Synthesis, characterization and hypolipidemic effects of urazine derivatives on rat: Study of molecular modeling and enzyme inhibition.

The prevalence of hyperlipidemia has increased dramatically worldwide. It is a major public health threat, characterized by the presence of an abnormal lipid profile, primarily with elevated serum total cholesterol (TC), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL) levels, and reduced high-density lipoprotein (HDL) level. Genetic factors, dietary and lifestyle habits play important roles in hyperlipidemia. It can increase the risk of chronic metabolic disorders, such as obesity, cardiovascular disease, and type II diabetes. The main objective of the present study was to evaluate the effect of urazine derivatives on serum triglyceride, cholesterol, LDL, HDL, and nitric oxide (NO) levels in high-fat diet (HFD)-induced hyperlipidemic rats. Synthetic compounds were prepared and confirmed by spectroscopic methods. Then, 88 male Sprague-Dawley rats were divided into 11 groups: control, HFD-treated group, HFD plus atorvastatin-treated group, and HFD plus 8 synthetic compounds-treated groups. The body weight, triglyceride, cholesterol, LDL, HDL, and NO levels were measured. The data with p < 0.05 were considered significant. The results indicated that HFD significantly increased cholesterol, triglyceride, and LDL levels and decreased NO concentration and HDL level compared to the control group (p < 0.05). However, HFD plus urazine derivatives significantly decreased NO, cholesterol, and triglyceride levels and increased HDL levels compared to the HFD-treated group (p < 0.05). Urazine derivatives may improve liver dysfunction in HFD-induced hyperlipidemic rats by modulation of detoxification enzymes and their anti-oxidant effects and also blood lipid profile.

In vitro evaluation of the anticancer activity of barbituric/thiobarbituric acid-based chromene derivatives.

BACKGROUND: Cancer is one of the most important reasons for mortality worldwide. Several synthetic products have shown valuable efficiency as an anticancer medicines. Chromene derivatives have long been used as the promising compounds which are potent in inhibition of the growth of tumors. METHODS AND RESULTS: In this study, we investigate an anticancer activity of barbituric/thiobarbituric acid-based chromene derivates. For this purpose, viability, antioxidant and apoptotic assays were conducted using three different cancer cell lines (A2780, MCF7, and A549). In most cases, the antiproliferative activity of barbituric acid-based derivatives was higher than that of thiobarbituric acid-based compounds. Among 14 compounds, compound 4g was the most potent one, which showed the highest effect on cells by increasing the accumulation of ROS (up to 540% increase), increasing the level of caspase-3 and caspase-9 (~ 35% increase), and decreasing the mitochondrial membrane potential (2.5 folds reduction). To characterize the type of cell death involved into our experiment Annexin V/PI double staining of compound 4g was performed. The results showed that the number of late apoptotic and/or necrotic cells (Ann V + /PI +) increased fourfold upon treatment with IC50 concentration of 4g. CONCLUSIONS: Overall, the anti-proliferative activity of barbituric acid-based derivatives was higher than that of thiobarbituric acid compounds, and compound 4g can be introduced as a potential candidate to prevent various cancers.

Antioxidant and glycohydrolase inhibitory behavior of curcumin-based compounds: Synthesis and evaluation of anti-diabetic properties in vitro.

Naturally occurring anti-diabetic compound curcumin can prevent diabetes complications due to antioxidant and anti-inflammatory properties as well as the attenuation of postprandial hyperglycemia. In this line, we have synthesized thirteen curcumin based derivatives (L1-L13) by multi-component reaction, characterized by IR, 1HNMR, 13C NMR, MS, elemental analysis and evaluated for possible antioxidant properties and α-glucosidase (α-Glu) and α-amylase (α-Amy) inhibitory potential. The curcumin-based pyrano[2,3-d]pyrimidine derivatives could inhibit α-Glu and α-Amy enzyme activity which showed desirable antioxidant activity. Furthermore, among the series, L5, L12, L9, L10, L8 and L11 were identified as more potent inhibitors of α-Glu enzyme than curcumin and the compounds of L12, L4, L9, L5, L10, L8, L13, and L11 were the stronger inhibitors of the α-Amy enzyme in vitro. Besides, among them, L12 had the lowest IC50 for the inhibition of both enzymes. Since strong inhibitors for pancreatic α-Amy result in the progression of severe gastrointestinal side effects, the inhibitors that show the lower α-Amy/α-Glu inhibitory ratio have attracted much attention in medicinal chemistry. Besides, considering antioxidant characteristics of synthesized compounds, the L7 derivative with the highest antioxidant activity and the lowest "α-Amy/α-Glu inhibitory" ratio could be an appropriate candidate for further study through the rational drug design to the exploration of a new class of powerful anti-diabetic drugs.

Synthesis and investigation of inhibitory activities of imidazole derivatives against the metallo-ß-lactamase IMP-1.

Mutations in bacteria can result in antibiotic resistance due to the overuse or abuse of ß-lactam antibiotics. One strategy which bacteria can become resistance toward antibiotics is secreting of metallo ß-lactamase enzymes that can open the lactam ring of the ß-lactam antibiotic and inactivate them. This issue is a threat for human health and one strategy to overcome this situation is co-administration of ß-lactam antibiotics with an inhibitor. So far, no clinically available inhibitors of metallo ß-lactamases (MBLs) reported and the clinically inhibitors of serine ß-lactamase are useless for MBLs. Accordingly, finding a potent inhibitor of the MBLs being very important. In this study, imidazole derivatives primarily were synthesized and their inhibitory activity were measured. Later in silico binding model was used to predict the configuration and conformation of the ligands into the active site of enzyme. Two molecules demonstrated with IC50 of 39 µM and 46 µM against MBL (IMP-1).

Synthesis and enzyme-based evaluation of analogues L-tyrosine thiol carboxylic acid inhibitor of metallo-ß-lactamase IMP-1.

The emergence of drug-resistant pathogenic bacteria is occurring due to the global overuse and misuse of ß-lactam antibiotics. Infections caused by some bacteria which secrete metallo-ß-lactamases (enzymes that inactivate ß-lactam antibiotics) are increasingly prevalent and have become a major worldwide threat to human health. These bacteria are resistant to ß-lactam antibiotics and MBL-inhibitor/ß-lactam antibiotic combination therapy can be a strategy to overcome this problem. So far, no clinically available inhibitors of metallo-ß-lactamases (MBLs) have been reported. In this study, L-benzyl tyrosine thiol carboxylic acid analogues (2a-2k) were synthesized after the study of computational simulation by adding of methyl, chloro, bromo and nitro groups to the benzyl ring for investigation of SAR analysis. Although the synthesized molecules 2a-k shows the potent inhibitory effects against metallo-ß-lactamase (IMP-1) with the range of Kic values of 1.04-4.77 µM, they are not as potent as the candidate inhibitor.

Diethylalkylsulfonamido(4-methoxyphenyl)methyl)phosphonate/phosphonic acid derivatives act as acid phosphatase inhibitors: synthesis accompanied by experimental and molecular modeling assessments.

Purple acid phosphatases (PAPs) are binuclear metallo-hydrolases that have been isolated from various mammals, plants, fungi and bacteria. In mammals, PAP activity is associated with bone resorption and can lead to bone metabolic disorders such as osteoporosis; thus human PAP is an attractive target to develop anti-osteoporotic drugs. The aim of the present study was to investigate inhibitory effect of synthesized diethylalkylsulfonamido(4-methoxyphenyl)methyl)phosphonate/phosphonic acid derivatives as potential red kidney bean PAP (rkbPAP) inhibitors accompanied by experimental and molecular modeling assessments. Enzyme kinetic data showed that they are good rkbPAP inhibitors whose potencies improve with increasing alkyl chain length. Hexadecyl derivatives, as most potent compounds (Ki = 1.1 µM), inhibit rkbPAP in the mixed manner, while dodecyl derivatives act as efficient noncompetitive inhibitor. Also, analysis by molecular modeling of the structure of the rkbPAP-inhibitor complexes reveals factors, which may be important for the determination of inhibition specificity.

Inhibition of guinea pig aldehyde oxidase activity by different flavonoid compounds: An in vitro study.

Aldehyde oxidase (AO), a cytosolic molybdenum-containing hydroxylase, is predominantly active in liver and other tissues of mammalian species and involved in the metabolism of extensive range of aldehydes and nitrogen-containing compounds. A wide range of natural components including polyphenols are able to interfere with AO-catalyzed reactions. Polyphenols and flavonoids are one of the extensive secondary plant metabolites ubiquitously present in plants considered an important part of the human diet. The aim of the present study was to investigate inhibitory effect of selected phenolic compounds from three subclasses of aurone, flavanone and phenolic lactone compounds on the activity of AO, spectrophotometrically. AO enzyme was partially purified from liver of guinea pig. Then, inhibitory effects of 10 flavonoid compounds including 8 derivatives of 2-benzylidenebenzofuran-3(2H)-ones, as well as naringenin and ellagic acid on the activity of aldehyde oxidase were assessed compared with the specific inhibitor of AO, menadione. Among the phenolic compounds with inhibitory effects on the enzyme, ellagic acid (IC50=14.47 µM) was the most potent agent with higher inhibitory action than menadione (IC50=31.84 µM). The mechanisms by which flavonoid compounds inhibit AO activity have been also determined. The inhibitory process of the assessed compounds occurs via either a non-competitive or mixed mode. Although flavonoid compounds extensively present in the nature, mainly in dietary regimen, aurones with promising biological properties are not widely distributed in nature, so synthesis of aurone derivatives is of great importance. Additionally, aurones seem to provide a promising scaffold in medicinal chemistry for the skeleton of new developing drugs, so the results of the current study can be valuable in order to better understanding drug-food as well as drug-drug interaction and also appears to be worthwhile in drug development strategies.

Synthesis of 4-sulfamoyl phenyl diazocarboxylic acid derivatives as novel non-classical inhibitors of human carbonic anhydrase II activity: an in vitro study.

The first class of carbonic anhydrase inhibitors (CAIs) discovered was sulfonamides, but their clinical use is limited due to side effects caused by their inhibition of multiple CA isoforms. To overcome this, researchers have focused on developing isoform-selective CAIs. This study involved the synthesis and characterization of novel carboxylic acid/sulfonamide derivatives. We investigated the interaction between these compounds and the human carbonic anhydrase II (hCA II) isoform using spectroscopic and computational methods. The synthesized compounds were evaluated based on their IC50, Kd and Ki values, and it was found that the inhibitory potency and binding affinity of the compounds increased with the number of carboxylic acids zinc binding groups. Specifically, the compound C4, with three carboxylic acid groups, showed the strongest inhibitory potency. Fluorescence measurements revealed that all compounds quenched the intrinsic fluorescence of hCA II through a dynamic quenching process, and each compound had one binding site in the hCA II structure. Thermodynamic analysis indicated hydrogen bonds and van der Waals interactions played key roles in the binding of these compounds to hCA II. Docking studies showed that the carboxylic acid groups directly attached to the zinc ion in the active site, displacing water/hydroxide ions and causing steric hindrance. Overall, the strengthening of inhibitory activity and the binding power of these carboxylic acid derivatives for the hCA II makes these compounds interesting for designing novel hCA II inhibitors.Communicated by Ramaswamy H. Sarma.

Synthesis and psychobiological evaluation of modafinil analogs in mice.

BACKGROUND AND THE PURPOSE OF THE STUDY: Modafinil, a novel wake-promoting agent with low potential for abuse and dependence, has a reliable structure to find some novel derivatives with better activity and lower potential for abuse and risk of dependency. This study was designed to evaluate psychobiological activity of some novel N-aryl modafinil derivatives. METHODS: Seven novel N-aryl modafinil derivatives were synthesized through three reactions: a) preparation of benzhydrylsulfanyl acetic acid through reaction of benzhydrol with thioglycolic acid, b) formation of desired amide by adding the substituted aniline to activated acid with EDC (1-ethyl-3-(3-dimethyl amino propyl) carbodiimide). This reaction was catalyzed by HOBt (N- hydroxylbenzotriazole), and c) oxidation of sulfur to sulfoxide group with H2O2. Then, their psychobiological effect on the performance of male albino mice were compared to that of modafinil as following: wakefulness by determining the effects of derivatives on phenobarbital-induced loss of the righting reflex (LOPR); exploratory activity by measuring activity in the open field test (OFT); depression by measuring immobility time (IT) during forced swimming test (FST) and the anxiogenic and anxiolytic like effects by using elevated plus-maze test (EPM). All tests were videotaped and analyzed for the frequency and duration of the behaviors during the procedures. CONCLUSIONS: 2-(Benzhydrylsulfonyl)-N-(4-chlorophenyl)acetamide (4c) showed comparable result in LOPR test. However, all analogs were found to be stimulant except 2-(benzhydrylsulfinyl)-N-phenylacetamide (4a). Also 4c led the most exploratory activity in mice among derivatives. FST results showed that 4a had the longest IT while modafinil, 2-(benzhydrylsulfinyl)-N-(3-chlorophenyl) acetamide (4b) and 2-(benzhydrylsulfinyl)-N-(4-ethylphenyl) acetamide (4d) had the shortest IT. In EPM, all derivatives showed anxiogenic-like behavior since they decreased open arms time and open arms entries and simultaneously increased close arms time.

Synthesis of 4-hydroxy-L-proline derivatives as new non-classical inhibitors of human carbonic anhydrase II activity: an in vitro study.

Carbonic anhydrase (CA) is a zinc metalloenzyme that facilitates the rapid conversion of water and carbon dioxide into proton and bicarbonate ion. CA isozymes have been broadly studied in many pathological/physiological processes. In the current research, a series of 4-hydroxy-L-proline derivatives were designed and chemically synthetized, and interaction of these carboxylic acid-based compounds with hCA II were evaluated. Results indicated that different derivatives had different potencies on hCAII inhibitory activity and among them, compounds 3 b and 3c had the lowest IC50 and Kd values than 4-hydroxy-L-proline and other derivatives and therefore had the most affinity to the hCA II enzyme. As a result, compounds 3 b and 3c were chosen for additional testing in this research. The Kinetic data demonstrated that 3 b and 3c inhibit the hCA II esterase activity in a linear competitive way, with Ki values in the low micromolar range. Fluorescence tests showed that the hCA II surface hydrophobicity is diminished in the presence of compounds 3 b and 3c, as confirmed by the decrease in ANS binding to hCA II in their presence. Docking results revealed that 3 b and 3c had more binding energy than 4-hydroxy-L-proline. Furthermore, these compounds could occupy the active site of hCA II, where they would interact with critical amino acid residues via non-covalent forces to inhibit hCA II. Overall, the strengthening of inhibitory activity and the binding power of these carboxylic acid derivatives (3 b and 3c) for the hCA II makes these compounds interesting for designing novel hCA II inhibitors.Communicated by Ramaswamy H. Sarma.

Synthesis and Characterization of Novel 4-aryl-4H-chromene Derivatives using Borax and Evaluation of their Anticancer Effects.

BACKGROUND/INTRODUCTION: 4-aryl-4H-chromenes have attracted attention as potential anticancer agents. OBJECTIVE: In an effort to discover effective compounds, we designed a new series of these chromenes with methoxy substitution at 2, 3, 4, 5, and 6 positions. METHODS: The synthesized compounds were tested for anticancer properties against two human cancer cell lines (MCF- 7 and PC3) as well as a normal cell line. Furthermore, induction of apoptosis was explored through various methods, such as flow cytometry analysis, morphological changes, activation of caspase 3, ROS, and MMP. RESULTS: The MTT assay showed that the 5g derivative, with methoxy groups at ortho and meta positions, exhibited the highest potency (IC50 = 40 µM) against the PC3 cell line. Our findings revealed that compound 5g induced apoptosis in the PC3 cell line, which was demonstrated by activation of caspase 3, an increase in ROS levels, and early apoptosis percentage. CONCLUSION: These results suggest that compound 5g holds promise as a potential therapeutic approach to cancer treatment.

Synthesis of benzylidene-indandione derivatives as quantification of amyloid fibrils.

The formation of amyloid fibrils due to its association with fatal diseases, including Alzheimer's, has been investigated by many researchers. These common diseases, mostly become verified when it is too late to be treated. Currently, no cure is available for neurodegenerative diseases, and the process of diagnosing amyloid fibrils in the early stages, while there are fewer amyloid fibrils, has become an issue of interest. To do so, determining new probes with the highest binding affinity to the lowest number of amyloid fibrils is necessary. In this study, we proposed to employ new synthesized benzylidene-indandione derivatives as amyloid fibrils fluorescent detection probes. Native soluble proteins of insulin, bovine serum albumin (BSA), BSA amorphous aggregation, and insulin amyloid fibrils were used to evaluate our compounds' specificity to the amyloid structure. While ten synthesized compounds were examined individually, four of them including 3d, 3g, 3i, and 3j showed a high binding affinity with selectivity and specificity to amyloid fibrils, and their binding properties were also confirmed with in silico analysis. The drug-likeness prediction results for selected compounds by Swiss ADME server shows a satisfactory percentage of blood-brain barrier (BBB) permeability and gastrointestinal (GI) absorption for the compounds 3g, 3i, and 3j. More evaluation is needed to determine all properties of compounds in vitro and in vivo.

Synthesis of benzylidene-benzofuranone derivatives as probes for detection of amyloid fibrils in cells.

Aggregated protein is the common cause of a wide variety of neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease, etc. It is proven that protein aggregation like amyloid ß (Aß) is one of the critical factors causing AD and, its diagnosis in the early stages of the disease is important for the treatment or prevention of AD. To have a better understanding of protein aggregation and its pathologies, there is a huge need to design and develop new and more trustworthy probe molecules for in vitro amyloid quantification and in vivo amyloid imaging. In this study, 17 new biomarker compounds, have been synthesized from benzofuranone derivatives, to detect and identify amyloid in vitro (dye-binding assay) as well as in the cell by staining method. According to the results, some of these synthetic derivatives can be considered suitable identifiers and quantifiers to detect amyloid fibrils in vitro. Compared to thioflavin T, 4 probes out of 17 probes have shown good results in selectivity and detectability of Aß depositions, and their binding properties were also confirmed with in silico analysis. The drug-likeness prediction results for selected compounds by the Swiss ADME server show a satisfactory percentage of blood-brain barrier (BBB) permeability and gastrointestinal (GI) absorption. Among all of them, compound 10 was able to show better binding properties than others, and in vivo study showed that this compound was capable of detecting intracellular amyloid.Communicated by Ramaswamy H. Sarma.

Design, synthesis, spectroscopic characterizations, antidiabetic, in silico and kinetic evaluation of novel curcumin-fused aldohexoses.

Curcumin (bis-α,ß-unsaturated ß-diketone) plays an important role in the prevention of numerous diseases, including diabetes. Curcumin, as an enzyme inhibitor, has ideal structural properties including hydrophobic nature, flexible backbone, and several available hydrogen bond (H-bond) donors and acceptors. In this study, curcumin-fused aldohexose derivatives 3(a-c) were synthesized and used as influential agents in the treatment of diabetes with inhibitory properties against two carbohydrate-hydrolyzing enzymes α-glucosidase (α-Gls) and α-amylase (α-Amy) which are known to be significant therapeutic targets for the reduction of postprandial hyperglycemia. These compounds were isolated, purified, and then spectrally characterized via FT-IR, Mass, 1H, and 13C NMR, which strongly confirmed the targeted product's formation. Also, their inhibitory properties against α-Gls and α-Amy were evaluated spectroscopically. The Results indicated that all compounds strongly inhibited α-Amy and α-Gls by mixed and competitive mechanisms, respectively. The intrinsic fluorescence of α-Amy was quenched by the interaction with compounds 1 and 3b through a dynamic quenching mechanism, and the 1 and 3b/α-Amy complexes were spontaneously formed, mainly driven by the hydrophobic interaction and hydrogen bonding. Fourier transform infrared spectra (FT-IR) comprehensively verified that the binding of compounds 1 and 3b to α-Amy would change the conformation and microenvironment of α-Amy, thereby inhibiting the enzyme activity. Docking and molecular dynamics (MD) simulations showed that all compounds interacted with amino acid residues located in the active pocket site of the proteins. In vivo studies confirmed the plasma glucose diminution after the administration of compound 3b to Wistar rats. Accordingly, the results of the current work may prompt the scientific communities to investigate the possibility of compound 3b application in the clinic.

Curcumin-loaded porous particles functionalized with pH-responsive cell-penetrating peptide for colorectal cancer targeted drug delivery.

The anticancer properties of curcumin have been broadly examined in several shapes, such as nanoparticles and nanocomposite structures. Despite its benefits, curcumin also has some disadvantages, including rapid metabolism, poor absorption, and rapid systemic excretion. Therefore, numerous strategies have been used to increase curcumin's bioavailability. One of these approaches is the use of porous particles like aerogels as drug carriers. Aerogels are special due to their peculiar physical structure. They have a high specific surface area, a significant amount of porosity, and a solid composition, which make them a good choice for drug delivery systems. In the present study, a pH-sensitive aerogel was constructed and evaluated for targeted drug delivery of curcumin to colon cancer. To control the release of curcumin, trehalose was used as a coating agent, and PLP (poly(l-lysine isophthalamide)) was used as a targeted drug delivery agent. PLP is a pseudo-peptidic polymer that increases the cell permeability. In order to investigate and compare the synthesized aerogel before and after loading curcumin and coating with trehalose, physicochemical characterization analyses were performed. Finally, the efficacy of the final formulation was evaluated on HT29 colon cells using the cell bioavailability test. The results indicated the successful synthesis of the aerogel with porous structure with solitary cavities. The trehalose coating performed well, preventing drug release at lower pH but allowing the drug to be released at its intended site. The designed curcumin-loaded porous particles functionalized with PLP showed significant efficacy due to increasing penetration of curcumin into cells, and has potential for use as a new drug carrier with dual effectivity in cancer therapy.

Synthesis, in silico, in vitro and in vivo studies of novel natural-based arylidenes curcumin as potential glycohydrolase digestive enzymes inhibitors.

Diabetes is one of the most common metabolic diseases in humans and the use of herbal medicines is of great clinical importance to inhibit carbohydrate-hydrolyzing enzymes and reduce blood glucose levels in diabetic patients. Inhibition of glycosidase activity is an effective way to treat and prevent diabetes. Therefore, in this study, curcumin-based benzaldehyde derivatives were synthesized and used as influential agents in the treatment of diabetes with inhibitory properties against two carbohydrate-hydrolyzing enzymes α-glucosidase (α-Glu) and α-amylase (α-Amy) as significant therapeutic targets for reducing postprandial hyperglycemia. Overall, the findings showed that due to the specific inhibitory activity against α-Glu in comparison with α-Amy, as well as more stability and antioxidant activity than curcumin, C5 and C8 derivatives are potentially important anti-diabetic drugs, not only to decrease glycemic index but also to limit the activity of the main production pathways of reactive oxygen species (ROS) in diabetic patients.Communicated by Ramaswamy H. Sarma.

Optimization of an α-aminonaphthylmethylphosphonic acid inhibitor of purple acid phosphatase using rational structure-based design approaches.

Purple acid phosphatases (PAPs) are ubiquitous binuclear metallohydrolases that have been isolated from various animals, plants and some types of fungi. In humans and mice, elevated PAP activity in osteoclasts is associated with osteoporosis, making human PAP an attractive target for the development of anti-osteoporotic drugs. Based on previous studies focusing on phosphonate scaffolds, as well as a new crystal structure of a PAP in complex with a derivative of a previously synthesized α-aminonaphthylmethylphosphonic acid, phosphonates 24-40 were designed as new PAP inhibitor candidates. Subsequent docking studies predicted that all of these compounds are likely to interact strongly with the active site of human PAP and most are likely to interact strongly with the active site of pig PAP. The seventeen candidates were synthesized with good yields and nine of them (26-28, 30, 33-36 and 38) inhibit in the sub-micromolar to nanomolar range against pig PAP, with 28 and 35 being the most potent mammalian PAP inhibitors reported with Ki values of 168 nM and 186 nM, respectively. This study thus paves the way for the next stage of drug development for phosphonate inhibitors of PAPs as anti-osteoporotic agents.

An in situ and in vitro investigation of cytoplasmic TDP-43 inclusions reveals the absence of a clear amyloid signature.

Introduction: Several neurodegenerative conditions are associated with a common histopathology within neurons of the central nervous system, consisting of the deposition of cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43). Such inclusions have variably been described as morphologically and molecularly ordered aggregates having amyloid properties, as filaments without the cross-ß-structure and dye binding specific for amyloid, or as amorphous aggregates with no defined structure and fibrillar morphology.Aims and Methods: Here we have expressed human full-length TDP-43 in neuroblastoma x spinal cord 34 (NSC-34) cells to investigate the morphological, structural, and tinctorial properties of TDP-43 inclusions in situ. We have used last-generation amyloid diagnostic probes able to cross the cell membrane and detect amyloid in the cytoplasm and have adopted Raman and Fourier transform infrared microspectroscopies to study in situ the secondary structure of the TDP-43 protein in the inclusions. We have then used transmission electron microscopy to study the morphology of the TDP-43 inclusions.Results: The results show the absence of amyloid dye binding, the lack of an enrichment of cross-ß structure in the inclusions, and of a fibrillar texture in the round inclusions. The aggregates formed in vitro from the purified protein under conditions in which it is initially native also lack all these characteristics, ruling out a clear amyloid-like signature.Conclusions: These findings indicate a low propensity of TDP-43 to form amyloid fibrils and even non-amyloid filaments, under conditions in which the protein is initially native and undergoes its typical nucleus-to-cell mislocalization. It cannot be excluded that filaments emerge on the long time scale from such inclusions, but the high propensity of the protein to form initially other types of inclusions appear to be an essential characteristic of TDP-43 proteinopathies.KEY MESSAGESCytoplasmic inclusions of TDP-43 formed in NSC-34 cells do not stain with amyloid-diagnostic dyes, are not enriched with cross-ß structure, and do not show a fibrillar morphology.TDP-43 assemblies formed in vitro from pure TDP-43 do not have any hallmarks of amyloid.

Molecular aspects on the interaction of isatin-3-isonicotinylhydrazone to deoxyribonucleic acid: model for intercalative drug-DNA binding.

Isatin-3-isonicotinylhydrazone was synthesized and characterized. The interaction of native calf thymus DNA with isatin-3-isonicotinylhydrazone (IINH) in 10 mM Tris-HCl aqueous solutions at neutral pH 7.4 has been investigated by spectrophotometric, circular dichroism (CD), melting temperature (T ( m )), spectrofluorimetric, and viscometric techniques. It is found that IINH molecules could intercalate between base pairs of DNA as are evidenced by: hypochromism in UV absorption band of IINH, induced CD spectral changes, sharp increase in specific viscosity of DNA, and increase in the fluorescence of methylene blue (MB)-DNA solutions in the presence of increasing amounts of IINH, which indicates that it is able to release the intercalated MB completely. The binding constants of IINH-DNA complex at four different temperatures (277, 288, 298, and 310 K) were calculated to be 4.7 × 10(4), 2.2 × 10(4), 1.75 × 10(4) and 1.1 × 10(4) M(-1), respectively. Furthermore, the enthalpy and entropy of the reaction between IINH and CT-DNA showed that the reaction is enthalpy-favored and entropy- disfavored (∆H = -30.187 kJ mol(-1); ∆S = -20.46 J mol(-1)K(-1)) which are other evidences to indicate the IINH is able to be intercalated in the DNA base pairs.

In Vitro Cytotoxicity and Apoptosis Inducing Evaluation of Novel Halogenated Isatin Derivatives.

BACKGROUND: Isatin (1H-indole-2,3-dione) and its derivatives have been utilized in a variety of biological activities. Anticancer compounds were the most extensively highlighted and explored among the range of beneficial properties. OBJECTIVE: Herein, we report the targeting effect of halogenated isatin derivatives on cancer cell mitochondria and their antiproliferative mechanism. METHODS: A series of novel 5-halo-Isatin derivatives consisting of the 5-Amino-1,3,4-thiadiazole-2-thiol scaffold were synthesized and easily conducted in good yields through a condensation reaction between keto groups of Isatin and primary amine under alcoholic conditions, followed by S-benzylation. The compounds were fully characterized using spectroscopic methods such as 1H-NMR, FTIR, mass spectroscopy and then tested in vitro towards three cancer cell lines HT-29 (colon cancer), MCF-7 (breast cancer), and SKNMC (neuroblastoma). Apoptosis induction was investigated through assessment of caspase 3 and mitochondrial membrane potential. RESULTS: The most potent compounds of 5b, 5r (IC50 = 18,13 µM), and 5n (IC50 = 20,17 µM) were found to show strong anticancer activity, especially for MCF7 cells. Further anticancer mechanism studies indicated that 5b and 5r induced apoptosis through the intrinsic mitochondrial pathway. CONCLUSION: This research demonstrated that 5b and 5r have an anticancer property via the modulation of oxidative stress-mediated mitochondrial apoptosis and immune response, which deserves further studies on their clinical applications.