Design, synthesis, in vitro and in silico evaluations of new isatin-triazine- aniline hybrids as potent anti- Alzheimer multi-target directed lead compounds.

Multi target directed ligands (MTDLs) are one of the promising tools for treatment of complex disease like Alzheimer's disease (AD). In this study, using rational design, we synthesized new 15 hybrids of the s-triazine, isatin and aniline derivatives as anti- AD compounds. The design was as way as that new compounds could had anti cholinesterase (ChE), antioxidant and biometal chelation ability. In vitro biological evaluation against ChE enzymes showed that these molecules were excellent inhibitors with IC50 values ranging from 0.2 nM to 734.5 nM for acetylcholinesterase (AChE), and 0.02 µM to 1.92 µM for butyrylcholinesterase (BChE). Among these compounds, 8 l with IC50 AChE = 0.7 nM, IC50 BChE = 0.09 µM and 8n with IC50 AChE = 0.2 nM, IC50 BChE = 0.03 µM were the most potent compounds. In silico studies showed that these molecules had key and effective interactions with the corresponding enzymes residues. The molecules with hydroxyl group on aniline moiety had also good antioxidant activity with EC50 values ranging from 64.2 µM to 103.6 µM. The UV-Vis spectroscopy study revealed that molecule 8n was also able to chelate biometals such as Zn2+, Cu2+and Fe2+ properly. It was concluded that these molecules could be excellent lead compounds for future studies.

Construction of a bacteriophage-derived vector with potential applications in targeted drug delivery and cell imaging.

There is a strong relationship between the dysregulation of epidermal growth factor receptor (EGFR) and the development of epithelial-derived cancers. Therefore, EGFR has usually been considered the desired target for gene therapy. Here, we propose an approach for targeting EGFR-expressing cells by phage particles capable of displaying EGF and GFP as tumor-targeting and reporting elements, respectively. For this purpose, the superfolder GFP-EGF (sfGFP-EGF) coding sequence was inserted at the N-terminus of the pIII gene in the pIT2 phagemid. The capability of the constructed phage to recognize EGFR-overexpressing cells was monitored by fluorescence microscopy, fluorescence-activated cell sorting (FACS), and cell-based ELISA experiments. FACS analysis showed a significant shift in the mean fluorescence intensity (MFI) of the cells treated with phage displaying sfGFP-EGF compared to phage displaying only sfGFP. The binding of phage displaying sfGFP-EGF to A-431 cells, monitored by fluorescence microscopy, indicated the formation of the sfGFP-EGF-EGFR complex on the surface of the treated cells. Cell-based ELISA experiments showed that phages displaying either EGF or sfGFP-EGF can specifically bind EGFR-expressing cells. The vector constructed in the current study has the potential to be engineered for gene delivery purposes as well as cell-based imaging for tumor detection.

Dispersive solid-phase extraction of risperidone from plasma samples using graphene oxide aerogels and determination with liquid chromatography.

A graphene oxide-based aerogel was synthesized and applied to the extraction and the determinations with the high-performance liquid chromatography-ultraviolet detector. After the characterization of the produced graphene-aerogel, it was utilized as a dispersive solid-phase extraction sorbent for risperidone extraction from plasma samples. Aerogels are materials with a large surface area-to-mass ratio and plenty of core with functional groups which can easily attach to the analytes to extract them to the second phase. The suggested method determined risperidone in plasma samples in the wide dynamic range from 20 ng/ml to 3 µg/ml. The limits of detection and quantification of the developed method were calculated as 2.4 and 8.2 ng/ml, respectively. As a novel feature, the developed method has no need to precipitate plasma proteins, improving the analytical performance of the analysis. Also, for the first time, the produced materials were utilized for the extraction of risperidone from the plasma samples. The obtained results revealed that the developed approach could be employed as an accurate method for the quantification of risperidone in real plasma samples.

Design, Synthesis, and Biological Evaluation of Novel Indanone Derivatives as Cholinesterase Inhibitors for Potential Use in Alzheimer's Disease.

Indanone derivatives containing meta/para-substituted aminopropoxy benzyl/benzylidene moieties were designed based on the structures of donepezil and ebselen analogs as the cholinesterase inhibitors. The designed compounds were synthesized and their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities were measured. Inhibitory potencies (IC50 values) for the synthesized compounds ranged from 0.12 to 11.92 µM and 0.04 to 24.36 µM against AChE and BChE, respectively. Compound 5 c showed the highest AChE inhibitory potency with IC50 value of 0.12 µM, whereas the highest BChE inhibition was achieved by structure 7 b (IC50 =0.04 µM). Structure-activity relationship (SAR) analysis revealed that there is no significant difference between meta and para-substituted derivatives in AChE and BChE inhibition. However, the most potent AChE inhibitor 5 c belongs to meta-substituted compounds, while the most active BChE inhibitor is para-substituted derivative 7 b. The order of enzyme inhibition potency based on the substituted amine group is dimethyl amine>piperidine>morpholine. Compounds containing C=C linkage are more potent AChE inhibitors than the corresponding saturated structures. Molecular docking studies indicated that 5 c interacts with AChE in a very similar way to that observed experimentally for donepezil. The introduced indanone-aminopropoxy benzylidenes could be used in drug-discovery against Alzheimer's disease.

Development of New Inhibitors of HDAC1-3 Enzymes Aided by In Silico Design Strategies.

Histone deacetylases (HDACs) are overexpressed in cancer, and their inhibition shows promising results in cancer therapy. In particular, selective class I HDAC inhibitors such as entinostat are proposed to be more beneficial in breast cancer treatment. Computational drug design is an inevitable part of today's drug discovery projects because of its unequivocal role in saving time and cost. Using three HDAC inhibitors trichostatin, vorinostat, and entinostat as template structures and a diverse fragment library, all synthetically accessible compounds thereof (∼3200) were generated virtually and filtered based on similarity against the templates and PAINS removal. The 298 selected structures were docked into the active site of HDAC I and ranked using a calculated binding affinity. Top-ranking structures were inspected manually, and, considering the ease of synthesis and drug-likeness, two new structures (3a and 3b) were proposed for synthesis and biological evaluation. The synthesized compounds were purified to a degree of more than 95% and structurally verified using various methods. The designed compounds 3a and 3b showed 65-80 and 5% inhibition on HDAC 1, 2, and 3 isoforms at a concentration of 10 µM, respectively. The novel compound 3a may be used as a lead structure for designing new HDAC inhibitors.

Design, synthesis and biological evaluation of novel indanone containing spiroisoxazoline derivatives with selective COX-2 inhibition as anticancer 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.

Investigation of intestinal transportation of peptide-displaying bacteriophage particles using phage display method.

To investigate whether peptide sequences with specific translocation across the gastrointestinal barrier can be identified as drug delivery vehicles, in vivo phage display was conducted. For this purpose, a random library of 12-mer peptides displayed on M13 bacteriophage was orally administered to mice followed by recovery of the phage particles from the blood samples after three consecutive biopanning rounds. The obtained peptide sequences were analyzed using bioinformatics tools and software. The results demonstrated that M13 bacteriophage bearing peptides translocate nonspecifically across the mice intestinal mucosal barrier deduced from random distribution of amino acids in different positions of the identified peptide sequences. The most probable reason for entering the phage particles into systemic circulation after oral administration of the peptide library can be related to the nanoscale nature of their structures which provides a satisfying platform for the purpose of designing nanocarriers in pharmaceutical applications.

Guided rational design with scaffold hopping leading to novel histamine H3 receptor ligands.

During the past decades, histamine H3 receptors have received widespread attention in pharmaceutical research due to their involvement in pathophysiology of several diseases such as neurodegenerative disorders. In this context, blocking of these receptors is of paramount importance in progression of such diseases. In the current investigation, novel histamine H3 receptor ligands were designed by exploiting scaffold-hopping drug-design strategy. We inspected the designed molecules in terms of ADME properties, drug-likeness, as well as toxicity profiles. Additionally molecular docking and dynamics simulation studies were performed to predict binding mode and binding free energy calculations, respectively. Among the designed structures, we selected compound d2 and its demethylated derivative as examples for synthesis and affinity measurement. In vitro binding assays of the synthesized molecules demonstrated that d2 has lower binding affinity (Ki = 2.61 µM) in radioligand displacement assay to hH3R than that of demethylated form (Ki = 12.53 µM). The newly designed compounds avoid of any toxicity predictors resulted from extended in silico and experimental studies, can offer another scaffold for histamine H3R antagonists for further structure-activity relationship studies.

Synthesis and Biological Evaluation of 1,3,5-Trisubstituted 2-Pyrazolines as Novel Cyclooxygenase-2 Inhibitors with Antiproliferative Activity.

A new series of 1,3,5-trisubstituted 2-pyrazolines for the inhibition of cyclooxygenase-2 (COX-2) were synthesized. The designed structures include a COX-2 pharmacophore SO2 CH3 at the para-position of the phenyl ring located at C-5 of a pyrazoline scaffold. The synthesized compounds were tested for in vitro COX-1/COX-2 inhibition and cell toxicity against human colorectal adenocarcinoma cell lines HT-29. The lead compound (4-chlorophenyl){5-[4-(methanesulfonyl)phenyl]-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl}methanone (16) showed significant COX-2 inhibition (IC50 =0.05±0.01 µM), and antiproliferative activity (IC50 =5.46±4.71 µM). Molecular docking studies showed that new pyrazoline-based compounds interact via multiple hydrophobic and hydrogen-bond interactions with key binding site residues of the COX-2 enzyme.

Hybridization-based design of novel anticholinesterase indanone-carbamates for Alzheimer's disease: Synthesis, biological evaluation, and docking studies.

Inspired by the structures of donepezil and rivastigmine, a novel series of indanone-carbamate hybrids was synthesized using the pharmacophore hybridization-based design strategy, and their biological activities toward acetylcholinesterase (AChE) and butyrylcholinesterase were evaluated. Among the synthesized compounds, 4d and 4b showed the highest AChE inhibitory activities with IC50 values in the micromolar range (compound 4d: IC50 = 3.04 µM; compound 4b: IC50 = 4.64 µM). Moreover, the results of the Aß1-40 aggregation assay revealed that compound 4b is a potent Aß1-40 aggregation inhibitor. The kinetics of AChE enzymatic activity in the presence of 4b was investigated, and the results were indicative of a reversible partial noncompetitive type of inhibition. A molecular docking study was conducted to determine the possible allosteric binding mode of 4b with the enzyme. The allosteric nature of AChE inhibition by these compounds provides the opportunity for the design of subtype-selective enzyme inhibitors. The presented indanone-carbamate scaffold can be structurally modified and optimized through medicinal chemistry-based approaches for designing novel multitargeted anti-Alzheimer agents.

Application of bioinformatics and molecular dynamics simulation approaches for identification of fibroblast growth factor 10 analogues with potentially improved thermostability.

Fibroblast growth factor 10 functions as a paracrine mesenchymal molecule to initiate signalling pathways regarding to cellular development and health. However, the low thermal stability restricts it's functionality in the human body and the shelf-life of FGF10-based formulations. The current study aimed to employ rational design and bioinformatics approaches to identify some point mutations which may improve the thermal stability of FGF10. Bioinformatics analyses resulted in N105D, C106F, K144R, K153M and I156R as the potential stability conferring mutations. The identified mutants were subjected to MD simulation indicating that all mutations are both structurally and energetically favoured. Finally, the effects of the identified mutations on receptor binding of FGF10 were predicted and the results showed that K144R and K153M mutations may increase the binding affinity relative to the wild type. The findings of the current study propose potentially improved FGF10 analogues for further experimental investigations.

Design, synthesis, biological evaluation, and docking study of novel dual-acting thiazole-pyridiniums inhibiting acetylcholinesterase and ß-amyloid aggregation for Alzheimer's disease.

New compounds containing thiazole and pyridinium moieties were designed and synthesized. The potency of the synthesized compounds as selective inhibitors of acetylcholinesterase (AChE), and ß-amyloid aggregation (Aß) 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 ß-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.

Structure-based discovery of novel small molecule inhibitors of platelet-derived growth factor-B.

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.

Dispersive solid-phase extraction adsorbent of methamphetamine using in-situ synthesized carbon-based conductive polypyrrole nanocomposite: focus on clinical applications in human urine.

Determination of methamphetamine is of great importance in clinical and forensic laboratories because there are low dosages of drugs in the biological media and social problems created due to the methamphetamine consumption. Polymeric carbon based-nano composites are reasonable candidates for dispersive solid phase extraction method due to facial and affordable synthesis process and high selectivity and sensitivity. Nano graphene oxide polypyrolle composite was synthesized and employed as dispersive solid-phase extraction adsorbent for methamphetamine extraction from complex urine matrix. Full characterization of the prepared nano graphene oxide polypyrolle composite was completed and the influential extraction parameters were investigated through one-parameter-at-a-time method. High-performance liquid chromatography detectors were applied as detection and quantification instrument. The optimized extraction parameters included 300 µL of methanol, 10 min of extraction and desorption time, 6000 stirring rate, urine pH value of 10, 60 mg of adsorbent, and 6 mL of urine volume. After outlining the calibration curve, the linear range of the method was considered as 30-800 ng/mL. The detection limit for the suggested method was 9 ng/mL. The analysis of addicted subjects with the proposed method confirmed the utility of the method in different analytical and clinical laboratories.

Empagliflozin Attenuates Renal and Urinary Markers of Tubular Epithelial Cell Injury in Streptozotocin-induced Diabetic Rats.

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.

Overview of CD24 as a new molecular marker in ovarian cancer.

Ovarian cancer (OC) is the fifth leading cause of cancer-related death among women. The high mortality rate is due to lack of early symptoms, late diagnosis, limited treatment options, and also emerging of drug resistance. Todays, molecular markers have become promising in tumor-targeted therapy. Several molecular markers have been known in OC immunotherapy. Identification of the specific molecular markers with prognostic significance is interested. CD24 is a small sialoglycoprotein which is localized in lipid rafts through its glycosylphosphatidylinositol (GPI) anchor. It has been reported that CD24 is overexpressed in many cancers including OC. Also, CD24 is identified as a cancer stem cell marker in OC. The CD24 expression is associated with the development, invasion, and metastasis of cancer cells. The exact role of CD24 in cancer cells is not clearly understood. Recently, CD24 has been identified as an independent prognostic marker of survival in patients with OC. In this study, we reviewed the molecular targets in OC immune-targeted therapy and also presented an overview of the new molecular marker CD24 and its association with the OC by reviewing the recent literature.

Affinity maturation and characterization of the ofatumumab monoclonal antibody.

CD20 molecule, a phosphoprotein with 297 amino acids and four transmembrane domains, is a member of MS4A protein family. Anti-CD20 antibodies such as ofatumumab, which have been developed for cancer treatment and has demonstrated efficacy in relapsed/refractory chronic lymphocytic leukemia, are among the most successful therapies to date. Rational engineering methods can be applied with reasonable success to improve functional characteristics of antibodies. Considering the importance of this issue, we have used in silico modeling approach for the improvement of ofatumumab monoclonal antibody. Four mutated variants of ofatumumab were developed and expressed in Chinese hamster ovary (CHO) cells along with the unmodified antibody. Analysis of affinity of the purified antibodies with CD20 showed significant improvement in antigen-binding characteristics of one of the variants compared with the control antibody. This study represents the first step toward development of the second generation ofatumumab antibody with improved affinity.

Aldehyde oxidase at the crossroad of metabolism and preclinical screening.

Human AOX1 is a member of the mammalian aldehyde oxidase (AOX) family of enzymes and it is an emerging cytosolic enzyme involved in phase I drug-metabolism, bio-transforming a number of therapeutic agents and xenobiotics. The current trend in drug-development is to design molecules which are not recognized and inactivated by CYP450 monooxygenases, the main drug-metabolizing system, to generate novel therapeutic agents characterized by optimal pharmacokinetic and pharmacodynamic properties. Unfortunately, this has resulted in a substantial enrichment in molecules which are recognized and metabolized by AOXs. The observation has raised interest in the generation of tools capable of predicting AOX-dependent drug-metabolism of novel molecules during the early phases of drug development. Such tools are likely to reduce the number of failures occurring at the clinical and late phase of the drug development process. The current review describes different in silico, in vitro and in vivo methods for the prediction of AOX metabolizing ability and focuses on the existing drawbacks and challenges associated with these approaches.

Characterization of the novel anti-TNF-α single-chain fragment antibodies using experimental and computational approaches.

Single-chain fragment variable (scFv) antibodies are antibody fragments consist of variable domains of full antibodies known to retain antigen binding properties while having much lower molecular weights granting some beneficial properties to them. In our previous study, three phage particles each displaying an individual scFv antibody (i.e. J43, J44, and J48) were identified as tumor necrosis factor alpha (TNF-α) binders. The current study aimed to produce previously identified anti-TNF-α scFv antibodies and to investigate their abilities to bind and inhibit TNF-α biological effect. The estimated free energy of folding determined using spectrofluorimetry method for the prepared scFv proteins was in the range of 6.35-12.45 kJ mol-1 indicating their proper folding in the solution. In ELISA experiment, the produced scFvs showed an appropriate affinity towards TNF-α with Kd values in the range of 0.5-2.18 µM. They also inhibited the TNF-α-induced cytotoxicity on L929 cells with sub-micromolar IC50 values (0.12 and 0.73 µM for J44 and J48, respectively). Molecular docking studies showed that J44 could mimic adalimumab interactions with TNF-α, confirming its relatively high TNF-α inhibitory effect compared to J43 and J48. It seems that the findings in the current study can be useful for designing more potent anti-TNF-α antibodies.

Recombinant M2e-HA2 fusion protein induced immunity responses against intranasally administered H9N2 influenza virus.

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.