BIN1 in the Pursuit of Ousting the Alzheimer's Reign: Impact on Amyloid and Tau Neuropathology.

Alzheimer's disease contributes to 60-70% of all dementia cases in the general population. Belonging to the BIN1/amphiphysin/RVS167 (BAR) superfamily, the bridging integrator (BIN1) has been identified to impact two major pathological hallmarks in Alzheimer's disease (AD), i.e., amyloid beta (Aß) and tau accumulation. Aß accumulation is found to increase by BIN1 knockdown in cortical neurons in late-onset AD, due to BACE1 accumulation at enlarged early endosomes. Two BIN1 mutants, KR and PL, were identified to exhibit Aß accumulation. Furthermore, BIN1 deficiency by BIN1-related polymorphisms impairs the interaction with tau, thus elevating tau phosphorylation, altering synapse structure and tau function. Even though the precise role of BIN1 in the neuronal tissue needs further investigation, the authors aim to throw light on the potential of BIN1 and unfold its implications on tau and Aß pathology, to aid AD researchers across the globe to examine BIN1, as an appropriate target gene for disease management.

The Design, Synthesis, and Evaluation of Diaminopimelic Acid Derivatives as Potential dapF Inhibitors Preventing Lysine Biosynthesis for Antibacterial Activity.

We created thiazole and oxazole analogues of diaminopimelic acid (DAP) by replacing its carboxyl groups and substituting sulphur for the central carbon atom. Toxicity, ADME, molecular docking, and in vitro antimicrobial studies of the synthesized compounds were carried out. These compounds displayed significant antibacterial efficacy, with MICs of 70-80 µg/mL against all tested bacteria. Comparative values of the MIC, MBC, and ZOI of the synthesized compound were noticed when compared with ciprofloxacin. At 200 µg/mL, thio-DAP (1) had a ZOI of 22.67 ± 0.58, while ciprofloxacin had a ZOI of 23.67 ± 0.58. To synthesize thio-DAP (1) and oxa-DAP (2), l-cysteine was used as a precursor for the L-stereocenter (l-cysteine), which is recognized by the dapF enzyme's active site and selectively binds to the ligand's L-stereocenter. Docking studies of these compounds were carried out using the programme version 11.5 Schrodinger to reveal the hydrophobic and hydrophilic properties of these complexes. The docking scores of compounds one and two were -9.823 and -10.098 kcal/mol, respectively, as compared with LL-DAP (-9.426 kcal/mol.). This suggests that compounds one and two interact more precisely with dapF than LL-DAP. Chemicals one and two were synthesized via the SBDD (structure-based drug design) approach and these act as inhibitors of the dapF in the lysine pathway of bacterial cell wall synthesis.

Radiolabeling of protected tryptophan with [18F]fluoromethyl tosylate: Formation of [18F]fluoromethyl ester of tryptophan instead of 1-N-[18F]fluoromethyl tryptophan methylester.

The reaction of [18F]fluoromethyl tosylate with methyl(tert-butoxycarbonyl)-l-tryptophanate results in formation the O-alkylated ester of the tryptophan instead of alkylation of the indole nitrogen of tryptophan as initially anticipated. Treatment of protected tryptophan with NaH in dimethyl formamide (DMF) along with [18F]fluoromethyl tosylate at 130°C results in the formation of [18F]fluoromethyl(tert-butoxycarbonyl)-l-tryptophanate. Preferential formation of the O-alkylated product is postulated to be due to the hydrolysis of the ester. Confirmation of the O-alkylation was obtained by synthesizing the [19F]fluoromethyl(tert-butoxycarbonyl)-l-tryptophanate insitu and examining its NMR characteristics using multiple NMR techniques. Similar results were also obtained when reacting Boc-tryptophan-N-carboxyanhydride precursor with fluoromethyl tosylate.

Computational investigation of binding mechanism of substituted pyrazinones targeting corticotropin releasing factor-1 receptor deliberated for anti-depressant drug design.

In spite of various research investigations towards anti-depressant drug discovery program, no one drug has not yet launched last 20 years. Corticotropin-releasing factor-1 (CRF-1) is one of the most validated targets for the development of antagonists against depression, anxiety and post-traumatic stress disorders. Various research studies suggest that pyrazinone based CRF-1 receptor antagonists were found to be highly potent and efficacious. In this research investigation, we identified the pharmacophore and binding pattern through 2D and 3D-QSAR and molecular docking respectively. Molecular dynamics studies were also performed to explore the binding pattern recognition. We establish the relationship between activity and pharmacophoric features to design new potent compounds. The best 2D-QSAR model was generated through multiple linear regression method with r2 value of 0.97 and q2 value of 0.89. Also 3D-QSAR model was obtained through k-nearest neighbor molecular field analysis method with q2 value of 0.52 and q2_se value of 0.36. Molecular docking and binding energy were also evaluated to define binding patterns and pharmacophoric groups, including (i) hydrogen bond with residue Asp284, Glu305 and (ii) π-π stacking with residue Trp9. Compound 11i has the highest binding affinity compared to reference compounds, so this compound could be a potent drug for stress related disorders. Most of the compounds, including reference compounds were found within acceptable range of physicochemical parameters. These observations could be provided the leads for the design and optimization of novel CRF-1 receptor antagonists. Communicated by Ramaswamy H. Sarma.

Challenges in the automated synthesis of [18F]-1-fluoroethyl tryptophan: Formation of both O- and N-alkylated products.

[18F]Fluoroethyl tosylate was synthesized using an automated "Synthra" module using ethylene di-tosylate and [18F]fluoride/K222/K2CO3 in acetonitrile. [18F]Fluoroethyl tosylate was purified by semi-preparative HPLC followed by reformulation using a C18 Sep-Pak cartridge and eluted with DMF. Using this [18F]fluoroethyl tosylate, we attempted to alkylate protected tryptophan aiming to obtain the N-[18F]fluoroethyl-t-Boc-tryptophan methyl ester. Initial attempts resulted in the formation of the O-alkylated, rather than N-alkylated product. Manual removal of the cartridge from the automated module, followed by an extended drying of the cartridge under high flow nitrogen, was required to form the desired N-alkylated product. This demonstrates that the drying process in automated modules requires modification for sensitive N-alkylation of compounds and may be essential for compounds like tryptophan methyl ester that have multiple potential sites of alkylation in their chemical structure.

NMR and DFT investigations of structure of colchicine in various solvents including density functional theory calculations.

A detailed NMR investigation of the chemical shifts of hydrogen and carbon atoms associated with the structure of the naturally occurring alkaloid colchicine was conducted using high field NMR. Initially, the experimental chemical shifts for colchicine in chloroform and DMSO were compared to the values calculated using density functional theory (DFT). There were significant deviations observed for the chloroform solvent, but these were only slight in the DMSO solution. Dilution of the chloroform solution changed the experimental chemical shifts and improved agreement with the DFT calculations, suggesting self-aggregation at higher concentrations. A dimeric model was proposed for which agreement with the DFT calculated chemical shifts was better than for corresponding monomeric structures. Three further solvents were studied to evaluate changes in chemical shift values at different dilutions. Chloroform, benzene and water showed significant chemical shift changes implying self-aggregation, whereas DMSO and acetone did not show significant change upon dilution.

Management of radioactive waste gases from PET radiopharmaceutical synthesis using cost effective capture systems integrated with a cyclotron safety system.

The emphasis on the reduction of gaseous radioactive effluent associated with PET radiochemistry laboratories has increased. Various radioactive gas capture strategies have been employed historically including expensive automated compression systems. We have implemented a new cost-effective strategy employing gas capture bags with electronic feedback that are integrated with the cyclotron safety system. Our strategy is suitable for multiple automated 18F radiosynthesis modules and individual automated 11C radiosynthesis modules. We describe novel gas capture systems that minimize the risk of human error and are routinely used in our facility.

Comparison of experimental and DFT-calculated NMR chemical shifts of 2-amino and 2-hydroxyl substituted phenyl benzimidazoles, benzoxazoles and benzothiazoles in four solvents using the IEF-PCM solvation model.

A comparative study of experimental and calculated NMR chemical shifts of six compounds comprising 2-amino and 2-hydroxy phenyl benzoxazoles/benzothiazoles/benzimidazoles in four solvents is reported. The benzimidazoles showed interesting spectral characteristics, which are discussed. The proton and carbon chemical shifts were similar for all solvents. The largest chemical shift deviations were observed in benzene. The chemical shifts were calculated with density functional theory using a suite of four functionals and basis set combinations. The calculated chemical shifts revealed a good match to the experimentally observed values in most of the solvents. The mean absolute error was used as the primary metric. The use of an additional metric is suggested, which is based on the order of chemical shifts. The DP4 probability measures were also used to compare the experimental and calculated chemical shifts for each compound in the four solvents. Copyright © 2015 John Wiley & Sons, Ltd.

Heteronuclear NMR spectroscopic investigations of hydrogen bonding in 2-(benzo[d]thiazole-2'-yl)-N-alkylanilines.

The 2-(benzo[d]thiazole-2'-yl)-N-alkylanilines have previously revealed the presence of a strong intramolecular hydrogen bond. This in turn gives rise to a more complicated multiplet for the protons attached to the carbon adjacent to the amino group. This intramolecular hydrogen bond was investigated by a deuterium exchange experiment using heteronuclear NMR spectroscopy (1H, 13C, 15N and 2H). We observed changes in the multiplet structure and chemical shifts providing further evidence that the deuterium replaces the hydrogen in the intramolecular hydrogen bond. A time course study of the D2O exchange confirmed the presence of a strong hydrogen bond. The comparison of the structures obtained by X-ray crystallography showed a very small difference in planarity between the two-substituted and four-substituted amino compounds. In both the cases, the phenyl ring is not absolutely coplanar with the thiazole unit. The existence of this intramolecular hydrogen bond in 2-(benzo[d]thiazole-2'-yl)-N-alkylanilines was further confirmed by single crystal X-ray crystallography.

Synthesis, characterization and (11) C-radiolabeling of aminophenyl benzothiazoles: structural effects on the alkylation of amino group.

Several aminophenyl benzothiazoles were prepared with a view to using them as amyloid binding agents for imaging ß-amyloid in Alzheimer's disease. These precursors were radiolabeled with (11) C-positron-emitting radioisotope using an automated synthesizer and selected radiolabeled compounds were further purified by HPLC. Our results demonstrate that changes in structure have a major influence on the radioactive yield and the ease with which the radiolabel can be introduced. Aminophenyl benzothiazoles with an attached isopropyl group resisted dialkylation perhaps due to steric hindrance caused by this group. Straight chain attachment of methyl, ethyl, butyl, and crotyl groups in the structure decreased the radiochemical yield. Notably, the o-aminophenyl benzothiazole derivatives were difficult to alkylate despite stringent experimental conditions. This reactivity difference is attributed to the hydrogen bonding characteristics of the o-amino group with the nitrogen atom of the thiazole ring.

A comparative study between para-aminophenyl and ortho-aminophenyl benzothiazoles using NMR and DFT calculations.

Ortho-substituted and para-substituted aminophenyl benzothiazoles were synthesised and characterised using NMR spectroscopy. A comparison of the proton chemical shift values reveals significant differences in the observed chemical shift values for the NH protons indicating the presence of a hydrogen bond in all ortho-substituted compounds as compared to the para compounds. The presence of intramolecular hydrogen bond in the ortho amino substituted aminophenyl benzothiazole forces the molecule to be planar which may be an additional advantage in developing these compounds as Alzheimer's imaging agent because the binding to amyloid fibrils prefers planar compounds. The splitting pattern of the methylene proton next to the amino group also showed significant coupling to the amino proton consistent with the notion of the existence of slow exchange and hydrogen bond in the ortho-substituted compounds. This is further verified by density functional theory calculations which yielded a near planar low energy conformer for all the o-aminophenyl benzothiazoles and displayed a hydrogen bond from the amine proton to the nitrogen of the thiazole ring. A detailed analysis of the (1)H, (13)C and (15)N NMR chemical shifts and density functional theory calculated structures of the compounds are described.

Synthesis, NMR structural characterization and molecular modeling of substituted thiosemicarbazones and semicarbazones using DFT calculations to prove the syn/anti isomer formation.

Thiosemicarbazones possessing electron-donating and electron-withdrawing groups were prepared, and their spectral characteristics determined. In all cases, the spectra showed that one isomer was formed, allowing further functionalization to molecules of biological interest. We provide NMR data for some of the thiosemicarbazones and semicarbazones. We also provide evidence that for 2-pyridyl thiosemicarbazone, the syn isomer slowly converts into the anti isomer in dimethyl sulfoxide solvent with first-order kinetics. Molecular modeling and density functional theory calculations confirmed these observations.

Synthesis and characterization of 1- and 2-cinnamoyloxyacetonaphthones.

The synthesis of 1- and 2-cinnamoyloxyacetonaphthones was achieved in one step using hydroxyl acetonaphthones and substituted cinnamic acids in the presence of a catalytic amount of phosphoroxychloride. Structural characterization was accomplished using high-resolution nuclear magnetic resonance (NMR) spectroscopy. Chemical shifts of the compounds were compared and the change in the chemical shifts relative to electron-donating and -withdrawing groups is presented. Introduction of a thiophene ring instead of phenyl-substituted analogs caused shielding of the olefinic proton.

Effect of alkyl groups on the cellular hydrolysis of stavudine phosphoramidates.

We examined the effect of cellular metabolism of three alkyl-substituted amino acid ester phosphoramidate derivatives of stavudine in different cell lines. Marked cell-to-cell differences were found in both the rate of hydrolysis and chiral selectivity. This selectivity implies that different enzymes may be involved in the metabolism of these compounds depending on the cell type involved. Notably, both the methyl and ethyl substituted derivatives underwent hydrolysis in presence of various cell lines, whereas the tert-butyl substituted compound was resistant to hydrolysis implying that steric hindrance associated with this group along with electron density may play a key role in the hydrolysis profile of these compounds. Additionally we found this mimicked the hydrolysis profiles obtained for bacterial enzymes. Furthermore, our results suggest that the site of attack of the cellular enzymes is confined to the ester side chain of the molecule. This result is also consistent with our earlier observation using bacterial enzymes as well as using 'd' isomers.

Synthesis and metabolism of naphthyl substituted phosphoramidate derivatives of stavudine.

The synthesis of naphthylphosphoramidate derivatives of stavudine was achieved using a four-step procedure. The derivatives were subjected to several different enzymes including lipase, esterase, Subtilisin Carlsberg, and Carica papaya, and their hydrolysis rates were determined. Based on the rates of hydrolysis, we were able to differentiate between the chiralities at the phosphorus center of the phosphoramidate compounds. In addition, lipase was found to distinguish between both alpha and beta forms of the compounds. The superior chiral selectivity shown by lipase toward the naphthyl substituted phosphoramidate derivatives is attributed to the restrictive binding pocket of the lipase.

Effect of change in nucleoside structure on the activation and antiviral activity of phosphoramidate derivatives.

Changing the nucleoside group of a series of phosphoramidate derivatives affects the enzyme mediated hydrolysis rate of the compounds. d4T and AZT-substituted analogs were activated by enzymes such as lipases, esterases, and proteases. On the other hand, 3dT-substituted derivatives were comparatively less prone to hydrolysis under similar experimental conditions. From the experimental results, we propose that the most preferable nucleoside group for enzyme activation is d4T rather than AZT or 3dT. Additionally, we also observed that depending on the enzymes used the chiral selectivity of the enzymes for the phosphorus center of these phosphoramidate derivatives differed, demonstrating the importance of the nucleoside structure for this class of compounds.

Protease-mediated enzymatic hydrolysis and activation of aryl phosphoramidate derivatives of stavudine.

Several proteases are capable of hydrolyzing the aryl substituted phosphoramidate derivatives of stavudine resulting in the formation of the active metabolite, alaninyl d4T monophosphate. Subtilisin Protease A, Subtilisin Griseus, Subtilisin Carlsberg, Papaya, Bacillus were amongst the most effective proteases in hydrolyzing stavudine derivatives and specificity of their activity was confirmed using several protease inhibitors to block the hydrolysis of these phosphoramidate derivatives. We found that these proteases exhibit chiral selectivity at the phosphorus center of stavudine derivatives. Our results indicate that cellular proteases may be responsible for the activation of these phosphoramidate derivatives. In addition, we show that the enzymatic hydrolysis takes place at the carboxymethyl ester side chain of these pro-drugs and the direct attack on the phosphorus center by these enzymes does not occur. Finally, we describe a novel activation pathway hitherto unknown for the activation and viral inhibitory characteristic shown by these phosphoramidate derivatives of stavudine.

In vitro anti-HIV potency of stampidine alone and in combination with standard anti-HIV drugs.

The purpose of the present study was compare the in vitro anti-HIV potency stampidine (CAS 217178-62-6), a novel aryl phosphate derivative of stavudine (CAS 3056-17-5), and drug combinations containing stampidine to the anti-HIV tency of the standard drugs zidovudine (CAS 30516-87-1), stavudine, lamivudine (CAS 134678-17-4), nelfinavir (CAS 159989-65-8), and nevirapine (CAS 129618-40-2) as well as their combinations. Stampidine inhibited the laboratory HIV-1 strain HTLV(IIIB) (B-envelope subtype) as well as the primary clinical HIV-1 isolates BR/92/025 (C-envelope subtype) and BR/93/20 (F-envelope sub-type) with subnanomolar IC50 values. Stampidine was as effective as zidovudine against HTLV(IIIB) and BR/92/025 and 3-logs more effective than zidovudine against BR/93/20. Stampidine was more effective than stavudine, lamivudine, nelfinavir, and nevirapine against all three HIV-1 isolates. The combination of stampidine with zidovudine + lamivudine was more effective than the combination of nelfinavir or nevirapine with zidovudine lamivudine against all three HIV-1 isolates. The combination of stampidine with nelfinavir was more effective than zidovudine + lamivudine as well as the combination of zidovudine + lamivudine with nelfinavir. The combination of stampidine with lamivudine + nelfinavir was more effective than the combination of zidovudine with lamivudine + nelfinavir. The combination of stampidine with lamivudine + nevirapine was more effective than the combination of stavudine with lamivudine + nevirapine. These findings demonstrate that (a) stampidine, as well as its combinations with the standard anti-HIV drugs zidovudine, lamivudine, nelfinavir or nevirapine, are potent inhibitors of HIV-1 replication in human peripheral blood mononuclear cells, and (b) replacement of either zidcovudine, zidovudine+lamivudine or stavudine in 3-drug cocktails with stampidine resulted in greater anti-HIV potency in vitro.

Zidampidine, an aryl phosphate derivative of AZT: in vivo pharmacokinetics, metabolism, toxicity, and anti-viral efficacy against hemorrhagic fever caused by Lassa virus.

The pharmacokinetics, metabolism, and toxicity of Zidampidine, an aryl phosphate derivative of AZT, 3'-azidothymidine-5'-[p-bromophenyl methoxyalaninyl phosphate] were investigated in CD-1 mice. Following iv injection, Zidampidine was rapidly converted to its metabolites Ala-AZT-MP and AZT. Zidampidine was not toxic to mice at doses up to 250mg/kg. We next examined the therapeutic effect of Zidampidine in CBA mice challenged with intracerebral injections of the Josiah strain of Lassa virus. Mice were treated either with vehicle or non-toxic doses of Zidampidine administered intraperitoneally 24h prior, 1h prior, and 24, 48, 72, and 96h after virus inoculation. The probability of survival following the Lassa challenge was significantly improved for Zidampidine-treated mice (Kaplan Meier, Log-Rank p value<0.0001). This pilot study provides the basis for future preclinical evaluation of Zidampidine and its potential as a new agent for the treatment of viral hemorrhagic fevers caused by Lassa virus.

Enzymatic hydrolysis of stampidine and other stavudine phosphoramidates in the presence of mammalian proteases.

Mammalian proteases have not been implicated in the metabolism of any nucleoside phosphoramidate prodrug. The results presented herein provide unprecedented and conclusive experimental evidence that mammalian proteases are capable of hydrolyzing stavudine phosphoramidates. Specifically, cathepsin B and Proteinase K are able to metabolize stampidine and other phosphoramidate derivatives of stavudine. Additionally, cathepsin B exhibits chiral selectivity at the phosphorus center. The elucidation of the metabolic pathways leading to activation of stampidine may provide the basis for pharmacologic interventions aimed at modulating the metabolism and thereby improving the therapeutic window of stampidine as an anti-HIV agent.