A-kinase anchoring proteins are enriched in the central pair microtubules of motile cilia in Chlamydomonas reinhardtii.

Cilia are microtubule-based sensory organelles present in a number of eukaryotic cells. Mutations in the genes encoding ciliary proteins cause ciliopathies in humans. A-kinase anchoring proteins (AKAPs) tether ciliary signaling proteins such as protein kinase A (PKA). The dimerization and docking domain (D/D) on the RIIα subunit of PKA interacts with AKAPs. Here, we show that AKAP240 from the central-pair microtubules of Chlamydomonas reinhardtii cilia uses two C-terminal amphipathic helices to bind to its partner FAP174, an RIIα-like protein with a D/D domain at the N-terminus. Co-immunoprecipitation using anti-FAP174 antibody with an enriched central-pair microtubule fraction isolated seven interactors whose mass spectrometry analysis revealed proteins from the C2a (FAP65, FAP70, and FAP147) and C1b (CPC1, HSP70A, and FAP42) microtubule projections and FAP75, a protein whose sub-ciliary localization is unknown. Using RII D/D and FAP174 as baits, we identified two additional AKAPs (CPC1 and FAP297) in the central-pair microtubules.

Design, synthesis and SAR of antitubercular benzylpiperazine ureas.

N-furfuryl piperazine ureas disclosed by scientists at GSK Tres Cantos were chosen as antimycobacterial hits from a phenotypic whole-cell screen. Bioisosteric replacement of the furan ring in the GSK Tres Cantos molecules with a phenyl ring led to molecule (I) with an MIC of 1 µM against Mtb H37Rv, low cellular toxicity (HepG2 IC50 ~ 80 µM), good DMPK properties and specificity for Mtb. With the aim of delineating the SAR associated with (I), fifty-five analogs were synthesized and screened against Mtb. The SAR suggests that the piperazine ring, benzyl urea and piperonyl moieties are essential signatures of this series. Active compounds in this series are metabolically stable, have low cellular toxicity and are valuable leads for optimization. Molecular docking suggests these molecules occupy the Q0 site of QcrB like Q203. Bioisosteric replacement of N-furfuryl piperazine-1-carboxamides yielded molecule (I) a novel lead with satisfactory PD, metabolism, and toxicity profiles.

Identification of potential antileishmanial 1,3-disubstituted-4-hydroxy-6-methylpyridin-2(1H)-ones, in vitro metabolic stability, cytotoxicity and molecular modeling studies.

We report the synthesis and in vitro evaluation of 1,3-disubstituted-4-hydroxy-6-methylpyridin-2(1H)-one derivatives against Leishmania donovani. Amongst the compound library synthesized, molecules 3d, 3f, 3h, 3i, 3l, and 3m demonstrated substantial dose-dependent killing of the promastigotes. Their IC50 values range from 55.0 to 77.0 µg/ml, with 3m (IC50 55.75 µg/ml) being equipotent with amphotericin B (IC50 50.0 µg/ml, used as standard). The most active compound 3m, is metabolically stable in rat liver microsomes. Furthermore, the molecules are highly specific against leishmania as shown by their weak antibacterial and antifungal activity. In vitro cytotoxicity studies show the compounds lack any cytotoxicity. Furthermore, molecular modeling studies show plausibility of binding to Leishmania donovani topoisomerase 1 (LdTop1). Structure activity relationships reveal bulky substitutions on the pyridone nitrogen are well-tolerated, and such compounds have better binding affinity. Intramolecular hydrogen bonds confer some rigidity to the molecules, rendering a degree of planarity akin to topotecan. Taken together, we emphasis the merits of molecules possessing the 1,3-disubstituted-4-hydroxy-6-methylpyridin-2(1H)-one skeleton as potential antileishmanial agents warranting further investigation.

Designing Antibacterial Peptides with Enhanced Killing Kinetics.

Antimicrobial peptides (AMPs) are gaining attention as substitutes for antibiotics in order to combat the risk posed by multi-drug resistant pathogens. Several research groups are engaged in design of potent anti-infective agents using natural AMPs as templates. In this study, a library of peptides with high sequence similarity to Myeloid Antimicrobial Peptide (MAP) family were screened using popular online prediction algorithms. These peptide variants were designed in a manner to retain the conserved residues within the MAP family. The prediction algorithms were found to effectively classify peptides based on their antimicrobial nature. In order to improve the activity of the identified peptides, molecular dynamics (MD) simulations, using bilayer and micellar systems could be used to design and predict effect of residue substitution on membranes of microbial and mammalian cells. The inference from MD simulation studies well corroborated with the wet-lab observations indicating that MD-guided rational design could lead to discovery of potent AMPs. The effect of the residue substitution on membrane activity was studied in greater detail using killing kinetic analysis. Killing kinetics studies on Gram-positive, negative and human erythrocytes indicated that a single residue change has a drastic effect on the potency of AMPs. An interesting outcome was a switch from monophasic to biphasic death rate constant of Staphylococcus aureus due to a single residue mutation in the peptide.

Preferential Formulation of Second Generation Antipsychotic Asenapine as Inclusion Complex with Sulphobutylether-ßCD (Captisol): In vitro and In vivo Evaluation.

BACKGROUND: Asenapine is an anti-psychotic agent approved by the US-FDA for treatment of acute schizophrenia and manic or bipolar I disorder in adults. It is poorly absorbed when administered orally, hence exhibits poor oral bioavailability, which limits its use in clinical practice. OBJECTIVE: Enhancement in solubility of asenapine through complexation with three different cyclodextrins, viz. ßCD, HPßCD and sulphobutylether-ßCD (Captisol®) was attempted and compared due to its poor bioavailability. METHOD: Kneading method was used for preparation of inclusion complexes which were characterized by FTIR, DSC, and XRD methods. Extent of binding and stability of the 1:1 inclusion complexes were evaluated by molecular modelling and phase solubility studies. Pharmacokinetic studies were also carried out of these inclusion complexes. RESULTS: Captisol® complex was the most stable amongst all complexes showing 4.9 times solubility enhancement of asenapine and 96% drug release at the end of 60 min, whereas asenapine maleate (uncomplexed drug) was released completely at the end of 120min. The Cmax and AUC values of Captisol® asenapine complex (AS-Captisol complex) were 2.8 and 2.3 times higher than the uncomplexed drug. CONCLUSION: This study thus demonstrated that Captisol® inclusion complex is an effective strategy for solubility and bioavailability enhancement of asenapine.

Discovery of new leads against Mycobacterium tuberculosis using scaffold hopping and shape based similarity.

BM212 [1,5-diaryl-2-methyl-3-(4-methylpiperazin-1-yl)-methyl-pyrrole] is a pyrrole derivative with strong inhibitory activity against drug resistant Mycobacterium tuberculosis and mycobacteria residing in macrophages. However, it was not pursued because of its poor pharmacokinetics and toxicity profile. Our goal was to design and synthesize new antimycobacterial BM212 analogs with lower toxicity and better pharmacokinetic profile. Using the scaffold hopping approach, three structurally diverse heterocycles - 2,3-disubstituted imidazopyridines, 2,3-disubstituted benzimidazoles and 1,2,4-trisubstituted imidazoles emerged as promising antitubercular agents. All compounds were synthesized through easy and convenient methods and their structures confirmed by IR, 1H NMR, 13C NMR and MS. In-vitro cytotoxicity studies on normal kidney monkey cell lines and HepG2 cell lines, as well as metabolic stability studies on rat liver microsomes for some of the most active compounds, established that these compounds have negligible cytotoxicity and are metabolically stable. Interestingly the benzimidazole compound (4a) is as potent as the parent molecule BM212 (MIC 2.3µg/ml vs 0.7-1.5µg/ml), but is devoid of the toxicity against HepG2 cell lines (IC50 203.10µM vs 7.8µM).

Soluble curcumin amalgamated chitosan microspheres augmented drug delivery and cytotoxicity in colon cancer cells: In vitro and in vivo study.

In present investigation, initially curcumin was complexed with 2-HP-ß-CD (curcumin-2-HP-ß-CD-complex) in 1:1 ratio and later amalgamated with chitosan microspheres (curcumin-2-HP-ß-CD-CMs) for selective delivery in colon only through oral route of administration. Various analytical, spectral and in-silico docking techniques revealed that the curcumin was deeply inserted in the 2-HP-ß-CD cavity with apparent stability constant of 3.35×10-3M. Furthermore, the mean particle size of 6.8±2.6µm and +39.2±4.1mV surface charge of curcumin-2-HP-ß-CD-complex-CMs in addition to encapsulation efficiency of about 79.8±6.3% exhibited that the tailored microspheres were optimum for colon delivery of curcumin. This was also demonstrated in dissolution testing and standard cell proliferation assay in which curcumin-2-HP-ß-CD-complex-CMs exhibited maximum release in simulated colonic fluid (SCF, pH ∼7.0-8.0, almond emulsion-ß-glucosidase) with improved therapeutic index in HT-29 cells. Consistently, curcumin-2-HP-ß-CD-complex-CMs successively enhanced the colonic bio-distribution of curcumin by ∼8.36 folds as compared to curcumin suspension in preclinical pharmacokinetic studies. In conclusion, curcumin-2-HP-ß-CD-complex-CMs warrant further in vivo tumor regression study to establish its therapeutic efficacy in experimental colon cancer.

A Computational Model for Docking of Noncompetitive Neuraminidase Inhibitors and Probing their Binding Interactions with Neuraminidase of Influenza Virus H5N1.

BACKGROUND: With cases of emergence of drug resistance to the current competitive inhibitors of neuraminidase (NA) such as oseltamivir and zanamavir, there is a present need for an alternative approach in the treatment of avian influenza. With this in view, some flavones and chalcones were designed based on quercetin, the most active naturally occurring noncompetitive inhibitor. OBJECTIVE: We attempt to understand the binding of quercetin to H5N1-NA, and synthetic analogs of quercetin namely flavones and its precursors the chalcones using computational tools. METHODS: Molecular docking was done using Libdock. Molecular dynamics (MD) simulations were performed using Amber14. We synthesized the two compounds; their structures were confirmed by infrared spectroscopy, 1H-NMR, and mass spectrometry. These molecules were then tested for H5N1-NA inhibition and kinetics of inhibition. RESULTS: Molecular docking studies yielded two compounds i.e., 4'-methoxyflavone and 2'-hydroxy-4-methoxychalcone, as promising leads which identified them as binders of the 150-cavity of NA. Furthermore, MD simulation studies revealed that quercetin and the two compounds bind and hold the 150 loop in its open conformation, which ultimately perturbs the binding of sialic acid in the catalytic site. Estimation of the free energy of binding by MM-PBSA portrays quercetin as more potent than chalcone and flavone. These molecules were then determined as non-competitive inhibitors from the Lineweaver-Burk plots rendered from the enzyme kinetic studies. CONCLUSION: We conclude that non-competitive type of inhibition, as shown in this study, can serve as an effective method to block NA and evade the currently seen drug resistance.

Characterization of pioglitazone cyclodextrin complexes: Molecular modeling to in vivo evaluation.

AIMS: The objective of present study was to study the influence of different ß-cyclodextrin derivatives and different methods of complexation on aqueous solubility and consequent translation in in vivo performance of Pioglitazone (PE). MATERIAL AND METHODS: Three cyclodextrins: ß-cyclodextrin (BCD), hydroxypropyl-ß-cyclodextrin (HPBCD) and Sulfobutylether-7-ß-cyclodextrin (SBEBCD) were employed in preparation of 1:1 Pioglitazone complexes by three methods viz. co-grinding, kneading and co-evaporation. Complexation was confirmed by phase solubility, proton NMR, Fourier Transform Infrared spectroscopy, Differential Scanning Calorimetry (DSC) and X-Ray diffraction (XRD). Mode of complexation was investigated by molecular dynamic studies. Pharmacodynamic study of blood glucose lowering activity of PE complexes was performed in Alloxan induced diabetic rat model. RESULTS: Aqueous solubility of PE was significantly improved in presence of cyclodextrin. Apparent solubility constants were observed to be 254.33 M(-1) for BCD-PE, 737.48 M(-1) for HPBCD-PE and 5959.06 M(-1) for SBEBCD-PE. The in silico predictions of mode of inclusion were in close agreement with the experimental proton NMR observation. DSC and XRD demonstrated complete amorphization of crystalline PE upon inclusion. All complexes exhibited >95% dissolution within 10 min compared to drug powder that showed <40% at the same time. Marked lowering of blood glucose was recorded for all complexes. CONCLUSION: Complexation of PE with different BCD significantly influenced its aqueous solubility, improved in vitro dissolution and consequently translated into enhanced pharmacodynamic activity in rats.

Quantifying ligand-receptor interactions for gorge-spanning acetylcholinesterase inhibitors for the treatment of Alzheimer's disease.

There is a need for continued development of acetylcholinesterase (AChE) inhibitors that could prolong the life of acetylcholine in the synaptic cleft and also prevent the aggregation of amyloid peptides associated with Alzheimer's disease. The lack of a 3D-QSAR model which specifically deconvulates the type of interactions and quantifies them in terms of energies has motivated us to report a CoRIA model vis-à-vis the standard 3D-QSAR methods, CoMFA and CoMSIA. The CoRIA model was found to be statistically superior to the CoMFA and CoMSIA models and it could efficiently extract key residues involved in ligand recognition and binding to AChE. These interactions were quantified to gauge the magnitude of their contribution to the biological activity. In order to validate the CoRIA model, a pharmacophore map was first constructed and then used to virtually screen public databases, from which novel scaffolds were cherry picked that were not present in the training set. The biological activities of these novel molecules were then predicted by the CoRIA, CoMFA, and CoMSIA models. The hits identified were purchased and their biological activities were measured by the Ellman's method for AChE inhibition. The predicted activities are in unison with the experimentally measured biological activities.