These results may be used to guide producers when choosing rootstocks for cultivating hydroponic tomatoes.

Phage Display Selection, Identification, and Characterization of Novel Pancreatic Cancer Targeting Peptides.

Pancreatic cancer is characterized by a 5-year survival rate of 3%, in part due to inadequate detection methods. The small size of peptides offers advantages regarding molecular targeting. Thus, peptides may be used in detection of pancreatic cancer. Here, peptides that target pancreatic cancer cells were selected using phage display technology using a 15-mer fUSE5 library. Phage were pre-cleared against immortalized pancreatic cells (hTERT-HPNE), followed by selections against pancreatic cancer (Mia Paca-2) cells. Next-generation sequencing identified two peptides, MCA1 and MCA2, with a Log2 fold change (Mia Paca-2/ hTERT-HPNE) >1.5. Modified ELISA and fluorescent microscopy showed that both peptides bound significantly higher to Mia Paca-2 cells, and not to hTERT-HPNE, embryonic kidney (HEK 293), ovarian (SKOV-3) and prostate cancer (LNCaP) cell lines. Further characterization of MCA1 and MCA2 revealed EC50 values of 16.11 µM (95% CI [9.69, 26.31 µM]) and 97.01 µM (95% CI [58.64, 166.30 µM]), respectively. Based on these results, MCA1 was selected for further studies. A competitive dose response assay demonstrated specific binding and an IC50 value of 2.15 µM (95% CI [1.28, 3.62 µM]). Taken together, this study suggests that MCA1 may be used as a pancreatic cancer targeting ligand for detection of the disease.

Ovarian Cancer Targeting Phage for In Vivo Near-Infrared Optical Imaging.

Ovarian cancer is often diagnosed at late stages due to current inadequate detection. Therefore, the development of new detection methods of ovarian cancer is needed. This may be achieved by phage nanoparticles that display targeting peptides for optical imaging. Here, two such phage clones are reported. Ovarian cancer binding and specificity of phage clones (pJ18, pJ24) and peptides (J18, J24) were investigated using fluorescent microscopy and modified ELISA. Further, AF680-labeled phage particles were subjected to biodistribution and optical imaging studies in SKOV-3 xenografted mice. Fluorescent microscopy and ELISA of phage and peptides showed significantly increased binding to SKOV-3 cells compared to controls. Additionally, these studies revealed that J18 exhibits specificity for ovarian cancer SKOV-3 and OVCAR-3 cell lines. Further, peptides displayed increased SKOV-3 binding compared to N35 (non-relevant peptide) with EC50 values of 22.2 ± 10.6 µM and 29.0 ± 6.9 (mean ± SE), respectively. Biodistribution studies of AF680-labeled phage particles showed tumor uptake after 4 h and excretion through the reticuloendothelial system. Importantly, SKOV-3 tumors were easily localized by optical imaging after 2 h and 4 h and displayed good tumor-to-background contrast. The fluorescent tumor signal intensity was significantly higher for pJ18 compared to wild type (WT) after 2 h.

In vitro high throughput phage display selection of ovarian cancer avid phage clones for near-infrared optical imaging.

Ovarian cancer is among the leading causes of cancer deaths in women, and is the most fatal gynecological malignancy. Poor outcomes of the disease are a direct result of inadequate detection and diagnostic methods, which may be overcome by the development of novel efficacious screening modalities. However, the advancement of such technologies is often time-consuming and costly. To overcome this hurdle, our laboratory has established a time and cost effective method of selecting and identifying ovarian carcinoma avid bacteriophage (phage) clones using high throughput phage display technology. These phage clones were selected from a filamentous phage fusion vector (fUSE5) 15-amino acid peptide library against human ovarian carcinoma (SKOV-3) cells, and identified by DNA sequencing. Two phage clones, pM6 and pM9, were shown to exhibit high binding affinity and specificity for SKOV-3 cells using micropanning, cell binding and fluorescent microscopy studies. To validate that the binding was mediated by the phage-displayed peptides, biotinylated peptides (M6 and M9) were synthesized and the specificity for ovarian carcinoma cells was analyzed. These results showed that M6 and M9 bound to SKOV-3 cells in a dose-response manner and exhibited EC50 values of 22.9 ± 2.0 µM and 12.2 ± 2.1µM (mean ± STD), respectively. Based on this, phage clones pM6 and pM9 were labeled with the near-infrared fluorophore AF680, and examined for their pharmacokinetic properties and tumor imaging abilities in vivo. Both phage successfully targeted and imaged SKOV-3 tumors in xenografted nude mice, demonstrating the ability of this method to quickly and cost effectively develop novel ovarian carcinoma avid phage.

In vivo phage display selection of an ovarian cancer targeting peptide for SPECT/CT imaging.

The often fatal outcome of ovarian cancer (OC) is related to inadequate detection methods, which may be overcome by development of nuclear imaging agents. Cancer targeting peptides have been identified using in vivo bacteriophage (phage) display technology; however, the majority of these ligands target tumor vasculature. To overcome this problem, a two-tier phage display method was employed to select an ovarian cancer targeting peptide with good pharmacokinetic and imaging properties. A fUSE5 15-amino acid peptide library was screened against xenografted human OC SKOV-3 tumors in mice, which was followed by selection against enriched SKOV-3 cells. The selected peptide RSLWSDFYASASRGP (J18) was synthesized with a GSG-spacer and a 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA) chelator and radiolabeled with (111)In. SKOV-3 xenografted mice were used to evaluate the biodistribution and single photon emission computed tomography (SPECT) imaging capabilities of the radiolabeled peptide. Competitive binding experiments using (111)In-DOTA-GSG-J18 indicated that the peptide displayed a half maximal inhibitory concentration (IC50) value of 10.5 ± 1.1 µM. Biodistribution studies revealed that tumor uptake was 1.63 ± 0.68, 0.60 ± 0.32, 0.31 ± 0.12 and 0.10 ± 0.02% injected dose/g at 30 min, 1 h, 2 h and 4 h post-injection of (111)In-DOTA-GSG-J18, respectively. SPECT/CT imaging demonstrated good tumor uptake and minimal background binding. This study demonstrated successful utilization of a two-tier phage display selection process to identify an ovarian cancer avid peptide with excellent SPECT/CT imaging capabilities.

The impact of loss of myrosinase on the bioactivity of broccoli products in F344 rats.

In vitro, animal, and epidemiological studies all show that broccoli products containing sulforaphane, the bioactive hydrolysis product of glucoraphanin (GRP), lower risk for cancer. As a result, GRP-rich extracts are appearing on the market as dietary supplements. However, these products typically have no hydrolyzing enzyme for sulforaphane (SF) formation. We evaluated safety and compared efficacy to other broccoli preparations. Four daily doses of 0.5 mmol GRP/kg BW, given by gavage to adult male F344 rats, caused temporary cecal inflammation that was essentially resolved four days later. A similar dose dispersed in the diet caused no inflammation. To compare efficacy, we fed rats 20% freeze-dried broccoli (heated or unheated), 3.5% broccoli seed meal, or 4.3% semipurified GRP, each balanced within an AIN93G semipurified diet, for 4 days. Diets lacking myrosinase (semipurified GRP and heated broccoli florets) caused upregulation of NAD(P)H-quinone oxidoreductase 1 (NQO1) in colon but not liver. Surprisingly, broccoli seed, rich in myrosinase and GRP, also caused NQO1 upregulation in colon but not liver. In contrast, unheated broccoli florets caused upregulation in both colon and liver. These data suggest that GRP supplements may not exert systemic effects. We hypothesize that within whole broccoli additional components enhanced sulforaphane-dependent upregulation of NQO1 in liver.