Molecular identification and functional analysis of Niemann-Pick type C2 protein in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae)

Niemann-Pick type C2 (NPC2) proteins in arthropods have been extensively differentiated and possibly duplicated according to environmental conditions and are probable to have different functions. The participation of NPC2 proteins in chemical communication in arthropods brings new objectives in environmental-friendly strategies for pest population control. In this study, NPC2 gene in Macrocentrus cingulum (McinNPC2) was newly identified by rapid amplification cDNA ends (RACE) technology. McinNPC2 amino acid sequence alignment with other representative NPC2 annotates to evaluate the highly conserved consensus amino acids, but with odorant binding proteins in M. cingulum show that only one consensus amino acid. Primary six-cysteine structures that are same to odorant binding proteins in M. cingulum were observed in McinNPC2. Phylogenetic analysis of McinNPC2 indicated that the nearest monophyletic group forming one clade with high posterior probability values clusters as Cyphomyrmex costatus (CcosNPC2) whereas the nearest evolutionary relation group as some odorant binding proteins. Moreover, quantitative real-time PCR (qPCR) measurements show that the McinNPC2 gene expression level in various tissues of the female is significantly and ubiquitously higher than in male, whereas the highest expression level in female antennae. We further explore the binding characterization of recombinant McinNPC2 to candidate odor molecules and did the modeling and docking simulations. The results showed ligands binding specificity and docking tests results indicate that β-ionone, an aroma compound commonly found in essential oils, can strongly bind with McinNPC2. In conclusion, we proposed that McinNPC2 may be involved in chemical communication and play roles in perception of plant volatiles.     

New indole based hybrid oxadiazole scaffolds with N-substituted acetamides: As potent anti-diabetic agents

Current study is based on the sequential conversion of indolyl butanoic acid (1) into ethyl indolyl butanoate (2), indolyl butanohydrazide (3), and 1,3,4-oxadiazole-2-thiol analogs (4) by adopting chemical transformations. In a parallel series of reactions, 2-bromo-N-phenyl/arylacetamides (7a-l) were synthesized by reacting different amines derivatives (5a-l) with 2-bromoacetyl bromide (6) to serve as electrophile. Then, the synthesized electrophiles (7a-l) were treated with nucleophilic 1,3,4-oxadiazole-2-thiol analog (4) to afford a range of N-substituted derivatives (8a-l). The structural confirmation of all the synthetic compounds was carried out by IR, ¹H-, ¹³C NMR, EI-MS, and CHN analysis data. All synthesized molecules (8a-l) were tested for their antidiabetic potential via inhibition of the α-glucosidase enzyme followed by their in silico study. Their cytotoxicity profile was also ascertained via hemolytic activity and all of them possessed very low cytotoxicity. Compounds 8h and 8l were found most active having IC₅₀ values 9.46 ± 0.03 µM and 9.37 ± 0.03 µM, respectively. However, all other molecules also exhibited good to moderate inhibition potential with IC₅₀ values between 12.68 ± 0.04–37.82 ± 0.07, compared to standard acarbose (IC₅₀ = 37.38 ± 0.12 µM), hence can be used as lead molecules for further research in order to get better antidiabetic agents.     

Revealing interaction between sulfobutylether‐β‐cyclodextrin and reserpine by chemiluminescence and site‐directed molecular docking

The host–guest interaction between sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD) and reserpine (RSP) is described using flow injection‐chemiluminescence (FI‐CL) and site‐directed molecular docking methods. It was found that RSP could inhibit the CL intensity produced by a luminol/SBE‐β‐CD system. The decrease in CL intensity was logarithmic over an RSP concentration range of 0.03 to 700.0 nM, giving a regression equation of ?I = 107.1lgC_RES + 186.1 with a detection limit of 10 pM (3σ). The CL assay was successfully applied in the determination of RSP in injection, saliva and urine samples with recoveries in the range 93.5–106.1%. Using the proposed CL model, the binding constant (K_CD‐R) and the stoichiometric ratio of SBE‐β‐CD/RSP were calculated to be 7.4 × 10⁶ M^‐1 and 1 : 1, respectively. Using molecular docking, it was confirmed that luminol binds to the small cavity of SBE‐β‐CD with a nonpolar interaction, while RSP targeted the larger cavity of SBE‐β‐CD and formed a 1 : 1 complex with hydrogen bonds. The proposed new CL method has the potential to become a powerful tool for revealing the host–guest interaction between CDs and drugs, as well as monitoring drugs with high sensitivity. Copyright © 2013 John Wiley & Sons, Ltd.     

Principles of docking: An overview of search algorithms and a guide to scoring functions

The docking field has come of age. The time is ripe to present the principles of docking, reviewing the current state of the field. Two reasons are largely responsible for the maturity of the computational docking area. First, the early optimism that the very presence of the "correct" native conformation within the list of predicted docked conformations signals a near solution to the docking problem, has been replaced by the stark realization of the extreme difficulty of the next scoring/ranking step. Second, in the last couple of years more realistic approaches to handling molecular flexibility in docking schemes have emerged. As in folding, these derive from concepts abstracted from statistical mechanics, namely, populations. Docking and folding are interrelated. From the purely physical standpoint, binding and folding are analogous processes, with similar underlying principles. Computationally, the tools developed for docking will be tremendously useful for folding. For large, multidomain proteins, domain docking is probably the only rational way, mimicking the hierarchical nature of protein folding. The complexity of the problem is huge. Here we divide the computational docking problem into its two separate components. As in folding, solving the docking problem involves efficient search (and matching) algorithms, which cover the relevant conformational space, and selective scoring functions, which are both efficient and effectively discriminate between native and non-native solutions. It is universally recognized that docking of drugs is immensely important. However, protein-protein docking is equally so, relating to recognition, cellular pathways, and macromolecular assemblies. Proteins function when they are bound to other molecules. Consequently, we present the review from both the computational and the biological points of view. Although large, it covers only partially the extensive body of literature, relating to small (drug) and to large protein-protein molecule docking, to rigid and to flexible. Unfortunately, when reviewing these, a major difficulty in assessing the results is the non-uniformity in the formats in which they are presented in the literature. Consequently, we further propose a way to rectify it here.     

Computer aided screening and evaluation of herbal therapeutics against MRSA infections

Methicillin resistant Staphylococcus aureus (MRSA), a pathogenic bacterium that causes life threatening outbreaks such as community-onset and nosocomial infections has emerged as 'superbug'. The organism developed resistance to all classes of antibiotics including the best known Vancomycin (VRSA). Hence, there is a need to develop new therapeutic agents. This study mainly evaluates the potential use of botanicals against MRSA infections. Computer aided design is an initial platform to screen novel inhibitors and the data finds applications in drug development. The drug-likeness and efficiency of various herbal compounds were screened by ADMET and docking studies. The virulent factor of most of the MRSA associated infections are Penicillin Binding Protein 2A (PBP2A) and Panton-Valentine Leukocidin (PVL). Hence, native structures of these proteins (PDB: 1VQQ and 1T5R) were used as the drug targets. The docking studies revealed that the active component of Aloe vera, β-sitosterol (3S, 8S, 9S, 10R, 13R, 14S, 17R) -17- [(2R, 5R)-5-ethyl-6-methylheptan-2-yl] -10, 13-dimethyl 2, 3, 4, 7, 8, 9, 11, 12, 14, 15, 16, 17- dodecahydro-1H-cyclopenta [a] phenanthren-3-ol) showed best binding energies of -7.40 kcal/mol and -6.34 kcal/mol for PBP2A and PVL toxin, respectively. Similarly, Meliantriol (1S-1-[ (2R, 3R, 5R)-5-hydroxy-3-[(3S, 5R, 9R, 10R, 13S, 14S, 17S)-3-hydroxy 4, 4, 10, 13, 14-pentamethyl-2, 3, 5, 6, 9, 11, 12, 15, 16, 17-decahydro-1H-cyclopenta[a] phenanthren-17-yl] oxolan-2-yl] -2- methylpropane-1, 2 diol), active compound in Azadirachta indica (Neem) showed the binding energies of -6.02 kcal/mol for PBP2A and -8.94 for PVL toxin. Similar studies were conducted with selected herbal compound based on pharmacokinetic properties. All in silico data tested in vitro concluded that herbal extracts of Aloe-vera, Neem, Guava (Psidium guajava), Pomegranate (Punica granatum) and tea (Camellia sinensis) can be used as therapeutics against MRSA infections.     

Analysis of the interacting surface of maurotoxin with the voltage‐gated Shaker B K+ channel

Maurotoxin (MTX) is a 34‐residue toxin that was isolated initially from the venom of the scorpion Scorpio maurus palmatus. Unlike the other toxins of the α‐KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C₁? C₅, C₂? C₆, C₃? C₄, and C₇? C₈ (instead of the conventional C₁? C₅, C₂? C₆, C₃? C₇, and C₄? C₈, herein referred to as Pi1‐like) that does not prevent its folding along the classic α/β scaffold of scorpion toxins. MTX_Pi1 is an MTX variant with a conventional pattern of disulfide bridging without any primary structure alteration of the toxin. Here, using MTX and/or MTX_Pi1 as models, we investigated how the type of folding influences toxin recognition of the Shaker B potassium channel. Amino acid residues of MTX that were studied for Shaker B recognition were selected on the basis of their homologous position in charybdotoxin, a three disulfide‐bridged scorpion toxin also active on this channel type. These residues favored either an MTX‐ or MTX_Pi1‐like folding. Our data indicate clearly that Lys²³ and Tyr³² (two out of ten amino acid residues studied) are the most important residues for Shaker B channel blockage by MTX. For activity on SKCa channels, the same amino acid residues also affect, directly or indirectly, the recognition of SK channels. The molecular modeling technique and computed docking indicate the existence of a correlation between the half cystine pairings of the mutated analogs and their activity on the Shaker B K⁺ channel. Overall, mutations in MTX could, or could not, change the reorganization of disulfide bridges of this molecule without affecting its α/β scaffold. However, changing of the peptide backbone (cross linking disulfide bridges from MTX‐like type vs MTX_Pi1‐like type) appears to have less impact on the molecule activity than mutation of certain key amino acids such as Lys²³ and Tyr³² in this toxin. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Molecular docking of potential inhibitors with the mTOR protein

The mTOR (mammalian or mechanistic Target of Rapamycin) is linked with oral cancer. Therefore, it is of interest to study the molecular docking-based binding of paclitaxel (a FDA approved drug for oral cancer) and its analogues with mTOR. Hence, we report the binding features of 10-Deacetyltaxol, 7-Epi-10-deacetyltaxol, 7-Epi-Taxol and 6alpha-Hydroxypaclitaxel with mTOR for further consideration.     

Shape-based virtual screening,docking, and molecular dynamics simulations to identify Mtb-ASADH inhibitors

Aspartate β-semialdehyde dehydrogenase (ASADH) is a key enzyme for the biosynthesis of essential amino acids and several important metabolites in microbes. Inhibition of ASADH enzyme is a promising drug target strategy against Mycobacterium tuberculosis (Mtb). In this work, in silico approach was used to identify potent inhibitors of Mtb-ASADH. Aspartyl β-difluorophosphonate (β-AFP), a known lead compound, was used to understand the molecular recognition interactions (using molecular docking and molecular dynamics analysis). This analysis helped in validating the computational protocol and established the participation of Arg99, Glu224, Cys130, Arg249, and His256 amino acids as the key amino acids in stabilizing ligand–enzyme interactions for effective binding, an essential feature is H-bonding interactions with the two arginyl residues at the two ends of the ligand. Best binding conformation of β-AFP was selected as a template for shape-based virtual screening (ZINC and NCI databases) to identify compounds that competitively inhibit the Mtb-ASADH. The top rank hits were further subjected to ADME and toxicity filters. Final filter was based on molecular docking analysis. Each screened molecule carries the characteristics of the highly electronegative groups on both sides separated by an average distance of 6?Å. Finally, the best predicted 20 compounds exhibited minimum three H-bonding interactions with Arg99 and Arg249. These identified hits can be further used for designing the more potent inhibitors against ASADH family. MD simulations were also performed on two selected compounds (NSC4862 and ZINC02534243) for further validation. During the MD simulations, both compounds showed same H-bonding interactions and remained bound to key active residues of Mtb-ASADH.     

Molecular docking analysis of stevioside with Akt and PPARγ

Stevioside is a diterpenoid glycoside consisting of an aglycone (steviol) and three glucose molecules. It is commonly used as an anti-hyperglycemic food because of its non-caloric property. Therefore, it is of interest to document the interactions of stevioside with AKT & PPAR-γ proteins using Autodock Vina PyRx docking techniques. Results of the docking studies indicate that stevioside had more than two hydrogen bond interactions with the AKT and PPAR γ protein for further consideration.     

Probing the interaction of anticancer drug temsirolimus with human serum albumin: molecular docking and spectroscopic insight

The binding interaction between temsirolimus, an important antirenal cancer drug, and HSA, an important carrier protein was scrutinized making use of UV and fluorescence spectroscopy. Hyper chromaticity observed in UV spectroscopy in the presence of temsirolimus as compared to free HSA suggests the formation of complex between HSA and temsirolimus. Fluorescence quenching experiments clearly showed quenching in the fluorescence of HSA in the presence of temsirolimus confirming the complex formation and also confirmed that static mode of interaction is operative for this binding process. Binding constant values obtained through UV and fluorescence spectroscopy reveal strong interaction; temsirolimus binds to HSA at 298 K with a binding constant of 2.9 × 10⁴ M^?1implying the strength of interaction. The negative Gibbs free energy obtained through Isothermal titration calorimetry as well as quenching experiments suggests that binding process is spontaneous. Molecular docking further provides an insight of various residues that are involved in this binding process; showing the binding energy to be -12.9 kcal/mol. CD spectroscopy was retorted to analyze changes in secondary structure of HSA; increased intensity in presence of temsirolimus showing changes in secondary structure of HSA induced by temsirolimus. This study is of importance as it provides an insight into the binding mechanism of an important antirenal cancer drug with an important carrier protein. Once temsirolimus binds to HSA, it changes conformation of HSA which in turn can alter the functionality of this important carrier protein and this altered functionality of HSA can be highlighted in variety of diseases.