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Recently, multi-target directed ligands have been of research interest for multifactorial disorders such as Alzheimer's disease (AD). Since H

Sphingosine 1-phosphate type 1 (S1P1) receptors are expressed on lymphocytes and regulate immune cells trafficking. Sphingosine 1-phosphate and its analogues cause internalization and degradation of S1P1 receptors, preventing the auto reactivity of immune cells in the target tissues. It has been shown that S1P1 receptor agonists such as fingolimod can be suitable candidates for treatment of autoimmune diseases. The current study aimed to generate GRIND-based 3D-QSAR predictive models for agonistic activities of 2-imino-thiazolidin-4-one derivatives on S1P1 to be used in virtual screening of chemical libraries. The developed model for the S1P1 receptor agonists showed appropriate power of predictivity in internal (r2acc 0.93 and SDEC 0.18) and external (r2 0.75 and MAE (95% data), 0.28) validations. The generated model revealed the importance of variables DRY-N1 and DRY-O in the potency and selectivity of these compounds towards S1P1 receptor. To propose potential chemical entities with S1P1 agonistic activity, PubChem chemicals database was searched and the selected compounds were virtually tested for S1P1 receptor agonistic activity using the generated models, which resulted in four potential compounds with high potency and selectivity towards S1P1 receptor. Moreover, the affinities of the identified compounds towards S1P1 receptor were evaluated using molecular dynamics simulations. The results indicated that the binding energies of the compounds were in the range of −39.31 to −46.18 and −3.20 to −9.75 kcal mol−1, calculated by MM-GBSA and MM-PBSA algorithms, respectively. The findings in the current work may be useful for the identification of potent and selective S1P1 receptor agonists with potential use in diseases such as multiple sclerosis., Graphical abstract Image 1, Highlights • A set of S1P1 and S1P3 receptor agonists was used to develop 3D-QSAR predictive models. • The predictivity of the generated models were validated using external and internal validation methods. • PubChem chemicals database was searched for identification of selective S1P1 receptor agonists. • Molecular dynamics simulations were used to calculate ligands binding energies to S1P1 receptor.

Platelet-derived growth factor (PDGF) is a family of growth factors with mitogenic and chemotactic activity. However, uncontrolled and overactivated PDGF signaling has been implicated in a variety of diseases, such as cancers and atherosclerosis. In this context, inhibition of PDGF-PDGFR signaling is of paramount importance in progression of such diseases. The purpose of the current study was to identify novel PDGF-B inhibitors using virtual screening methods. To this end, a combination of molecular modeling techniques such as molecular docking and dynamics simulation, as well as drug likeness filtering criteria, was applied to select anti-PDGF peptidomimetic candidates based on crystallography solved structure of an anti-PDGF-B monoclonal antibody named, MOR8457. In vitro biological assays of the selected compounds revealed two of them being active at micromolar IC50 concentrations. The presented work can provide a framework for systematic peptidomimetic identification for anti-PDGF-B agents from large chemical libraries.

Dry powder inhalers (DPI) are widely used systems for pulmonary delivery of therapeutics. The inhalation performance of DPIs is influenced by formulation features, inhaler device and inhalation pattern. The current review presents the affecting factors with great focus on powder characteristics which include particle size, shape, surface, density, hygroscopicity and crystallinity. The properties of a formulation are greatly influenced by a number of physicochemical factors of drug and added excipients. Since available particle engineering techniques result in particles with a set of modifications, it is difficult to distinguish the effect of an individual feature on powder deposition behavior. This necessitates developing a predictive model capable of describing all influential factors on dry powder inhaler delivery. Therefore, in the current study, a model was constructed to correlate the inhaler device properties, inhalation flow rate, particle characteristics and drug/excipient physicochemical properties with the resultant fine particle fraction. The r2 value of established correlation was 0.74 indicating 86% variability in FPF values is explained by the model with the mean absolute errors of 0.22 for the predicted values. The authors believe that this model is capable of predicting the lung deposition pattern of a formulation with an acceptable precision when the type of inhaler device, inhalation flow rate, physicochemical behavior of active and inactive ingredients and the particle characteristics of DPI formulations are considered.

New compounds containing indanone and carbamate moieties were designed based on the hybrid pharmacophore approach. The designed compounds were synthesized and fully characterized by using different methods such as IR, Mass, 1H NMR, 13C NMR, and HPTLC. In vitro inhibitory activities of all synthesized compounds were evaluated against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The compound 3-[(5,6-dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl) methyl] phenyl (4-methoxyphenyl) carbamate (4h) showed the best inhibitory activities with IC50 values of 1.20 and 0.30 μM against AChE and BChE, respectively. In addition, this compound showed a β-amyloid self-aggregation inhibitory effect (86.8%), stronger than that of donepezil (45.5%). Moreover, significant neuroprotective activity on PC12 cells against H2O2 induced cell death was demonstrated for compound 4h at 10 and 100 µM concentrations. Kinetic studies using the 4h confirmed a partial non-competitive mixed type of inhibition mechanism on both enzymes. Considering the suggested inhibition mechanism, molecular docking was used to predict the possible allosteric binding mode of 4h with both AChE and BChE enzymes. The presented indanone-carbamate scaffold can be structurally modified and optimized through medicinal chemistry-based approaches for designing novel multi-target anti-Alzheimer agents.

Objective A new family of 3′-(Mono, di or tri-substituted phenyl)-4′-(4-(methylsulfonyl) phenyl) spiroisoxazoline derivatives containing indanone spirobridge was designed, synthesized, and evaluated for their selective COX-2 inhibitory potency and cytotoxicity on different cell lines. Methods A synthetic reaction based on 1,3-dipolar cycloaddition mechanism was applied for the regiospecific formation of various spiroisoxazolines. The activity of the newly synthesized compounds was determined using in vitro cyclooxygenase inhibition assay. The toxicity of the compounds was evaluated by MTT assay. In addition, induction of apoptosis, and expression levels of Bax, Bcl-2 and caspase-3 mRNA in MCF-7 cells were evaluated following exposure to compound 9f. The docking calculations and molecular dynamics simulation were performed to study the most probable modes of interactions of compound 9f upon binding to COX-2 enzyme. Results The docking results showed that the synthesized compounds were able to form hydrogen bonds with COX-2 involving methyl sulfonyl, spiroisoxazoline, meta-methoxy and fluoro functional groups. Spiroisoxazoline derivatives containing methoxy group at the C-3′ phenyl ring meta position (9f and 9g) showed superior selectivity with higher potency of inhibiting COX-2 enzyme. Furthermore, compound 9f, which possesses 3,4-dimethoxyphenyl on C-3′ carbon atom of isoxazoline ring, exhibited the highest COX-2 inhibitory activity, and also displayed the most potent cytotoxicity on MCF-7 cells with an IC50 value of 0.03 ± 0.01 µM, comparable with that of doxorubicin (IC50 of 0.062 ± 0.012 µM). The results indicated that compound 9f could promote apoptosis. Also, compared to the control group, the mRNA expression of Bax and caspase-3 significantly increased, while that of Bcl-2 significantly decreased upon exposure to compound 9f which may propose the activation of mitochondrial-associated pathway as the mechanism of observed apoptosis. Conclusion In vitro biological evaluations accompanied with in silico studies revealed that indanone tricyclic spiroisoxazoline derivatives are good candidates for the development of new anti-inflammatory and anticancer (colorectal and breast) agents.

The aim of this study was to identify novel TNF-α blocking peptide(s) using phage display technology. Two novel 7-mer TNF-α binding peptides P51 and P52 with Kd values of 1.47 and 0.51nM were identified. Phage particles displaying P51 and P52 peptides at 0.318nM concentration prevent cytotoxic effect of TNF-α on L929 cells by 8.2% and 16.15%, respectively. Synthesized P51 and P52 peptides also inhibited TNF-α induced cytotoxicity with IC50 values of 25.15±2.18 and 7.08±2.24μM, respectively. The result of RT-PCR also supports the inhibitory activity of the identified peptides, where P51 and P52 significantly inhibit the inductive effect of TNF-α on IκB-α mRNA levels. The inhibitory effects of the peptides were attributed to their abilities of binding at the inter-subunit interfaces leading to TNF-α dissociation. The results of molecular docking studies revealed that the peptides-TNF-α complexes are mostly stabilized by hydrophobic contacts.

Purpose: Robust pharmaceutical and industrial enzymes from extremophile microorganisms are main source of enzymes with tremendous stability under harsh conditions which make them potential tools for commercial and biotechnological applications. Methods: The genome of a Gram-positive halo-thermotolerant Bacillus sp. SL1, new isolate from Saline Lake, was investigated for the presence of genes coding for potentially pharmaceutical enzymes. We determined gene sequences for the enzymes laccase (CotA), l-asparaginase (ansA3, ansA1), glutamate-specific endopeptidase (blaSE), l-arabinose isomerase (araA2), endo-1,4-β mannosidase (gmuG), glutaminase (glsA), pectate lyase (pelA), cellulase (bglC1), aldehyde dehydrogenase (ycbD) and allantoinases (pucH) in the genome of Bacillus sp. SL1. Results: Based on the DNA sequence alignment results, six of the studied enzymes of Bacillus sp. SL-1 showed 100% similarity at the nucleotide level to the same genes of B. licheniformis 14580 demonstrating extensive organizational relationship between these two strains. Despite high similarities between the B. licheniformis and Bacillus sp. SL-1 genomes, there are minor differences in the sequences of some enzyme. Approximately 30% of the enzyme sequences revealed more than 99% identity with some variations in nucleotides leading to amino acid substitution in protein sequences. Conclusion: Molecular characterization of this new isolate provides useful information regarding evolutionary relationship between B. subtilis and B. licheniformis species. Since, the most industrial processes are often performed in harsh conditions, enzymes from such halo-thermotolerant bacteria may provide economically and industrially appealing biocatalysts to be used under specific physicochemical situations in medical, pharmaceutical, chemical and other industries.

The aim of this study is to prepare sulfated carboxymethyl cellulose (SCMC) nanofilter membrane using sulfur trioxide pyridine complex (SO3/pyridine) as sulfating agent and glutaraldehyde (GA) as a crosslinking agent onto polysulfone supporting membrane. The prepared nanofilter was characterized by Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, atomic force microscopy and zeta potential. To evaluate the prepared nanofilter, various amounts of SO3/Pyridine were used and efficiency of them was investigated. The results showed that increasing the sulfate groups raised the flux from 13.87 to 29.54 L/(m2·h−1), whereas percentage rejection was increased during the separation of salt aqueous solutions and then decreased. It can be concluded that, SCMC-GA-2 (with molar ratio of SO3/pyridine to CMC of 1) shows high separation efficiency in acidic conditions and improves the hydrophilicity and charge density of the filter.

Introduction: Nephrotoxicity as a side effect caused by the immunosuppressive drug, cyclosporine-A (CsA), can be a major problem in transplant medicine. Oxidative stress may play an important role in the CsA-induced nephrotoxicity. It has been shown that the antihypertensive drug, valsartan (Val), has also renoprotective effects but, its molecular mechanism is largely unknown. In the present study, it was aimed to evaluate the Val effect in the alleviation of CsA nephrotoxicity via probable renal glutathione peroxidase (GPx) upregulation and oxidative stress decrease. Methods: Thirty-two Sprague-Dawley rats were divided into four groups based on CsA and/or Val administration: group A (Control, 1 mL/kg/day of olive oil as vehicle), group B (CsA, 30 mg/kg/day), group C (CsA+Val, 30+30 mg/kg/day), and group D (Val, 30 mg/kg/day). After the administration period (six weeks), renal GPx expression was evaluated by real-time polymerase chain reaction (PCR). Plasma levels of GPx and 8-Hydroxydeoxyguanosine (8-OHdG) were measured by enzyme-linked immunosorbent assay (ELISA). Malondialdehyde (MDA) and protein carbonyl groups (PCG) were measured by spectrophotometer. Plasma levels of urea and creatinine were measured by an autoanalyzer. Results: CsA treatment led to the decrease in renal expression and plasma levels of GPx in comparison to other study groups. Rats received CsA were detected to have significantly (p

In this study, guinea pig aldehyde oxidase inhibitory activities of flavonoids were investigated using in silico, quantitative structure–activity relationships, molecular modeling, and experimental techniques, in order to understand more about their mode of interactions. The aldehyde oxidase inhibitory activity values determined experimentally in this work or collected from our previous report were used to derive mathematical models for the prediction purposes employing combined genetic algorithm and partial least square method, as well as multiple linear regression analysis. The statistical parameters for the developed models and the results of leave-one-out internal cross-validation were indicative of the validity of the models. To further investigate the mechanism of interaction between flavonoid inhibitors and guinea pig aldehyde oxidase enzyme, the structural model of the enzyme was built and the inhibitors were docked manually into the binding site. The model for quercetin-aldehyde oxidase complex was validated based on its appropriate stability during 10 ns molecular dynamics simulation, and hence the positioning procedure for the rest of flavonoids was guided based on the manually docked position of quercetin. The identified interactions were compared with those of flavonoids previously reported for rat aldehyde oxidase and the results showed a substantial commonality between the modes of interactions predicted for flavonoids positioned into the binding site of aldehyde oxidase from guinea pig and rat. This commonality is also reflected by the quantitative structure–activity relationships models. The results presented in this work may provide useful information where the structural requirements for aldehyde oxidase inhibition are sought, such as designing novel aldehyde oxidase inhibitors or investigating drug interaction involving aldehyde oxidase mediated biotransformation.

Cancer is one of the most important life-threatening diseases in the world. The current efforts to combat cancer are being focused on molecular-targeted therapies. The main purpose of such approaches is based on targeting cancer cell-specific molecules to minimize toxicity for the normal cells. RON (Recepteur d'Origine Nantais) tyrosine kinase receptor is one of the promising targets in cancer-targeted therapy and drug delivery.In this review, we will summarize the available agents against extracellular domain of RON with potential antitumor activities.The presented antibodies and antibody drug conjugates against RON in this review showed wide spectrum of in vitro and in vivo antitumor activities promising the hope for them entering the clinical trials.Due to critical role of extracellular domain of RON in receptor activation, the development of therapeutic agents against this region could lead to fruitful outcome in cancer therapy.

An efficient method was introduced for soluble expression of recombinant laccase (rpCotA(SL-1)) from a newly isolated halo-thermotolerant Bacillus sp. SL-1 in modified Escherichia coli, trxB2/gor2 mutant (Origami™ B (DE3)). The yield of purified soluble laccase in Origami strain under micro-aerobic condition was ∼20mg/L of bacterial culture, showing significant improvement over the laccase produced in E.coli BL21 strain under aerobic condition. The specific activity of 13U/mg for purified laccase produced in micro-aerobic condition was higher than that of 1.07U/mg observed for the purified enzyme obtained in aerobic condition in Origami. The kinetic Km and kcat parameters for laccase-induced oxidation reactions were 46μM and 23s(-1) for ABTS (2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), and 19.6μM and 24s(-1) for SGZ (syringaldazine) substrates, respectively. The rpCotA(SL-1) displayed thermostability at 70°C and tolerance to specified concentrations of NaCl, NaN3, EDTA and SDS as inhibitors. The enzyme was relatively stable in the presence of different concentration of organic solvents, however the residual activity was adversely affected as the dipole moment of the solvents increase. Here we successfully report the production of soluble and functional laccase in Origami at the expression level suitable for industrial application.

Leishmaniasis is a disease caused by a protozoan parasites belonging to the genus Leishmania. It causes morbidity and mortality in the tropical and subtropical regions. Current drugs are toxic, expensive, and require long-term treatment. Thus, identification and development of novel, cheap, efficient, and safe antileishmanial compounds as drug candidates are important from pharmaceutical point of view. Quantitative structure–activity relationship (QSAR) methods are used to predict the pharmaceutically relevant properties of drug candidates whenever it is applicable. The aim of this study was to use two different techniques, namely multiple linear regression (MLR) and artificial neural networks (ANNs) in predicting the antileishmanial activity (i.e. pIC50) of 5-(5-nitroheteroaryl-2-y1)-1,3,4-thiadiazole derivatives. To this end, genetic algorithm-coupled partial least square and backward multiple regression method were used to select a number of calculated molecular descriptors to be used in MLR and ANN-based QSAR studies. The predictive power of the models was also assessed using leave-one-out and leave-group-out cross validation methods. Also, molecular modeling studies were conducted based on DNA topoisomerase I to identify the binding interactions responsible for antileishmanial activity of those analogs. The results suggest that hydrogen bonding interactions and several hydrophobic interactions of ligands with the active site of Leishmania major topoisomerase IB are responsible for their potent antileishmanial activity. These results can be exploited for structure-based computer-aided drug designing of new and selective leishmania topoisomerase inhibitors. Copyright © 2016 John Wiley & Sons, Ltd.

A new series of 3′-(4-substitutedphenyl)-4′-(4-(methylsulfonyl)phenyl) spiroisoxazoline derivatives containing naphthalenone and chromanonespiro-bridge were synthesized for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. A synthetic reaction based on the 1,3-dipolar cycloaddition mechanism was used for the regiospecific formation of various spiroisoxazolines. One of the analogs, i.e., compound 7h, as the representative of the series was recrystallized and characterized structurally by single-crystal X-ray diffraction method. Moreover, the 3D structures of the synthesized compounds were docked into the COX-2 binding site to determine their most probable binding modes once the drug-receptor complexes are formed.

New compounds containing thiazole and pyridinium moieties were designed and synthesized. The potency of the synthesized compounds as selective inhibitors of acetylcholinesterase (AChE), and β-amyloid aggregation (Aβ) was evaluated. Compounds 7d and 7j showed the best AChE inhibitory activities at the submicromolar concentration range (IC50 values of 0.40 and 0.69 μM, respectively). Most of the novel compounds showed moderate to low inhibition of butyrylcholinesterase (BChE), which is indicative of their selective inhibitory effects towards AChE. Kinetic studies using the most potent compounds 7d and 7j confirmed a mixed-type of AChE inhibition mechanism in accordance with the docking results, which shows their interactions with both catalytic active (CAS) and peripheral anionic (PAS) sites. The specific binding of 7a, 7j, and 7m to PAS domain of AChE was also confirmed experimentally. In addition, 7d and 7j were able to show β-amyloid self-aggregation inhibitory effects (20.38 and 42.66% respectively) stronger than donepezil (14.70%) assayed at 10 μM concentration. Moreover, compounds 7j and 7m were shown to be effective neuroprotective agents in H2O2-induced oxidative stress on PC12 cells almost similar to those observed for donepezil. The ability of 7j to pass blood-brain barrier was demonstrated using the PAMPA method. The results presented in this work provide useful information about designing novel anti-Alzheimer agents.

WOS: 000504448700055, In the present study, the design and development of an efficient and green method for the synthesis of dialkyl 4-hydroxy-4H-chromene-2,3-dicarboxylate derivatives together with their biological evaluation are reported. A series of 4H-chromenes were synthesized in the presence of 1-hexyl-3-methylimidazolium bromide ([HMIM]Br) as an environmentally friendly media, without using any organic and toxic solvent and catalyst. The reaction was rapid and was conducted at room temperature with high-to-excellent yields. The antiproliferative potential of the synthesized compounds was evaluated against human lung (A549), breast (MCF-7), and colon (HT-29) cancerous cell lines by adopting MTT method. The tested chromenes showed cytotoxicity in the range of 8.8-82.3% against A549 cells at 200 mu g/mL. Also, chromene derivatives were assessed for tyrosinase and alpha-glucosidase inhibitory activities. Based on IC50 values (2.99-4.39 mM), all chromenes exhibited significant alpha-glucosidase inhibition compared with acarbose (IC50 = 7.90 mM). Furthermore, the ability of the studied compounds to inhibit tyrosinase was evaluated and found to be moderate (IC50 = 3.50-12.20 mM). In silico studies were performed to explore the binding modes of the chromenes at the binding site of alpha-glucosidase and tyrosinase. Molecular docking results revealed the importance of hydrogen bonding, hydrophobic, pi-pi stacking, pi-cation, and metal interactions between the target enzymes and the synthesized compounds. Collectively, the results obtained in the current work indicated that the studied chromenes may be regarded as lead compounds for designing new chemicals potentially effective in conditions such as skin disorders and diabetes mellitus. (C) 2019 Elsevier B.V. All rights reserved., Ministry of Health and Medical Education; Biotechnology Research Center at Tabriz University of Medical Sciences, The authors would like to acknowledge the Ministry of Health and Medical Education, and also, Biotechnology Research Center at Tabriz University of Medical Sciences for the financial support.

A specific and unique sequence probe was designed for detection of donkey adulteration in cooked sausages and its species specificity was confirmed bioinformatically in the common software and website (ClustalX and NCBI). Subsequently, a novel species-specific electrochemical DNA probe (locked nucleic acid, LNA) was synthesized and implemented in a construction of DNA-based electrochemical genosensor for sensitive, convenient and selective detection of donkey adulteration. The electrochemical behavior of the fabricated genosensor was studied by linear sweep, square wave, differential pulse voltammetry and electrochemical impedance spectroscopy techniques. Due to inherent optimal hybridization conditions, the lower limit of quantification (LLOQ) was obtained as 148 pM with a relative standard deviation of 0.16%. Eventually, as a proof of concept, the designed biosensor was successfully used for detection of donkey genetic element in consumable beef sausages preparations, as a real sample. It is predicted that the proposed biosensor will provide a sensitive, inexpensive, fast, and reliable bioassay for application in food analysis, forensic investigations, genetic screening and biodiagnostics. As a prominent feature of this study, the recorded results were confirmed by quantitative real time-polymerase chain reaction (QRT-PCR) as a standard method in adulteration analysis. Our future perspective is minutralization of the development bioassay for making on-desk device and specially merging the designed system by microfluidic systems for accelerating the analysis time.

Since the discovery of the histamine H

A tumor is defined as a group of cancer cells and 'surrounding' stromal bio-entities. Alongside the extracellular matrix (ECM) in the tumor microenvironment (TME), the stromal cells play key roles in cancer affliction and progression. Carcinoma-associated fibroblasts (CAFs) in the area of the tumor, whether activated or not, dictate the future of tumor cells. The CAFs and corresponding secreted growth factors (GFs), which mediate the crosstalk within the TME, can be targeted in therapies directed at the stroma. The impact of the fibroblast growth factor-fibroblast growth factor receptor (FGF-FGFR) signaling pathway in different kinds of tumors has been explored. Several tyrosine kinase inhibitors (TKIs), monoclonal antibodies (mAbs), and ligand traps targeting the formation of FGF-FGFR complex are in preclinical or early development phases. Moreover, there are numerous studies in the literature reporting the application of phage display technology for the development of peptides and proteins capable of functioning as FGF mimetics or traps, which are able to modulate FGF-related signaling pathways. In this review, prominent research in relation to phage display-assisted ligand identification for the FGF/FGFR system is discussed.

Aldehyde oxidase (AOX) is an important molybdenum-containing enzyme with high similarity with xanthine oxidase (XO). AOX involved in the metabolism of a large array of aldehydes and N-heterocyclic compounds and its activity is highly substrate-dependent. The aim of this work was to study the effect of five important phenothiazine drugs on AOX activity using benzaldehyde and phenanthridine as aldehyde and N-heterocyclic substrates, respectively. The effect of trifluperazine, chlorpromazine, perphenazine, thioridazine and promethazine on rat liver AOX was measured spectrophotometrically. To predict the mode of interactions between the studied compounds and AOX, a combination of homology modeling and a molecular docking study was performed. All phenothiazines could inhibit AOX activity measured either by phenanthridine or benzaldehyde with almost no effect on XO activity. In the case of benzaldehyde oxidation, the lowest and highest half-maximal inhibitory concentration (IC50) values were obtained for promethazine (IC50 = 0.9 µM), and trifluoperazine (IC50 = 3.9 µM), respectively; whereas perphenazine (IC50 = 4.3 µM), and trifluoperazine (IC50 = 49.6 µM) showed the strongest and weakest inhibitory activity against AOX-catalyzed phenanthridine oxidation, respectively. The in silico findings revealed that the binding site of thioridazine is near the dimer interference, and that hydrophobic interactions are of great importance in all the tested phenothiazines. The five studied phenothiazine drugs showed dual inhibitory effects on AOX activity towards aldehydes and N-heterocycles as two major classes of enzyme substrates. Most of the interactions between the phenothiazine-related drugs and AOX in the binding pocket showed a hydrophobic nature.

Empagliflozin, a SGLT-2 inhibitor, improves diabetic nephropathy through its pleiotropic anti-inflammatory effects. The present study aims to evaluate empagliflozin effects on renal and urinary levels of tubular epithelial cell injury markers in streptozotocin-induced diabetic rats. Empagliflozin at 10 mg/kg (p.o.) was administered for 4 weeks, beginning 8 weeks after induction of diabetes. Renal function as well as markers of renal tubular epithelial cell injury were assessed in kidney tissue homogenates and urine. Empagliflozin was able to ameliorate diabetes induced elevations in serum cystatin C levels. It also alleviated renal KIM-1/NGAL levels and urinary albumin, α-GST, and RBP excretions. In addition to decreasing urinary levels of cell cycle arrest indices i.e. TIMP-2 and IGFBP7, empagliflozin mitigated acetylated NF-κB levels in renal tissues of diabetic rats. As a whole, these findings reveal empagliflozin capability in improving diabetic nephropathy via ameliorating indices of renal inflammation, injury, and cell cycle arrest on streptozotocin-induced diabetic rats.

Carboxylated carbon porous adsorbent was derived from zeolite imidazole framework (ZIF-8) via carbonization of ZIF-8 under a nitrogen atmosphere. The synthesized carboxylated adsorbent was fully characterized by various techniques including Fourier transform spectroscopy (FTIR), powder X-ray diffraction (XRD), scanning electron microscopy (SEM), and zeta potential analysis. The carboxylated adsorbent was applied as dispersive solid phase extraction (DSPE) adsorbent for efficient extraction of methamphetamine (MET) from biological urine samples. Several extraction parameters influencing the extraction efficiency were investigated and the calibration curve was plotted under optimized conditions in urine media. The method showed a good linearity in the range of 50–2500 ng/mL. The limit of detection (LOD) and limit of quantification (LOQ) was 10 and 35.80 ng/mL, respectively. A satisfactory analysis of the positive real samples with the recovery of 99.83% confirms the applicability of the proposed method in different clinical and forensic laboratories.

Introduction: The bacterial enzyme has gained more attention in therapeutic application because of the higher substrate specificity and longer half-life. L-asparaginase is an important enzyme with known antineoplastic effect against acute lymphoblastic leukemia (ALL). Methods: Novel L-asparaginase genes were identified from a locally isolated halo-thermotolerant Bacillus strain and the recombinant enzymes were overexpressed in modified E. coli strains, OrigamiTM B and BL21. In addition, the biochemical properties of the purified enzymes were characterized, and the enzyme activity was evaluated at different temperatures, pH, and substrate concentrations. Results: The concentration of pure soluble enzyme obtained from Origami strain was ~30 mg/L of bacterial culture, which indicates the significant improvement compared to L-asparaginase produced by E. coli BL21 strain. The catalytic activity assay on the identified L-asparaginases (ansA1 and ansA3 genes) from Bacillus sp. SL-1 demonstrated that only ansA1 gene codes an active and stable homologue (ASPase A1) with high substrate affinity toward L-asparagine. The Kcat and Km values for the purified ASPase A1 enzyme were 23.96s-1 and 10.66 µM, respectively. In addition, the recombinant ASPase A1 enzyme from Bacillus sp. SL-1 possessed higher specificity to L-asparagine than L-glutamine. The ASPase A1 enzyme was highly thermostable and resistant to the wide range of pH 4.5–10. Conclusion: The biochemical properties of the novel ASPase A1 derived from Bacillus sp. SL-l indicated a great potential for the identified enzyme in pharmaceutical and industrial applications.

Purpose: TNF-α is an inflammatory cytokine with a key role in initiation of inflammatory responses. Anti-TNF-α antibodies are being used in clinic for the purpose of diagnosis and treatment due to their high specificity. The objective of the current study was to express and purify an anti-TNF-α scFv antibody identified by phage display technology. Methods: The DNA coding sequence of the identified scFv was cloned into pET28a vector and the corresponding protein was expressed as 6×His tagged using E.coli BL21 (DE3) pLysS expression system followed by affinity purification on Ni-Sepharose affinity column. Results: The J44 scFv antibody was cloned into the expression vector and successfully expressed and purified. The purity of the scFv fraction was confirmed using SDS-PAGE analysis. Western blotting technique was used to detect expression of 6×His tagged protein. Conclusion: In the current study an anti-TNF-α scFv antibody was successfully expressed in bacterial expression system and purified on affinity column. The purified protein can be used in different in vitro and in vivo experiments in order to elucidate its functionality.

Purpose: Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine, involved in both physiological and pathological pathways. Because of central role of TNF-α in pathogenesis of inflammatory diseases, in the current study, we aimed to identify novel scFv antibodies against TNF-α using phage display technology. Methods: Using libraries composed of phagemid displaying scFv antibodies, four rounds of biopanning against TNF-α were carried out, which led to identification of scFvs capable of binding to TNF-α. The scFv antibody with appropriate binding affinity towards TNF-α, was amplified and used in ELISA experiment. Results: Titration of phage achieved from different rounds of biopanning showed an enrichment of specific anti-TNF-α phages during biopanning process. Using ELISA experiment, a binding constant (Kd) of 1.11 ± 0.32 nM was determined for the phage displaying J48 scFv antibody. Conclusion: The findings in the current work revealed that the identified novel scFv antibody displayed at the N-terminal of minor coat proteins of phagemid binds TNF-α with suitable affinity. However, the soluble form of the antibody is needed to be produced and evaluated in more details regarding its binding properties to TNF-α.

 Single-chain antibodies  Recombinant protein expression  Inclusion bodies  Growth optimization  Escherichia coli  Tumor necrosis factor-alpha Abstract Purpose: The bacterial cultivation conditions for obtaining anti-TNF-α single chain variable fragment (scFv) antibody as the soluble product in E. coli was investigated. Methods: To avoid the production of inclusion bodies, the effects of lactose, IPTG, incubation time, temperature, shaking protocol, medium additives (Mg +2 , sucrose), pH, osmotic and heat shocks were examined. Samples from bacterial growth conditions with promising results of soluble expression of GST-hD2 scFv were affinity purified and quantified by SDS-PAGE and image processing for further evaluation. Results: The results showed that cultivation in LB medium under induction by low concentrations of lactose and incubation at 10 °C led to partial solubilization of the expressed anti-TNF-α scFv (GST-hD2). Other variables which showed promising increase in soluble expression of GST-hD2 were osmotic shock and addition of magnesium chloride. Furthermore, addition of sucrose to medium suppressed the expression of scFv completely. The other finding was that the addition of sorbitol decreased the growth rate of bacteria. Conclusion: It can be concluded that low cultivation temperature in the presence of low amount of inducer under a long incubation time or addition of magnesium chloride are the most effective environmental factors studied for obtaining the maximum solubilization of GST-hD2 recombinant protein.

Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine, involved in both physiological and pathological pathways. Although there have been various attempts to express and purify human TNF-α, the current work introduces a simple, rapid, and efficient method for its production without loss of biological activity. The protein was expressed based on GST-tagged fusion system in Escherichia coli under optimized condition. The expressed GST fusion protein was applied to glutathione affinity column and then, TNF-α was cleaved off the GST using thrombin protease. The purity of the product was more than 95% and further size exclusion chromatography slightly improved the purity. The purified human TNF-α was tested for its biological activity and structural analysis, using MTT assay (EC(50) of 4.1 ×10E-12 M in L929 cell death assay) and circular dichroism spectropolarimetry, respectively. The results showed that the method used in this study enables successful production of highly purified and fully functional TNF-α.

G protein-coupled receptors (GPCRs) constitute the largest superfamily of integral membrane proteins (IMPs) and they tremendously contribute in the flow of information into cells. In this study, the random forest (RF) and the subtractive fuzzy c-means clustering (SBC) methods have been used to determine the importance of input variables and discriminate GPCRs from non-GPCRs using twenty amino acid and fifty pseudo amino acid compositions derived from GPCR sequences. The studied dataset was retrieved from the UniProt/SWISSPROT database and consists of 1000 GPCR and 1000 non-GPCR reviewed sequences. The top ranked RF-SBC-based model discriminates GPCRs and non-GPCRs successfully with the accuracy, sensitivity, specificity and Matthew's coefficient correlation (MCC) rates of 99.15%, 99.60%, 98.70% and 0.983%, respectively. These rates were obtained from averaged values of 5-fold cross validation using only twenty four out of fifty pseudo amino acid composition features. The results show that the proposed RF-SBC-based model outperforms other existing algorithms in terms of the evaluated performance criteria. The webserver for the proposed algorithm is available at http://brcinfo.shinyapps.io/GPCRIden.

Influenza is a highly contagious respiratory tract disease and is considered a serious community health problem. Influenza viruses possess multiple conserved epitopes which are used for designing universal vaccines. To this aim, the gene coding for N-terminal part of M2e (SLLTEVET) and HA2 (GLFGAIAGF), was synthesized, linked by a (Gly4Ser)4 peptide linker, and cloned into pGS-21a vector. Afterwards, the construct was transferred into E. coli BL21 (DE3) cells to produce the designed antigenic protein called M2e-HA2. Immunization of mice with these peptides significantly induced humoral immune responses against the influenza virus. Three weeks after the last booster, mice were inoculated intranasally with 1 × 106 EID50 of H9N2 virus. The results indicated that the recombinant M2e-HA2 fusion protein could protect mice against H9N2 virus.

Fibroblast growth factor 7 (FGF7) is a member of the fibroblast growth factor (FGF) family of proteins. FGF7 is of stromal origin and produces a paracrine effect on epithelial cells. In the current investigation, we aimed to identify new single-domain antibodies (sdAbs) against FGF7 using phage display technology. The vector harboring the codon-optimized DNA sequence for FGF7 protein was transformed into Escherichia coli BL21 (DE3) pLysS, and then the protein was expressed at the optimized condition. Enzyme-linked immunosorbent assay, circular dichroism spectropolarimetry, and in vitro scratch assay experiments were used to confirm the proper folding and functionality of the purified FGF7 protein. The purity of the produced FGF7 was 92%, with production yield of 3.5 mg/L of culture. Panning against the purified FGF7 was performed, and the identified single-domain antibodies showed significant affinity. Further investigation on one of the selected sdAb displaying phage clones showed concentration-dependent binding to FGF7. The selected sdAb can be used for developing novel tumor-suppressing agents where inhibition of FGF7 is required.

Currently, modified Fe3O4 magnetic nanoparticles frequently are used as nanocarriers for proteins and enzymes purification. In the present study, Fe3O4 magnetic nanoparticles were prepared, and their surfaces were modified with p-aminophenol affinity ligand immobilized by different linkers. The modified nanocarriers were used for the purification of α-amylase from the bovine milk (after precipitation the casein) by affinity purification. To evaluate the effectiveness of the p-aminophenol modified magnetic nanocarriers, the three different types of nanocarriers with different linkers having varying lengths were prepared. All nanocarriers were characterized and validated using FT-IR, SEM, EDX, VSM and XRD analysis methods According to our results, p-aminophenol ligand attached to the nanocarrier by long linkers better separates the α-amylase from the casein free skim milk with 49.66% recovery and 48.18-fold purification efficiency. The results of this study showed that our novel magnetic nanocarriers have the capacity to be used for fast, reproducible and cost-effective purification of α-amylase.

BACKGROUND Nephrotoxicity side effect of the immunosuppressive drug, cyclosporine A (CsA), can be a major issue in transplantation medicine. Cyclosporine A-induced nephrotoxicity is multifactorial but oxidative stress has a critical role in this process. It has been demonstrated that Valsartan (Val) as an angiotensin receptor blocker has renoprotective effects, but the molecular mechanisms responsible for the renal protection, independent from its blood pressure lowering effect, have not yet been fully understood. The present study is aim at evaluating the Val effect in alleviation of CsA nephrotoxicity by probable increase in renal Klotho expression and/or reducing oxidative stress. METHODS Thirty-two Sprague-Dawley rats were divided into 4 groups based on the administration of CsA and/or Val: group A (control, 1 mL/kg per day of olive oil as vehicle), group B (CsA, 30 mg/kg per day), group C (CsA + Val, 30 + 30 mg/kg per day), and group D (Val, 30 mg/kg per day). Real-time polymerase chain reaction and Western blotting were used to evaluate Renal Klotho expression. Serum Klotho level was measured by enzyme-linked immunosorbent assay. 8-Hydroxy-deoxy guanosine and malondialdehyde levels as markers of oxidative stress were measured by enzyme-linked immunosorbent assay and spectrophotometrically, respectively. RESULTS Cyclosporine A treatment reduced renal expression and serum levels of Klotho, improved malondialdehyde and 8-hydroxy-deoxy guanosine levels, and also deteriorated renal function. Valsartan prevented CsA-induced oxidative stress as well as Klotho downregulation and could alleviate CsA-induced renal histological changes and function. CONCLUSIONS Administration of Val might lead to amelioration of CsA nephrotoxicity by probably diminishing CsA-induced renal Klotho downregulation and oxidative stress.

DNA is a major target for a number of anticancer substances. Interaction studies between small molecules and DNA are essential for rational drug designing to influence main biological processes and also introducing new probes for the assay of DNA. Tschimgine (TMG) is a monoterpene derivative with anticancer properties. In the present study we tried to elucidate the interaction of TMG with calf thymus DNA (CT-DNA) using different spectroscopic methods. UV-visible absorption spectrophotometry, fluorescence and circular dichroism (CD) spectroscopies as well as molecular docking study revealed formation of complex between TMG and CT-DNA. Binding constant (Kb) between TMG and DNA was 2.27×104M-1, that is comparable to groove binding agents. The fluorescence spectroscopic data revealed that the quenching mechanism of fluorescence of TMG by CT-DNA is static quenching. Thermodynamic parameters (ΔH

CD24 is a heavy glycosylated protein that is overexpressed in numerous cancer and cancer stem cells. It is involved in the development, invasion, and metastasis of the cancer cells. CD24 can be considered in targeted cancer therapy as a new target. Here, phage display technology was used for the identification of novel scFv antibodies against CD24. To do so, the CD24 protein was expressed and purified from a stable transgenic CHO cell line. The cells were developed by using a CD24 encoding construct, which targets the 18S rRNA gene. The recombinant CD24 was used in the biopanning process. Four rounds of biopanning were performed with the Tomlinson J library. The polyclonal phage ELISA and DNA sequencing of the selected colonies confirmed that specific binders have been enriched during the biopanning process. Based on the DNA sequencing results, three phage-displaying scFv were obtained and their specificity to CD24 was verified in the monoclonal phage ELISA. The results showed that clone 3 (called Jd3) has the highest affinity to CD24 and also is the most abundant clone (80%) among the three clones. Therefore, Jd3 was selected for further analyses. In order to produce soluble Jd3-scFv, the phage was transfected to E. coli BL21 pLysS. Soluble Jd3 scFv was expressed and purified successfully. The soluble Jd3 scFv showed appropriate affinity to recombinant CD24 in the ELISA analysis. Also, the immunocytochemistry experiment showed that the purified Jd3 scFv can bind to the CD24 expressing A549 cells. So, the scFv may be useful in the targeted delivery of drugs or diagnostic nanoparticles to the CD24 expressing cancer cells. To the best of our knowledge, this study is the first report to identify an scFv antibody against CD24, using the phage display method.

Todays, after four decades from the discovery of monoclonal antibodies by Kohler and Milstein in 1975, a dozen of antibodies are used in cancer targeted therapy with different strategies. The success of these antibodies depends on the specificity of antigens expressed on the cancer cells. Epithelial Cell Adhesion Molecule (EpCAM), a homophilic cell-cell adhesion glycoprotein is a well- known tumor antigen, which expresses on epithelial tumors and circulating tumor cells as well as cancer stem cells. The EpCAM signaling pathway is associated with proliferation, differentiation and adhesion of epithelial cancer cells. Here we review EpCAM structure, expression profile and its signaling pathway in cancer cells. In addition, we focused on structure, mechanism of action and success of anti EpCAM antibodies which have been used in different clinical trials. Based on literatures, Edrecolomab showed limited efficacy in the phase III studies. The wholly human monoclonal antibody Adecatumumab is dose- and target-dependent in metastatic breast cancer patients expressing EpCAM. The chimeric antibody, Catumaxomab, has been approved for the treatment of malignant ascites; however, this Mab showed considerable results in intrapleural administration in cancer patients. Anti EpCAM toxin conjugated antibodies like, Oportuzumab Monatox (scFv antibody and Pseudomonas exotoxin A (ETA)), Citatuzumab Bogatox (Fab fragment with bouganin toxin) and immono-conjugate antibody Tucotuzumab (monoclonal antibody with IL2), have shown acceptable results in different clinical trials. Almost, all of the antibodies were well-tolerated; however, still more clinical trials are needed for the approval of antibodies for the treatment of specific tumors.

RON (Recepteur d’Origine Nantais) tyrosine kinase receptor is a promising target for therapeutic intervention in cancer therapy. The aim of this work was identification of RON-binding peptides using phage display and computational modeling their mode of binding. A 12-mer peptide phage library was utilized to perform biopanning against RON. The RON-binding ability of the selected peptide-displaying phage and their possible binding sites were examined by ELISA. Binding modes and affinities were also predicted by docking and molecular dynamics (MD) simulation. The results of ELISA experiment showed that P6 peptide displaying phage has higher affinity for RON compared to others and its binding site is located out of ligand binding site. Docking and MD simulation results also indicated higher affinity of P6 to RON as well as its exosite-binding feature. Taken together, our data suggest a capacity for P6 peptide (FEHSLYKEMTHL) to be utilized as RON binding agent, and hence be used for various purposes, including design of drug delivery systems for transferring cytotoxic agents to RON-positive cancer cells, interfering with RON signaling, peptidomimetics design, and diagnostic imaging.

CD24 (cluster of differentiation 24) is a small heavy glycosylated protein, which is overexpressed in many cancer and some cancer stem cells and is associated with the development, invasion, and metastasis of cancer cells. The exact role of CD24 in these processes is not fully understood, however, in this article, it has been tried to present a collection of cancer-related mechanisms attributed to CD24. Based on the literature, CD24 dis-regulates different signaling pathways in various cancer cells, including; Src kinases, STAT3, EGFR, Wnt/β-catenin and MAPK. Src kinases play an important role in the signaling pathways which activate p38 MAPK and STAT3 pathways. Akt and ERK are downstream effectors of CD24-activated EGFR, which promote cell proliferation, invasion and metastasis. CD24 increases the expression of HER2 by the activation of NF-κB transcription factor. Moreover, CD24 up-regulates the expression of miR-21 oncomir through the activation of Src kinases. Identification of the details of these pathways and also new pathways will help researchers to explore new CD24 targeted therapies.

Sphingosine 1-phosphate type 1 (S1P1) receptors are expressed on lymphocytes and regulate immune cells trafficking. Sphingosine 1-phosphate and its analogues cause internalization and degradation of S1P1 receptors, preventing the auto reactivity of immune cells in the target tissues. It has been shown that S1P1 receptor agonists such as fingolimod can be suitable candidates for treatment of autoimmune diseases. The current study aimed to generate GRIND-based 3D-QSAR predictive models for agonistic activities of 2-imino-thiazolidin-4-one derivatives on S1P1 to be used in virtual screening of chemical libraries. The developed model for the S1P1 receptor agonists showed appropriate power of predictivity in internal (r2acc 0.93 and SDEC 0.18) and external (r2 0.75 and MAE (95% data), 0.28) validations. The generated model revealed the importance of variables DRY-N1 and DRY-O in the potency and selectivity of these compounds towards S1P1 receptor. To propose potential chemical entities with S1P1 agonistic activity, PubChem chemicals database was searched and the selected compounds were virtually tested for S1P1 receptor agonistic activity using the generated models, which resulted in four potential compounds with high potency and selectivity towards S1P1 receptor. Moreover, the affinities of the identified compounds towards S1P1 receptor were evaluated using molecular dynamics simulations. The results indicated that the binding energies of the compounds were in the range of −39.31 to −46.18 and −3.20 to −9.75 kcal mol−1, calculated by MM-GBSA and MM-PBSA algorithms, respectively. The findings in the current work may be useful for the identification of potent and selective S1P1 receptor agonists with potential use in diseases such as multiple sclerosis.