Santosh Kumar Upadhyay

NMR and molecular modelling studies on the interaction of fluconazole with β-cyclodextrin

BackgroundFluconazole (FLZ) is a synthetic, bistriazole antifungal agent, effective in treating superficial and systemic infections caused by Candida species. Major challenges in formulating this drug for clinical applications include solubility enhancement and improving stability in biological systems. Cyclodextrins (CDs) are chiral, truncated cone shaped macrocyles, and can easily encapsulate fluconazole inside their hydrophobic cavity. NMR spectroscopy has been recognized as an important tool for the interaction study of cyclodextrin and pharmaceutical compounds in solution state.ResultsInclusion complex of fluconazole with β-cyclodextrins (β-CD) were investigated by applying NMR and molecular modelling methods. The 1:1 stoichiometry of FLZ:β-CD complex was determined by continuous variation (Jobs plot) method and the overall association constant was determined by using Scotts method. The association constant was determined to be 68.7 M-1 which is consistent with efficient FLZ:β-CD complexation. The shielding of cavity protons of β-CD and deshielding of aromatic protons of FLZ in various1H-NMR experiments show complexation between β-CD and FLZ. Based on spectral data obtained from 2D ROESY, a reasonable geometry for the complex could be proposed implicating the insertion of the m-difluorophenyl ring of FLZ into the wide end of the torus cavity of β-CD. Molecular modelling studies were conducted to further interpret the NMR data. Indeed the best docked complex in terms of binding free energy supports the model proposed from NMR experiments and the m-difluorophenyl ring of FLZ is observed to enter into the torus cavity of β-CD from the wider end.ConclusionVarious NMR spectroscopic studies of FLZ in the presence of β-CD in D2O at room temperature confirmed the formation of a 1:1 (FLZ:β-CD) inclusion complex in which m-difluorophenyl ring acts as guest. The induced shift changes as well as splitting of most of the signals of FLZ in the presence of β-CD suggest some chiral differentiation of guest by β-CD.

Structural Insights into the Acyl Intermediates of the Plasmodium falciparum Fatty Acid Synthesis Pathway THE MECHANISM OF EXPANSION OF THE ACYL CARRIER PROTEIN CORE

Acyl carrier protein (ACP) plays a central role in fatty acid biosynthesis. However, the molecular machinery that mediates its function is not yet fully understood. Therefore, structural studies were carried out on the acyl-ACP intermediates of Plasmodium falciparum using NMR as a spectroscopic probe. Chemical shift perturbation studies put forth a new picture of the interaction of ACP molecule with the acyl chain, namely, the hydrophobic core can protect up to 12 carbon units, and additional carbons protrude out from the top of the hydrophobic cavity. The latter hypothesis stems from chemical shift changes observed in Cα and Cβ of Ser-37 in tetradecanoyl-ACP. 13C,15N-Double-filtered nuclear Overhauser effect (NOE) spectroscopy experiments further substantiate the concept; in octanoyl (C8)- and dodecanoyl (C12)-ACP, a long range NOE is observed within the phosphopantetheine arm, suggesting an arch-like conformation. This NOE is nearly invisible in tetradecanoyl (C14)-ACP, indicating a change in conformation of the prosthetic group. Furthermore, the present study provides insights into the molecular mechanism of ACP expansion, as revealed from a unique side chain-to-backbone hydrogen bond between two fairly conserved residues, Ile-55 HN and Glu-48 O. The backbone amide of Ile-55 HN reports a pKa value for the carboxylate, ∼1.9 pH units higher than model compound value, suggesting strong electrostatic repulsion between helix II and helix III. Charge-charge repulsion between the helices in combination with thrust from inside due to acyl chain would energetically favor the separation of the two helices. Helix III has fewer structural restraints and, hence, undergoes major conformational change without altering the overall-fold of P. falciparum ACP.

Resolving the functions of peptidylprolyl isomerases: insights from the mutagenesis of the nuclear FKBP25 enzyme

Peptidylprolyl isomerases have been implicated in chromatin regulation through their association with histones, chromatin-modifying enzymes and DNA-binding transcription factors. As with other post-translational modifications to proteins, a mechanistic understanding of the regulation of biological processes is fostered by loss-of-function studies both inxa0vitro and inxa0vivo. For peptidylprolyl isomerases, this can be accomplished with small-molecule inhibitors with high affinity for the isomerase active site or by mutation of amino acid residues that contribute to catalysis. In the present article, we review caveats to each of these approaches, and place emphasis on the thorough characterization of loss-of-function mutations in FKBPs (FK506-binding proteins). Using a case study of mutagenesis of the nuclear FKBP25 peptidylprolyl isomerase enzyme, we demonstrate that certain mutations generate a loss-of-function phenotype because they induce a complete loss of the FKBP domain fold, whereas other mutations are surgical in that they ablate catalytic isomerase activity, while maintaining domain structure. Peptidylprolyl isomerases are thought to have both catalytic and non-catalytic functions, but differentiating between these mechanisms has proved to be challenging. The domain-destabilizing and surgical mutants described will facilitate the characterization of these two reported functions of peptidylprolyl isomerases.

Transcriptome sequencing of a thalloid bryophyte; Dumortiera hirsuta (Sw) Nees: assembly, annotation, and marker discovery

Bryophytes are the first land plants but are scarcely studied at the molecular level. Here, we report transcriptome sequencing and functional annotation of Dumortiera hirsuta, as a representative bryophyte. Approximately 0.5 million reads with ~195u2009Mb data were generated by sequencing of mRNA using 454 pyrosequencer. De novo assembly of reads yielded 85,240 unigenes (12,439 contigs and 72,801 singletons). BlastX search at NCBI-NR database showed similarity of 33,662 unigenes with 10-10 e-value. A total of 23,685 unigenes were annotated at TAIR10 protein database. The annotated unigenes were further classified using the Gene Ontology. Analysis at Kyoto Encyclopedia of Genes and Genomes pathway database identified 95 pathways with significant scores, among which metabolic and biosynthesis of secondary metabolite were the major ones. Phenylpropanoid pathway was elucidated and selected genes were characterized by real time qPCR. We identified 447 transcription factors belonging to 41 families and 1594 eSSRs in 1479 unigenes. D. hirsuta unigenes showed homology across the taxa from algae to angiosperm indicating their role as the connecting link between aquatic and terrestrial plants. This could be a valuable genomic resource for molecular and evolutionary studies. Further, it sheds light for the isolation and characterization of new genes with unique functions.

NMR spectroscopic study of inclusion complexes of cetirizine dihydrochloride and β-cyclodextrin in solution

Cetirizine dihydrochloride (CTZ), an antihistamine, forms two 1:1 inclusion complexes with β-cyclodextrin (β-CD) in aqueous solution as confirmed by detailed 1 H NMR, COSY and ROESY spectroscopic studies. The stoichiometry and overall binding constant of the complexes were determined by the treatment of 1 H NMR chemical shift change (∆δ) data. Most of the CTZ protons exhibited splitting in the presence of β-CD.

In-depth 2-DE reference map of Aspergillus fumigatus and its proteomic profiling on exposure to itraconazole

Aspergillus fumigatus (A. fumigatus) is a medically important opportunistic fungus that may lead to invasive aspergillosis in humans with weak immune system. Proteomic profiling of this fungus on exposure to itraconazole (ITC), an azole antifungal drug, may lead to identification of its molecular targets and better understanding on the development of drug resistance against ITC in A. fumigatus. Here, proteome analysis was performed using 2-DE followed by mass spectrometric analysis which resulted in identification of a total of 259 unique proteins. Further, proteome profiling of A. fumigatus was carried out on exposure to ITC, 0.154 μg/ml, the minimum inhibitory concentration (MIC50). Image analysis showed altered levels of 175 proteins (66 upregulated and 109 downregulated) of A. fumigatus treated with ITC as compared to the untreated control. Peptide mass fingerprinting led to the identification of 54 proteins (12 up-regulated and 42 down-regulated). The differentially expressed proteins include proteins related to cell stress, carbohydrate metabolism and amino acid metabolism. We also observed four proteins, including nucleotide phosphate kinase (NDK), that are reported to interact with calcineurin, a protein involved in regulation of cell morphology and fungal virulence. Comparison of differentially expressed proteins on exposure to ITC with artemisinin (ART), an antimalarial drug with antifungal activity(1), revealed a total of 26 proteins to be common among them suggesting that common proteins and pathways are targeted by these two antifungal agents. The proteins targeted by ITC may serve as important leads for development of new antifungal drugs.

Backbone chemical shift assignments of the acyl-acyl carrier protein intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum

We report the backbone chemical shift assignments of the acyl-acyl carrier protein (ACP) intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum. The acyl-ACP intermediates butyryl (C4), -octanoyl (C8), -decanoyl (C10), -dodecanoyl (C12) and -tetradecanoyl (C14)-ACPs display marked changes in backbone HN, Cα and Cβ chemical shifts as a result of acyl chain insertion into the hydrophobic core. Chemical shift changes cast light on the mechanism of expansion of the acyl carrier protein core.

The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module

Abstract Prolyl isomerases are defined by a catalytic domain that facilitates the cis–trans interconversion of proline residues. In most cases, additional domains in these enzymes add important biological function, including recruitment to a set of protein substrates. Here, we report that the N-terminal basic tilted helix bundle (BTHB) domain of the human prolyl isomerase FKBP25 confers specific binding to double-stranded RNA (dsRNA). This binding is selective over DNA as well as single-stranded oligonucleotides. We find that FKBP25 RNA-association is required for its nucleolar localization and for the vast majority of its protein interactions, including those with 60S pre-ribosome and early ribosome biogenesis factors. An independent mobility of the BTHB and FKBP catalytic domains supports a model by which the N-terminus of FKBP25 is anchored to regions of dsRNA, whereas the FKBP domain is free to interact with neighboring proteins. Apart from the identification of the BTHB as a new dsRNA-binding module, this domain adds to the growing list of auxiliary functions used by prolyl isomerases to define their primary cellular targets.

Structural insights into the inclusion complexes between clomiphene citrate and β-cyclodextrin: The mechanism of preferential isomeric selection

A major challenge in pharmaceuticals for clinical applications is to alter the solubility, stability, and toxicity of drug molecules in living systems. Cyclodextrins (CDs) have the ability to form host-guest inclusion complexes with pharmaceuticals for further development of new drug formulations. The inclusion complex of clomiphene citrate (CL), a poorly water-soluble drug, with native β-cyclodextrin (β-CD) was characterized by a one and two-dimensional nuclear magnetic resonance (NMR) spectroscopic approach and also by molecular docking techniques. Here we report NMR and a computational approach in preferential isomeric selection of CL, which exists in two stereochemical isomers, enclomiphene citrate (ENC; E isomer) and zuclomiphene citrate (ZNC; Z isomer) with β-CD. β-CD cavity protons, namely, H-3 and H-5, experienced shielding in the presence of CL. The aromatic ring protons of the CL molecule were observed to be deshielded in the presence of β-CD. The stoichiometric ratio of the β-CD:CL inclusion complex was observed by NMR and found to be 1:1. The overall binding constant of β-CD:CL inclusion complexes was based on NMR chemical shifts and was calculated to be 50.21xa0M-1 . The change in Gibbs free energy (∆G) was calculated to be -9.80 KJ mol-1 . The orientation and structure of the β-CD:CL inclusion complexes are proposed on the basis of NMR and molecular docking studies. 2D 1 H-1 H ROESY confirmed the involvement of all three aromatic rings of CL in the inclusion complexation with β-CD in the solution, confirming the multiple equilibria between β-CD and CL. Molecular docking and 2D 1 H-1 H ROESY provide insight into the inclusion complexation of two isomers of CL into the β-CD cavity. A molecular docking technique further provided the different binding affinities of the E and Z isomers of CL with β-CD and confirmed the preference of the Z isomer binding for β-CD:CL inclusion complexes. The study indicates that the formation of a hydrogen bond between -O- of CL and the hydrogen atom of the hydroxyl group of β-CD was the main factor for noncovalent β-CD:CL inclusion complex formation and stabilization in the aqueous phase.

De novo characterization of Phenacoccus solenopsis transcriptome and analysis of gene expression profiling during development and hormone biosynthesis

The cotton mealybug Phenacoccus solenopsis is a devastating pest of cotton causing tremendous loss in the yield of crops each year. Widespread physiological and biological studies on P. solenopsis have been carried out, but the lack of genetic information has constrained our understanding of the molecular mechanisms behind its growth and development. To understand and characterize the different developmental stages, RNA-Seq platform was used to execute de-novo transcriptome assembly and differential gene expression profiling for the eggs, first, second, third instar and adult female stages. About 182.67 million reads were assembled into 93,781 unigenes with an average length of 871.4u2009bp and an N50 length of 1899 bp. These unigenes sequences were annotated and classified by performing NCBI non-redundant (Nr) database, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Clusters of Orthologous Groups (COG), Gene ontology (GO), the Swiss-Prot protein database (Swiss-Prot), and nearest related organism Acyrthosiphon pisum (pea aphid) database. To get more information regarding the process of metamorphosis, we performed a pairwise comparison of four developmental stages and obtained 29,415 differentially expressed genes. Some of the differentially expressed genes were associated with functional protein synthesis, anti-microbial protection, development and hormone biosynthesis. Functional pathway enrichment analysis of differentially expressed genes showed the positive correlation with specific physiological activities of each stage, and these results were confirmed by qRT-PCR experiments. This study gives a valuable genomics resource of P. solenopsis covering all its developmental stages and will promote future studies on biological processes at the molecular level.