Discovery of potential FGFR3 inhibitors via QSAR, pharmacophore modeling, virtual screening and molecular docking studies against bladder cancer.

BACKGROUND: Fibroblast growth factor receptor 3 is known as a favorable aim in vast range of cancers, particularly in bladder cancer treatment. Pharmacophore and QSAR modeling approaches are broadly utilized for developing novel compounds for the determination of inhibitory activity versus the biological target. In this study, these methods employed to identify FGFR3 potential inhibitors. METHODS: To find the potential compounds for bladder cancer targeting, ZINC and NCI databases were screened. Pharmacophore and QSAR modeling of FGFR3 inhibitors were utilized for dataset screening. Then, with regard to several factors such as Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties and Lipinski's Rule of Five, the recognized compounds were filtered. In further step, utilizing the flexible docking technique, the obtained compounds interactions with FGFR3 were analyzed. RESULTS: The best five compounds, namely ZINC09045651, ZINC08433190, ZINC00702764, ZINC00710252 and ZINC00668789 were selected for Molecular Dynamics (MD) studies. Off-targeting of screened compounds was also investigated through CDD search and molecular docking. MD outcomes confirmed docking investigations and revealed that five selected compounds could make steady interactions with the FGFR3 and might have effective inhibitory potencies on FGFR3. CONCLUSION: These compounds can be considered as candidates for bladder cancer therapy with improved therapeutic properties and less adverse effects.

A quartz crystal microbalance biosensor based on polyethylenimine-modified gold electrode to detect hepatitis B biomarker.

Biomarkers-based QCM-biosensors are suitable tools for the label-free detection of infectious diseases. In the current study, a QCM-biosensor was developed for the detection of HBsAg. Briefly, anti-HBsAg antibodies were covalently bound to the primary amines after PEI and thiolated-PEI surface modifications of gold-electrode. After RSM optimization, the statistical analysis revealed no significant difference between the immobilization yields of modified layers. Therefore, the PEI-modified QCM-biosensor was selected for further analysis. The PEI-surface was evaluated by FESEM, AFM, ATR-FTIR, and CA measurement. The surface hydrophilicity and its roughness were increased after PEI-coating. Also, FTIR confirmed the PEI-layering on the gold-surface. RSM optimization increased the antibody immobilization yield up to 80%. The QCM-biosensor showed noteworthy results with a wide dynamic range of 1-1 × 103 ng/mL, LOD of 3.14 ng/mL, LOQ of 9.52 ng/mL, and detection capability in human-sera, which were comparable with the ELISA. The mean accuracy of the QCM-biosensor was obtained at 91% when measured by the spike recovery test using human-sera. The biosensor was completely regenerated using 50 mM NaOH and 1% SDS. The benefits provided by the developed biosensor such as broad dynamic range, sensitivity, selectivity, stability, regenerate ability, and low cost suggest its potential application for the non-invasive and timely monitoring of HBV-biomarker.

pH-Responsive PEGylated Niosomal Nanoparticles as an Active-Targeting Cyclophosphamide Delivery System for Gastric Cancer Therapy.

A PEGylated niosomal formulation of cyclophosphamide (Nio-Cyclo-PEG) was prepared using a central composite design and characterized in terms of drug loading, size distribution, and average size. The stability of formulations was also studied at different conditions. In vitro cytotoxicity of drug delivery formulations was assessed on gastric cancer cells using MTT assay. The mechanism of cytotoxicity was studied at the transcriptional level by real-time PCR on Caspase3, Caspase9, CyclinD, CyclinE, MMP-2, and MMP-9 genes, while apoptosis was investigated with flow cytometry. The anti-metastatic property was evaluated using the scratch method. Propidium iodide staining was used to study the cell cycle. The results indicated that the as-designed nanocarrier exhibited a controlled drug release pattern with improved nanoparticle stability. It was found that the living cancer cells treated with Nio-Cyclo-PEG showed a significant decrease in number when compared with the niosomal carrier without PEG (Nio-Cyclo) and free drug (Cyclo). Moreover, the drug-loaded nanocarrier induced planned death (apoptosis) in the cancer cells through the regulation of Caspase3, Caspase9, CyclinD, CyclinE, MMP-9, and MMP-2 gene expression, indicating that the Nio-Cyclo-PEG formulation could significantly inhibit the cell cycle at the sub G1 phase as well as prevent the migration of cancer cells. In conclusion, Nio-Cyclo-PEG as developed in this study could serve as an active-targeting drug delivery nanocarriers for gastric cancer therapy with high efficacy and minimal side effects on healthy tissues/cells.

Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system.

BACKGROUND: Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27). RESULTS: Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze-thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking. CONCLUSION: The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales.

Lipopolysaccharide removal affinity matrices based on novel cationic amphiphilic peptides.

Lipopolysaccharide (LPS) is one of the most challenging contaminants in biopharmaceutical industries. Cationic amphiphilic peptides (CAPs) -based affinity matrices can be potent tools for LPS removal in such situations. In this study, two newly designed CAPs derived from the LPS binding site of factor C of horseshoe crab S3E3 and S3E3A were immobilized chemo-selectively on diaminodipropylamine (DADPA) and iodoacetyl functionalized Sepharose beads. Both peptides were immobilized via their carboxyl or sulfhydryl (thiol) groups by amide or thioether bonds, respectively. The generated four affinity matrices were used to remove LPS from bovine serum albumin (BSA). The effects of different influential factors including pH, NaCl, Ethylenediaminetetraacetic acid (EDTA), and LPS concentrations on LPS removal efficiency and protein recovery were investigated by Plackett Burman (PB) method.Statistical analysis revealed that immobilized S3E3 removed LPS more effectively than immobilized S3E3A. Increasing pH and LPS concentration had negative effects on LPS removal efficiency and protein recovery. Increasing NaCl concentration improved protein recovery but reduced LPS removal efficiency. Other factors such as EDTA and type of buffer had no significant effect on the measured responses.

Challenges and advancements in the pharmacokinetic enhancement of therapeutic proteins.

Nowadays, proteins are frequently administered as therapeutic agents in human diseases. However, the main challenge regarding the clinical application of therapeutic proteins is short circulating plasma half-life that leads to more frequent injections for maintaining therapeutic plasma levels, increased therapy costs, immunogenic reactions, and low patient compliance. So, the development of novel strategies to enhance the pharmacokinetic profile of therapeutic proteins has attracted great attention in pharmaceuticals. So far, several techniques, each with their pros and cons, have been developed including chemical bonding to polymers, hyper glycosylation, Fc fusion, human serum albumin fusion, and recombinant PEG mimetics. These techniques mainly classify into three strategies; (i) the endosomal recycling of neonatal Fc receptor which is observed for immunoglobulins and albumin, (ii) decrease in receptor-mediated clearance, and (iii) increase in hydrodynamic radius through chemical and genetic modifications. Recently, novel PEG mimetic peptides like proline/alanine/serine repeat sequences are designed to overcome pitfalls associated with the previous technologies. Biodegradability, lack of or low immunogenicity, product homogeneity, and a simple production process, currently make these polypeptides as the preferred technology for plasma half-life extension of therapeutic proteins. In this review, challenges and pitfalls in the pharmacokinetic enhancement of therapeutic proteins using PEG-mimetic peptides will be discussed in detail.

Niosomal delivery of simvastatin to MDA-MB-231 cancer cells.

OBJECTIVE: The objective of this study was to use nano-niosomal formulations to deliver simvastatin as a poor-water soluble drug into breast cancer cells. SIGNIFICANCE: Our study focused on the problem associated with poor water-soluble drugs which have significant biological activity in vivo. METHODS: Different niosomal formulations of simvastatin were prepared and characterized in terms of morphology, size, encapsulation efficiency (EE), and release kinetic. Antiproliferative activity and the mechanism were assessed by quantitative real-time PCR and flow cytometry. Moreover, confocal microscopy was employed to analyze the cell uptake of simvastatin loaded niosomes to the cancerous cells. RESULTS: Size, polydispersity index (PDI), and EE of the best formulation were obtained as 164.8 nm, 0.232, and 97%, respectively. The formulated simvastatin had a spherical shape and showed a slow release profile of the drug after 72 h. Stability data elucidated an increase in mean diameter and PDI which was lower for 4 °C than 25 °C. Confocal microscopy showed the localization of drug loaded niosomes in the cancer cells. The MTT assay revealed both free drug and drug loaded niosomes exhibited a dose-dependent cytotoxicity against breast cancer cells (MDA-MB-231 cells). Flow cytometry and qPCR analysis revealed drug loaded niosomes exert their cytotoxicity on cancerous cells via regulation of apoptotic and anti-apoptotic genes. CONCLUSION: The prepared niosomal simvastatin showed good physicochemical and biological properties than free drug. Our study suggests that niosomal delivery could be considered as a promising strategy for the delivery of poor water-soluble drugs to cancer cells.

Effect of glutamic acid elimination/substitution on the biological activities of S3 cationic amphiphilic peptides.

Cationic amphiphilic peptides (CAPs) are usually classified as bacterial membrane targeting molecules. Rational design and modification of cationic and amphiphilic properties of CAPs have made them to be used in new medical and biotechnological applications. However, CAPs modification and development strategies are challenging issues due to the risk of cytotoxicity or hemolytic activity. In this research, modified variants of S3 peptide were introduced. S3 is a linear 34 amino acid peptide derived from the lipopolysaccharide (LPS) binding site of factor C in horseshoe crab's hemolymph. Net positive charges of variants (S3E3 and S3E3A) increased by either eliminating negatively charged residues of the peptides or substituting them with alanine. Different biological activities of new variants including LPS binding affinity, antimicrobial activity, cytotoxicity against human breast tumor cell line, and hemolytic property were studied and compared to those of S3 peptide. S3E3 variant showed 68.5% higher LPS binding affinity, 40.4% stronger anti-microbial activity, conserved hemolytic property with the same anti-cancer activity compared to S3peptide. These results revealed that elimination/substitution of negatively charged residues will be a proper strategy for modification of S3 peptide.

Rational affinity enhancement of fragmented antibody by ligand-based affinity improvement approach.

Antibody engineering is now a noteworthy area in biopharmaceuticals as the next generation of marketed antibodies is engineered antibodies such as affinity- or stability-improved antibodies, fragmented or fused antibodies, antibody drug conjugates (ADCs), and PEGylated antibody fragments. In the current study, affinity enhancement of Nb against PlGF was performed by an in silico affinity maturation and molecular dynamics (MD) simulation. First, 300 single-point mutants were designed by identifying the residues involved in interaction with PlGF and different energy distributions. An energy based screening was performed to select best single-point mutants. Additionally, one variant containing two mutations was designed based on the selected single-point mutants. Finally, mutants-PlGF complexes were analyzed in details by all atom MD simulation. Trajectory analysis revealed that in both single (L112H, S31D, A97K, and R45E) and double (S31D & R45E) mutants, the free binding energies and the stability of complexes were significantly improved. The highest increment in affinity was observed for S31D mutant due to substantial increase in polar and electrostatic interactions. The secondary structure of Nb was intact in all variants and a shrinkage of PlGF over Nb was observed in all mutant-PlGF complexes during simulation. In addition, contact area and hydrogen-bond analysis as well as distance measurement in mutants-PlGF complexes also confirmed the affinity enhancement of variants relative to the native form. Our study showed that ligand-based affinity improvement could be considered as a promising approach for designing high affinity fragmented antibodies.

Identification of methionine oxidation in human recombinant erythropoietin by mass spectrometry: Comparative isoform distribution and biological activity analysis.

BACKGROUND: Oxidative degradation of human recombinant erythropoietin (hrEPO) may occur in manufacturing process or therapeutic applications. This unfavorable alteration may render EPO inefficient or inactive. We investigated the effect of methionine/54 oxidative changes on the amino acid sequences, glycoform distribution and biological activity of hrEPO. METHODS: Mass spectrometry was applied to verify the sequence and determine the methionine oxidation level of hrEPO. Isoform distribution was studied by capillary zone electrophoresis method. In vivo normocythemic mice assay was used to assess the biological activity of three different batches (A, B, and C) of the proteins. RESULTS: Nano-LC/ESI/MS/MS data analyses confirmed the amino acid sequences of all samples. The calculated area percent of three isoforms (2-4 of the 8 obtained isoforms) were decreased in samples of C, B, and A with 27.3, 16.7, and 6.8% of oxidation, respectively. Specific activities were estimated as 53671.54, 95826.47, and 112994.93 mg/mL for the samples of A, B, and C, respectively. CONCLUSION: The observed decrease in hrEPO biological activity, caused by increasing methionine oxidation levels, was rather independent of its amino acid structure and mainly associated with the higher contents of acidic isoforms.

Nanosilver based anionic linear globular dendrimer with a special significant antiretroviral activity.

HIV is commonly caused to a very complicated disease which has not any recognized vaccine, so designing and development of novel antiretroviral agents with specific application of nanomedicine is a globally interested research subject worldwide. In the current study, a novel structure of silver complexes with anionic linear globular dendrimer was synthesized, characterized and then assessed against HIV replication pathway in vitro as well. The results showed a very good yield of synthesis (up to 70%) for the nano-complex as well as a very potent significant (P < 0.05) antiretroviral activity with non-severe toxic effects in comparison with the Nevirapine as standard drug in positive control group. According to the present data, silver anionic linear globular dendrimers complex may have a promising future to inhibit replication of HIV viruse in clinical practice.

A novel nanobody-based immunocytokine of a mutant interleukin-2 as a potential cancer therapeutic.

The immunotherapeutic application of interleukin-2 (IL-2) in cancer treatment is limited by its off-target effects on different cell populations and insufficient activation of anti-tumor effector cells at the site of the tumor upon tolerated doses. Targeting IL-2 to the tumor microenvironment by generating antibody-cytokine fusion proteins (immunocytokine) would be a promising approach to increase efficacy without associated toxicity. In this study, a novel nanobody-based immunocytokine is developed by the fusion of a mutant (m) IL-2 with a decreased affinity toward CD25 to an anti-vascular endothelial growth factor receptor-2 (VEGFR2) specific nanobody, denoted as VGRmIL2-IC. The antigen binding, cell proliferation, IFN-γ-secretion, and cytotoxicity of this new immunocytokine are evaluated and compared to mIL-2 alone. Furthermore, the pharmacokinetic properties are analyzed. Flow cytometry analysis shows that the VGRmIL2-IC molecule can selectively target VEGFR2-positive cells. The results reveal that the immunocytokine is comparable to mIL-2 alone in the stimulation of Primary Peripheral Blood Mononuclear Cells (PBMCs) and cytotoxicity in in vitro conditions. In vivo studies demonstrate improved pharmacokinetic properties of VGRmIL2-IC in comparison to the wild or mutant IL-2 proteins. The results presented here suggest VGRmIL2-IC could be considered a candidate for the treatment of VEGFR2-positive tumors.

Development of a bivalent protein-based vaccine candidate against invasive pneumococcal diseases based on novel pneumococcal surface protein A in combination with pneumococcal histidine triad protein D.

Extensive efforts have been made toward improving effective strategies for pneumococcal vaccination, focusing on evaluating the potential of multivalent protein-based vaccines and overcoming the limitations of pneumococcal polysaccharide-based vaccines. In this study, we investigated the protective potential of mice co-immunization with the pneumococcal PhtD and novel rPspA proteins against pneumococcal sepsis infection. The formulations of each antigen alone or in combination were administered intraperitoneally with alum adjuvant into BALB/c mice three times at 14-day intervals. The production of antigen-specific IgG, IgG1 and IgG2a subclasses, and IL-4 and IFN-γ cytokines, were analyzed. Two in vitro complement- and opsonophagocytic-mediated killing activities of raised antibodies on day 42 were also assessed. Finally, the protection against an intraperitoneal challenge with 106 CFU/mouse of multi-drug resistance of Streptococcus pneumoniae ATCC49619 was investigated. Our findings showed a significant increase in the anti-PhtD and anti-rPspA sera IgG levels in the immunized group with the PhtD+rPspA formulation compared to each alone. Moreover, the results demonstrated a synergistic effect with a 6.7- and 1.3- fold increase in anti-PhtD and anti-rPspA IgG1, as well as a 5.59- and 1.08- fold increase in anti-PhtD and anti-rPspA IgG2a, respectively. Co-administration of rPspA+PhtD elicited a mixture of Th-2 and Th-1 immune responses, more towards Th-2. In addition, the highest complement-mediated killing activity was observed in the sera of the immunized group with PhtD+rPspA at 1/16 dilution, and the opsonophagocytic activity was increased from 74% to 86.3%. Finally, the survival rates showed that mice receiving the rPspA+PhtD formulation survived significantly longer (100%) than those receiving protein alone or PBS and exhibited the strongest clearance with a 2 log10 decrease in bacterial load in the blood 24h after challenge compared to the control group. In conclusion, the rPspA+PhtD formulation can be considered a promising bivalent serotype-independent vaccine candidate for protection against invasive pneumococcal infection in the future.

A Novel Immunotoxin Targeting Epithelial Cell Adhesion Molecule Using Single Domain Antibody Fused to Diphtheria Toxin.

Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in a variety of cancers such as colon, stomach, pancreas, and prostate adenocarcinomas. Inhibition of EpCAM is considered as a potential target for cancer therapy. In current study, anti-EpCAM immunotoxin (α-EpCAM IT) was developed using genetic fusion of α-EpCAM single domain antibody (nanobody) (α-EpCAM Nb) to truncated form of diphtheria toxin. The expression of recombinant α-EpCAM IT was induced by Isopropyl ß-d-1-thiogalactopyranoside (IPTG) and confirmed by SDS-PAGE and western blot. Recombinant α-EpCAM IT was purified from the inclusion bodies and refolded using urea gradient procedure. The cytotoxicity and apoptosis activity of α-EpCAM IT on EpCAM over-expressing (MCF7), low-expressing (HEK293), and no-expressing (HUVEC) cells were evaluated by 3-4,5-Dimethylthiazol-2-yl (MTT) assay and annexin V-FITC-PI assay as well. In addition, anti-tumor activity of α-EpCAM IT was evaluated on nude mice bearing MCF7 tumor cells. Results showed success expression and purification of α-EpCAM IT. The α-EpCAM IT showed time and dose-dependent anti-proliferative activity on MCF-7 cells. However, α-EpCAM IT did not show any anti-proliferative activity on HEK293 and HUVEC cells as well. In addition, the annexin V-FITC-PI assay results showed that α-EpCAM IT significantly increased apoptotic rate in MCF-7 cells with no effect on HEK293 and HUVEC as well. Moreover, α-EpCAM IT significantly reduced tumor size in vivo study. The achieved results indicate the potential of designing α-EpCAM IT as a novel therapeutic for cancer therapy.

Synthesis and characterization of Gd3+-loaded hyaluronic acid-polydopamine nanoparticles as a dual contrast agent for CT and MRI scans.

Magnetic resonance imaging and computed tomography (CT) suffer from low contrast sensitivity and potential toxicity of contrast agents. To overcome these limitations, we developed and tested a new class of dual contrast agents based on polydopamine nanoparticles (PDA-NPs) that are functionalized and targeted with hyaluronic acid (HA). These nanoparticles (NPs) are chelated with Gd3+ to provide suitable contrast. The targeted NPs were characterized through ultraviolet-visible spectroscopy (UV-vis), scanning electron microscopy (SEM), infrared Fourier transform (FTIR), and dynamic light scattering (DLS). The cytotoxicity was investigated on HEK293 cells using an MTT assay. The contrast property of synthesized Gd3+/PDA/HA was compared with Barium sulfate and Dotarem, as commercial contrast agents (CAs) for CT and MRI, respectively. The results illustrated that synthesized PDA-NPs have a spherical morphology and an average diameter of 72 nm. A distinct absorption peak around 280 nm in the UV-vis spectrum reported the self-polymerization of PDA-NPs. The HA coating on PDA-NPs was revealed through a shift in the FTIR peak of C=O from 1618 cm-1 to 1635 cm-1. The Gd3+ adsorption on PDA/HA-NPs was confirmed using an adsorption isotherm assay. The developed CA showed low in vitro toxicity (up to 158.98 µM), and created a similar contrast in MRI and CT when compared to the commercial agents. The r1 value for PDA/HA/Gd3+ (6.5 (mg/ml)-1 s-1) was more than Dotarem (5.6 (mg/ml)-1 s-1) and the results of the hemolysis test showed that at concentrations of 2, 4, 6, and 10 mg/ml, the hemolysis rate of red blood cells is very low. Additionally, the results demonstrated that PDA/HA/Gd3+ could better target the CD44+-expressing cancer cells than PDA/Gd3+. Thus, it can be concluded that lower doses of developed CA are needed to achieve similar contrast of Dotarem, and the developed CA has no safety concerns in terms of hemolysis. The stability of PDA/HA/Gd3+ has also been evaluated by ICP-OES, zeta potential, and DLS during 3 days, and the results suggested that Gd-HA NPs were stable.

Development of Streptococcus equisimilis Group G Mutant Strains with Ability to Produce Low Polydisperse and Low-Molecular-Weight Hyaluronic Acid

Background: Background: Hyaluronic acid (HA), a natural polymer with wide applications in biomedicine and cosmetics, is mainly produced by Streptococcal fermentation at industrial scale. In the present study, chemical random mutagenesis was used for development of Streptococcus equisimilis group G mutant strains with high HA productivity. Methods: Methods: The optimum of the pH of culture condition and cultivation time for HA production by wild strain group G were assessed. At first, two rounds of mutation at different concentrations of NTG was used for mutagenesis. Then, the nonhemolytic and hyaluronidase-negative mutants were screened on the blood and HA agar. HA productivity and molecular weight were determined by carbazole assay, agarose gel electrophoresis and specific staining. Moreover, stability of the high producer mutants was evaluated within 10 generations. Results: Results: The results showed that the wild-type strain produced 1241 ± 2.1 µg/ml of HA at pH 5.5 and 4 hours of cultivation, while the screened mutants showed a 16.1-45.5% increase in HA production. Two mutant strains, named Gm2-120-21-3 (2470 ± 8.1 µg/ml) and Gm2-120-21-4 (2856 ± 4.2 µg/ml), indicated the highest titer and a consistent production. The molecular weight (Mw) of HA for the mutants was less than 160 kDa, considering as a low Mw HA. Conclusion: Conclusion: The mutant strains producing a low polydisperse, as well as low Mw of HA with high titer might be regarded as potential industrial strains for HA production after further safety investigations.

Novel serratiopeptidase exhibits different affinities to the substrates and inhibitors.

The clinical application of serratiopeptidase as an anti-biofilm and anti-inflammatory agent is restricted due to the enzyme sensitivity to the environmental conditions. In our previous study, six enzyme variants were designed by introducing different mutations and truncations that exhibited higher thermal stability. In the present study, the interaction pattern and affinity of variants to substrates and inhibitors were studied using molecular docking and in vitro studies. CABS-dock and Swiss-dock servers were used for substrate (Bradykinin and Substance-P) and inhibitor (Lisinopril and EDTA) docking, respectively. The interactions were analyzed using LigPlot, UCSF Chimera, and visual molecular dynamics packages. Free energy calculations were performed using PRODIGY. Finally, the native enzyme and the best variant in terms of interaction pattern and binding score were selected for in-vitro affinity analysis toward Bradykinin and EDTA using HPLC and casein hydrolysis test, respectively. Molecular docking revealed that T344 [8-339ss] variant showed a different pattern for both substrates and inhibitors in the way that none of the native active site residues were involved in the receptor binding. As revealed by in vitro studies, T344 [8-339ss] displayed the highest number of hydrogen bond formation in docking with Bradykinin and remarkable decrement in the binding affinity for EDTA. This was the first report on the design of novel serratiopeptidase with higher activity to Bradykinin and improved resistance to EDTA as an inhibitor.

Biochemical and Biological Evaluation of an L-Asparaginase from Isolated Escherichia coli MF-107 as an Anti-Tumor Enzyme on MCF7 Cell Line

Background: One of the most widely used anticancer agents is microbial L-ASNase. Herein, we assessed the biochemical and biological properties of an isolated L-ASNase from a Gram-negative bacteria strain, Escherichia coli MF-107. Methods: Using garden asparagus, we obtained several bacterial isolates. These strains were further screened for L-ASNase activity. A promising bacterial isolate was selected for L-ASNase production and subsequent purification. The molecular weight of purified L-ASNase was determined. The MTT assay was applied to assess the cytotoxic effect of the purified enzyme. Also, for caspase activity determination and the apoptotic effect of purified enzyme on in cells, we conducted a real-time PCR method. Results: The molecular weight of the enzyme was approximately 37 kDa. In the pH range of 7.5 to 8, the enzyme had considerable stability. At 35 °C, the purified L-ASNase optimum activity was recorded. The cytotoxic effect of the enzyme on treated cells was dose-dependent with an IC50 value of 5.7 IU/ml. The Bax gene expression considerably raised by 5.75-fold (p < 0.001) upon L-ASNase treatment. On the other hand, the anti-apoptotic Bcl-2 gene expression showed a 2.63-fold increase compared to the control (p < 0.05). It was detected that the mRNA levels of caspase-3 and p53 were considerably upregulated (5.93 and 1.85-fold, respectively). We did not find any alternation in the caspase-8 activity of the treated cells compared to untreated cells. Conclusion: In this research, the proliferation of the breast cancer cells remarkably inhibited via the cytotoxic effect of isolated L-ASNase from microbial sources.

Development of a Multiepitope Vaccine Against SARS-CoV-2: Immunoinformatics Study.

Background: Since the first appearance of SARS-CoV-2 in China in December 2019, the world witnessed the emergence of the SARS-CoV-2 outbreak. Due to the high transmissibility rate of the virus, there is an urgent need to design and develop vaccines against SARS-CoV-2 to prevent more cases affected by the virus. Objective: A computational approach is proposed for vaccine design against the SARS-CoV-2 spike (S) protein, as the key target for neutralizing antibodies, and envelope (E) protein, which contains a conserved sequence feature. Methods: We used previously reported epitopes of S protein detected experimentally and further identified a collection of predicted B-cell and major histocompatibility (MHC) class II-restricted T-cell epitopes derived from E proteins with an identical match to SARS-CoV-2 E protein. Results: The in silico design of our candidate vaccine against the S and E proteins of SARS-CoV-2 demonstrated a high affinity to MHC class II molecules and effective results in immune response simulations. Conclusions: Based on the results of this study, the multiepitope vaccine designed against the S and E proteins of SARS-CoV-2 may be considered as a new, safe, and efficient approach to combatting the COVID-19 pandemic.

Efficacy and antitumor activity of a mutant type of interleukin 2.

Interleukin-2 (IL-2) is an important cytokine in survival, expansion, function of CD8+ T cells and natural killer cells in immunotherapy of melanoma and renal cell carcinomas. Its severe toxicity following binding to its high affinity IL-2 receptor alpha (IL-2Rα) has restricted its application in cancer patients. In the present study, we investigated the antitumor efficacy and cytotoxicity of a mutated human IL-2 previously designed by selective amino acid substitutions, and its reduced affinity towards high-affinity IL-2Rα (CD25) was approved compared to the wild type IL-2 (wtIL-2). Furthermore, their ability to induce PBMC cell proliferation, and interferon-gamma secretion was compared. The mutant IL-2 also represented higher antitumor activity and more efficient cytotoxicity than wild type hIL-2. The developed mutant IL-2 can be an alternative tool in IL-2 associated immunotherapy of various cancers.