Molecular Modeling and Optimization of Type II E.coli l-Asparginase Activity by in silico Design and in vitro Site-directed Mutagenesis.

INTRODUCTION: L-asparaginase (also known as L-ASNase) is a crucial therapeutic enzyme that is widely used in treatment of ALL (acute lymphoblastic leukemia) as a chemotherapeutic drug. Besides, this enzyme is used in the food industry as a food processing reagent to reduce the content of acrylamide in addition to the clinical industry. The improvement of activity and kinetic parameters of the L-ASNase enzyme may lead to higher efficiency resulting in practical achievement. In order to achieve this goal, we chosen glycine residue in position 88 as a potential mutation with advantageous outcomes. METHOD: In this study, firstly to find the appropriate mutation on glycine 88, various in silico analyses, such as MD simulation and molecular docking, were carried out. Then, the rational design was adopted as the best strategy for molecular modifications of the enzyme to improve its enzymatic properties. RESULT: Our in silico findings show that the four mutations G88Q, G88L, G88K, and G88A may be able to increase L-ASNase's asparaginase activity. The catalytic efficiency of each enzyme (kcat/Km) is the most important feature for comparing the catalytic activity of the mutants with the wild type form. The laboratory experiments showed that the kcat/Km for the G88Q mutant is 36.32% higher than the Escherichia coli K12 ASNase II (wild type), which suggests that L-ASNase activity is improved at lower concentration of L-ASN. Kinetic characterization of the mutants L-ASNase activity confirmed the high turnover rate (kcat) with ASN as substrate relative to the wild type enzyme. CONCLUSION: In silico analyses and laboratory experiments demonstrated that the G88Q mutation rather than other mutation (G88L, G88K, and G88A) could improve the kinetics of L-ASNase.

Impact of SNPs, off-targets, and passive permeability on efficacy of BCL6 degrading drugs assigned by virtual screening and 3D-QSAR approach.

B-cell lymphoma 6 (BCL6) regulates various genes and is reported to be overexpressed in lymphomas and other malignancies. Thus, BCL6 inhibition or its tagging for degradation would be an amenable therapeutic approach. A library of 2500 approved drugs was employed to find BCL6 inhibitory molecules via virtual screening. Moreover, the 3D core structure of 170 BCL6 inhibitors was used to build a 3D QSAR model and predict the biological activity. The SNP database was analyzed to study the impact on the destabilization of BCL6/drug interactions. Structural similarity search and molecular docking analyses were used to assess the interaction between possible off-targets and BCL6 inhibitors. The tendency of drugs for passive membrane permeability was also analyzed. Lifitegrast (DB11611) had favorable binding properties and biological activity compared to the BI-3802. Missense SNPs were located at the essential interaction sites of the BCL6. Structural similarity search resulted in five BTB-domain containing off-target proteins. BI-3802 and Lifitegrast had similar chemical behavior and binding properties against off-target candidates. More interestingly, the binding affinity of BI-3802 (against off-targets) was higher than Lifitegrast. Energetically, Lifitegrast was less favorable for passive membrane permeability. The interaction between BCL6 and BI-3802 is more prone to SNP-derived variations. On the other hand, higher nonspecific binding of BI-3802 to off-target proteins could bring about higher undesirable properties. It should also be noted that energetically less desirable passive membrane translocation of Lifitegrast would demand drug delivery vehicles. However, further empirical evaluation of Lifitegrast would unveil its true potential.

Evaluating the Performance of PPE44, HSPX, ESAT-6 and CFP-10 Factors in Tuberculosis Subunit Vaccines.

Mycobacterium tuberculosis (M. tuberculosis) is an intracellular pathogen causing long-term infection in humans that mainly attacks macrophages and can escape from the immune system with the various mechanisms. The only FDA-approved vaccine against M. tuberculosis (MTB) is Mycobacterium bovis bacillus Calmette-Guérin (BCG). The protection of this vaccine typically lasts 10-15 years. Due to the increasing number of people becoming ill with MTB each year worldwide, the need to develop a new effective treatment against the disease has been increased. During the past two decades, the research budget for TB vaccine has quadrupled to over half a billion dollars. Most of these research projects were based on amplifying and stimulating the response of T-cells and developing the subunit vaccines. Additionally, these studies have demonstrated that secretory and immunogenic proteins of MTB play a key role in the pathogenesis of the bacteria. Therefore, these proteins were used to develop the new subunit vaccines. In this review, based on the use of these proteins in the successful new subunit vaccines, the PPE44, HSPX, CFP-10 and ESAT-6 antigens were selected and the role of these antigens in designing and developing new subunit vaccines against TB and for the prevention of TB were investigated.

Prediction of Genes Involved in Lung Cancer with a Systems Biology Approach Based on Comprehensive Gene Information.

Over the past few years, hundreds of genes have been reported in relation to lung cancer. Systems biology studies can help validate this association and find the most valid genes to use in the diagnosis and treatment. We reviewed the candidate genes for lung cancer in 120 published articles from September 1, 1993, to September 1, 2020. We obtained 134 up- and 36 downregulated genes for lung cancer in this article. The genes extracted from the articles were imported to Search Tool for the Retrieval of Interacting genes/proteins (STRING) to construct the protein-protein interaction (PPI) Network and pathway enrichment. GO ontology and Reactome databases were used for describing the genes, average length of survival, and constructing networks. Then, the ClusterONE plugin of Cytoscape software was used to analyze and cluster networks. Hubs and bottleneck nodes were defined based on their degree and betweenness. Common genes between the ClusterONE plugin and network analysis consisted of seven genes (BRCA1-TP53-CASP3-PLK1-VEGFA-MDM2-CCNB1 and PLK1), and two genes (PLK1 and TYMS) were selected as survival factors. Our drug-gene network showed that CASP3, BRCA1, TP53, VEGFA, and MDM2 are common genes that are involved in this network. Also, among the drugs recognized in the drug-gene network, five drugs such as paclitaxel, oxaliplatin, carboplatin, irinotecan, and cisplatin were examined in different studies. It seems that these seven genes, with further studies and confirmatory tests, could be potential markers for lung cancer, especially PLK1 that has a significant effect on the survival of patients. We provide the novel genes into the pathogenesis of lung cancer, and we introduced new potential biomarkers for this malignancy.

Engineering of Cytolethal Distending Toxin B by Its Reducing Immunogenicity and Maintaining Stability as a New Drug Candidate for Tumor Therapy; an In Silico Study.

The cytolethal distending toxin (CDT), Haemophilus ducreyi, is one of the bacterial toxins that have recently been considered for targeted therapies, especially in cancer therapies. CDT is an A-B2 exotoxin. Its catalytic subunit (CdtB) is capable of inducing DNA double strand breaks, cell cycle arrest and apoptosis in host eukaryotic cells. The sequence alignment indicates that the CdtB is structurally homologyr to phosphatases and deoxyribonucleases I (DNase I). Recently, it has been found that CdtB toxicity is mainly related to its nuclease activity. The immunogenicity of CDT can reduce its effectiveness in targeted therapies. However, the toxin can be very useful if its immunogenicity is significantly reduced. Detecting hotspot ectopic residues by computational servers and then mutating them to eliminate B-cell epitopes is a promising approach to reduce the immunogenicity of foreign protein-based therapeutics. By the mentioned method, in this study, we try to reduce the immunogenicity of the CdtB- protein sequence. This study initially screened residue of the CdtB is B-cell epitopes both linearly and conformationally. By overlapping the B-cell epitopes with the excluded conserve residues, and active and enzymatic sites, four residues were allowed to be mutated. There were two mutein options that show reduced antigenicity probability. Option one was N19F, G74I, and S161F with a VaxiJen score of 0.45 and the immune epitope database (IEDB) score of 1.80, and option two was N19F, G74I, and S161W with a VaxiJen score of 0.45 and IEDB score of 1.88. The 3D structure of the proposed sequences was evaluated and refined. The structural stability of native and mutant proteins was accessed through molecular dynamic simulation. The results showed that the mutations in the mutants caused no considerable changes in their structural stability. However, mutant 1 reveals more thermodynamic stability during the simulation. The applied approaches in this study can be used as rough guidelines for finding hot spot immunogen regions in the therapeutic proteins. Our results provide a new version of CdtB that, due to reduced immunogenicity and increased stability, can be used in toxin-based drugs such as immunotoxins.

Genetic Diversity of ctxB Gene Among Classical O1 and El Tor Strains of Vibrio cholerae using High-Resolution Melting Curve Analysis.

BACKGROUND & OBJECTIVE: Vibrio cholerae is a natural inhabitant of the environment and causes severe diarrhea ailments (cholera) that affects thousands of people each year worldwide. The most important virulence factors of this pathogen are cholera toxin (cholera toxin CT) and Type IV pili (toxin co-regulated pili TCP), which are encoded within the genome of the filamentous bacteriophage CTXφ. In the present study, according to researchers' report on genotypic variations of cholera toxin, we evaluated the sequence of ctxB subunit obtained from 100 strains of patients infected with cholera in Iran. METHODS: The evaluation of genotype variations of cholera toxin was made by high-resolution melting curve analysis illustrating a single nucleotide change. Then, ctxB gene sequencing was performed. Through this analysis and the sequencing process, two standard samples were studied. RESULTS: Using serologic tests, all the strains analyzed in this study were identified to be in O1 serotype. However, there have been differences in sequences of ctxB as some were similar to V . cholerae O1 biovar El Tor str. N16961 while others were similar to the genotype of V . cholerae ATCC 14035. We did not observe any particular pattern within the process of mutation. CONCLUSION: The analysis of the new samples of ctxB showed that they were potentially different. It seems that these complicated species were affected by a new genetic exchange of El Tor and classic genotypes.

CdS nanocrystals/graphene oxide-AuNPs based electrochemiluminescence immunosensor in sensitive quantification of a cancer biomarker: p53.

An ultrahigh sensitive, simple and reliable Electrochemiluminescence (ECL) immunosensor for selective quantification of p53 protein was designed according to the enhancement effects of AuNPs on ECL emission of CdS nanocrystals (CdS NCs). CdS NCs were immobilized on the glassy carbon electrode and AuNPs introduced to the process through formation of a sandwich-type immunocomplex between first anti-p53/p53/ secondary anti-p53. ECL of CdS NCs firstly evoked the SPR of AuNPs which in return amplified the CdS NCs ECL intensity. By using graphene oxide in immunosensor fabrication procedure, and attaching more AuNPs on the surface of the electrode, the ECL intensity was further increased resulting in much higher sensitivity. After applying the optimum conditions, the linear range of the developed immunosensor was found between 20 and 1000 fg/ml with a calculated limit of detection of 4 fg/ml. Moreover, the interference, reproducibility and storage stability studies of the immunosensor were investigated. Finally, immunosensor's authenticity was evaluated by detecting the p53 protein in human spikes which offers it as a potential in early detection of cancer, monitoring the cancer progress and clinical prognosis.