Sandipan Chakraborty

Conformational transition in the substrate binding domain of β-secretase exploited by NMA and its implication in inhibitor recognition: BACE1–myricetin a case study

BACE1 is a key protease involved in the proteolysis of amyloid precursor protein (APP) that generates a toxic peptide amyloid beta (Aβ), a pathological feature of Alzheimers disease (AD). The enzyme is believed to possess an open and a closed conformation that corresponds to its free and inhibitor-bound form respectively. Here, we study the dynamic transition of BACE1 employing normal mode analysis (NMA) using a simplified elastic network model (ENM). Estimation of the catalytic cavity volume on the structures of BACE1 encoded by the lowest frequency normal mode reveals the dynamical transition of the enzyme from the open to the closed conformer. Detailed analysis reveals that concerted movement of different loop segments in the active site of the protein, namely flap regions, 10s loop, A loop and F loop, squeeze the catalytic cavity between the N-terminal and C-terminal lobe of the substrate binding domain of BACE1. We also propose that the NMA encoded multiple receptor conformations (MRC) of BACE1 elucidate the pharmacophoric feature necessary to inhibit the enzyme by a polyphenol, myricetin. van der Waals interaction is found to be the main driving force that guides the ligand induced conformational switching to the closed conformer. We suggest that NMA derived MRC of BACE1 is an efficient way to treat the receptor flexibility in docking and thus can be further applied in virtual screening and structure based drug design.

Binding and antioxidant properties of therapeutically important plant flavonoids in biomembranes: Insights from spectroscopic and quantum chemical studies

Plant flavonoids are emerging as novel therapeutic drugs for free radical mediated diseases, for which cell membranes mainly serve as targets for lipid peroxidation and related deleterious effects. Screening and characterization of these ubiquitous, therapeutically potent polyphenolic compounds require a clear understanding regarding their binding and possible locations in membranes, as well as quantitative estimates of relevant parameters such as partition coefficients, antioxidant and radical scavenging capacities. In this article we present perspectives emphasizing novel uses of the exquisitely sensitive two color intrinsic fluorescence of plant flavonoids (which arise due to highly efficient photoinduced excited state intramolecular proton transfer (ESIPT) reactions) to explore their binding to model biomembranes consisting of phosphatidylcholine liposomes. Extension of such studies to natural biomembranes of relevant interest is also exemplified. Spectrophotometric assays reveal that typical mono- as well as poly-hydroxy substituted flavonoids have remarkable inhibitory actions on lipid peroxidation, and are significantly more potent antioxidants (2.5-4 times higher) compared to the reference compound Trolox (an water soluble derivative of vitamin E). The structure-activity relationships emerging from such studies are consistent with theoretical predictions based on quantum chemical computations.

Contrasting binding of fisetin and daidzein in γ-cyclodextrin nanocavity.

Steady state and time resolved fluorescence along with anisotropy and induced circular dichroism (ICD) spectroscopy provide useful tools to observe and understand the behavior of the therapeutically important plant flavonoids fisetin and daidzein in γ-cyclodextrin (γ-CDx) nanocavity. Benesi-Hildebrand plots indicated 1:1 stoichiometry for both the supramolecular complexes. However, the mode of the binding of fisetin significantly differs from daidzein in γ-CDx, as is observed from ICD spectra which is further confirmed by docking studies. The interaction with γ-CDx proceeds mainly by the phenyl ring and partly by the chromone ring of fisetin whereas only the phenyl ring takes part for daidzein. A linear increase in the aqueous solubility of the flavonoids is assessed from the increase in the binding of the flavonoids with the γ-CDx cavity, which are determined by the gradual increase in the ICD signal, fluorescence emission as well as increase in fluorescence anisotropy with increasing (γ-CDx). This confirms γ-CDx as a nanovehicle for the flavonoids fisetin and daidzein in improving their bioavailability.

Effect of β-cyclodextrin on the molecular properties of myricetin upon nano-encapsulation: Insight from optical spectroscopy and quantum chemical studies

Myricetin, a bioactive plant flavonol, readily forms inclusion complex with the drug delivery vehicle beta-cyclodextrin (β-CD). Appearance of typical dual emission, consisting of normal (470 nm) and ESIPT tautomer (530 nm) bands, with concomitant rise in fluorescence intensity and dramatically blue shifted normal fluorescence of myricetin with increasing β-CD concentration, indicates facile entry of myricetin into the cavity of β-CD. The stoichiometry of the inclusion complex has been established to be equimolar (1:1), with an equilibrium constant of 439 ± 18 M(-1) at 25 °C. The driving force of inclusion is attributed to strong van der Waals interaction and formation of hydrogen bond between host (β-CD) and guest (myricetin). Both experimental and theoretical studies indicate that myricetin possibly incorporates within β-CD through its benzoyl moiety. Inclusion in β-CD increases the antioxidant potency of myricetin which has been attributed to the less delocalised HOMO and reduced HOMO-LUMO energy gap in the confined state.

A mechanistic insight into the amyloidogenic structure of hIAPP peptide revealed from sequence analysis and molecular dynamics simulation

A collective approach of sequence analysis, phylogenetic tree and in silico prediction of amyloidogenecity using bioinformatics tools have been used to correlate the observed species-specific variations in IAPP sequences with the amyloid forming propensity. Observed substitution patterns indicate that probable changes in local hydrophobicity are instrumental in altering the aggregation propensity of the peptide. In particular, residues at 17th, 22nd and 23rd positions of the IAPP peptide are found to be crucial for amyloid formation. Proline25 primarily dictates the observed non-amyloidogenecity in rodents. Furthermore, extensive molecular dynamics simulation of 0.24 μs have been carried out with human IAPP (hIAPP) fragment 19-27, the portion showing maximum sequence variation across different species, to understand the native folding characteristic of this region. Principal component analysis in combination with free energy landscape analysis illustrates a four residue turn spanning from residue 22 to 25. The results provide a structural insight into the intramolecular β-sheet structure of amylin which probably is the template for nucleation of fibril formation and growth, a pathogenic feature of type II diabetes.

Insight into the anti-amyloidogenic activity of polyphenols and its application in virtual screening of phytochemical database

Alzheimer’s Disease (AD) is the most frequent form of neurodegenerative disorder pathologically characterized by the presence of aggregates of Aβ in the region of the brain involved in memory and cognition. Polyphenols commonly found in several daily consumed food items and beverages have been demonstrated to inhibit Aβ aggregation using various biophysical or cell culture-based techniques. Here, insight into the anti-amyloidogenic activity of polyphenols has been explored using 2-D QSAR methodology implemented in CODESSA packages. Interestingly, hydrophobic interactions emerge as a significant descriptor for the recognition of amyloid fibril by polyphenols. Thus, it may be interpreted that highly potent polyphenols capably bind within the hydrophobic core region of the amyloid fibril and, thereby, destabilize the fibril by interrupting the hydrophobic interactions present within the fibril. Highly branched substituents and substituents with high degree of conformational flexibility on the polyphenolic structural scaffold enhance the fibril-destabilizing activity of compounds. In addition, specific electrostatic interaction between amyloid fibril and polyphenol plays axa0crucial role for the recognition of the fibril by polyphenols. The “application domain” of the derived QSAR model also has been defined, and the derived model has been used to screen a small in-house developed phytochemicals database comprising 200 compounds. 59 phytochemicals have been predicted as highly active anti-amyloidogenic phytochemicals. The modeling strategy is an efficient way to reduce the chemical search space and can selectively mine novel lead anti-amyloidogenic molecule from large chemical databases.

Sensing of hydrophobic cavity of serum albumin by an adenosine analogue: fluorescence correlation and ensemble spectroscopic studies.

Adenosine is a naturally occurring purine nucleoside that plays important role in various biochemical processes. We have studied the binding of TNP-Ado (trinitrophenylated-adenosine), a fluorescent analogue of adenosine (which itself is a weak fluorophore), with a model transport protein, bovine serum albumin (BSA). The binding affinity was determined using Fluorescence correlation spectroscopy (FCS) and compared with its value obtained from macroscopic fluorescence spectroscopic studies. Fluorescence and circular dichroism (CD) spectroscopies were employed together with molecular docking study to locate the probable binding site of TNP-Ado on BSA and its effect on the conformation and stability of BSA. Fluorescence studies showed that TNP-Ado binds to BSA in 1:1 stoichiometry via an entropically favoured process. Induced CD spectra revealed that a chiro-optical switching of TNP-Ado occurs upon binding to BSA. Results on urea-induced denaturation of BSA and docking study suggested that the binding site for the ligand is in the hydrophobic subdomain IIA of BSA, consistent with the results of other measurements. This study establishes TNP-Ado as a sensor of hydrophobic regions in proteins like serum albumin, having the capability of detecting a minimum concentration of 140ng/ml protein. FCS measurement of binding interaction of rhodamine-labeled TNP-Ado (RTNP-Ado) with BSA yielded an association constant of KFCS=(1.03±0.06) × 10(4)M(-1). The association constants (Ka) obtained for binding of BSA with rhodamine-free (i.e. TNP-Ado) and rhodamine-labeled (RTNP-Ado) ligands, obtained using the ensemble spectroscopic technique, were (2.3±0.06) × 10(5)M(-1) and (3.4±0.03) × 10(4)M(-1), respectively. The difference between the values of Ka for the free and labeled ligands suggests that fluorescent labeling of small molecules perceptibly interferes with the binding process. On the other hand, the difference in Ka obtained by FCS and ensemble techniques is due to the fact that while the former measures the change in the diffusion constant (i.e. size) of RTNP-Ado upon binding to BSA, the latter focuses on the change of tryptophan emission properties of BSA due to the presence of bound RTNP-Ado.

Mechanistic insight into the radical scavenging activity of polyphenols and its application in virtual screening of phytochemical library: an in silico approach

Polyphenols are amply present in fruits, vegetables, beverages and thus very common items in our diet. They are better characterized as antioxidants and are able to scavenge various radicals and singlet oxygen. The present study aims to provide structural and electronic insight into the antioxidant activity of polyphenols and identify novel natural antioxidant. With systematic searches of the large descriptor spaces available in CODESSA, we found that number of benzene ring (constitutional descriptors), electrostatic descriptors related to the hydrogen bonding ability and the lowest unoccupied molecular orbital (LUMO) energy of the polyphenols are very important descriptors related to the electron donation ability of polyphenols, evident from t test values and principal component analysis. Decreasing LUMO energy facilitates electron transfer and stability of the resulting polyphenolic radical increases with the number of attached aromatic ring due to enhanced resonance. Hydrogen bonding present in the parent polyphenol also facilitates electron donation and stabilizes the phenolic radical. Using multiple linear regressions, we have derived two quantitative structure activity relation (QSAR) models with four and five descriptors, respectively, and validated using cross-validation techniques. The calculated square correlation coefficient for four and five descriptor models is R2xa0=xa00.89 and R2xa0=xa00.9, respectively, with cross-validated squared correlation coefficient

Fragment-based Designing for the Generation of Novel Leads Against BACE1

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