Enhanced basepair dynamics pre-disposes protein-assisted flips of key bases in DNA strand separation during transcription initiation.

Localized separation of strands of duplex DNA is a necessary step in many DNA-dependent processes, including transcription and replication. Little is known about how these strand separations occur. The strand-separated E.coli RNA polymerase-promoter open-complex structure showed four bases of the non-template strand, the master base -11A, -7, -6 and +2, in a flipped state and inserted into protein pockets. To explore whether any property of these bases in the duplex state pre-disposes them to flipping, NMR studies were performed on a wild-type promoter in the duplex state. Measurement of relaxation times indicates that a limited number of base pairs, including the flipped ones, have faster opening rates than the rest. Molecular dynamics studies also show an inherently high dynamic character of the -11A:T base pair in the wild-type strand-paired state. In order to explore the role of the RNA polymerase in the flipping process, we have used 2-aminopurine as a fluorescent probe. Slower kinetics of the increase of 2-aminopurine fluorescence was observed with RNA polymerases containing several mutant σ70s. This may be interpreted as the protein playing an important role in enhancing the flipping rate. These results suggest that flipping of -11A, and perhaps other flipped bases observed in the open-complex, is facilitated by its inherent proclivity to open-up with further assistance from the protein, thus leading to a strand-open state. Other DNA-based processes that require strand-separation may use similar pathways for strand separation. We conclude that not only basepair stability, but also dynamics may play an important role in the strand-separation.

Chelerythrine-lysozyme interaction: spectroscopic studies, thermodynamics and molecular modeling exploration.

The binding of the iminium and alkanolamine forms of chelerythrine to lysozyme (Lyz) was investigated by spectroscopy and docking studies. The thermodynamics of the binding was studied by calorimetry. Spectroscopic evidence suggested that Trp-62 and Trp-63 in the ß-domain of the protein are closer to the binding site; moreover, the binding site was at a distance of 2.27 and 2.00 nm from the iminium and alkanolamine forms, respectively, according to the Forster theory of non-radiation energy transfer. The equilibrium binding constants for the iminium and alkanolamine forms at 298 K were evaluated to be 1.29 × 10(5) and 7.79 × 10(5) M(-1), respectively. The binding resulted in an alteration of the secondary structure of the protein with a distinct reduction of the helical organization. The binding of iminium was endothermic, involving electrostatic and hydrophobic interactions, while that of alkanolamine form was exothermic and dominated by hydrogen bonding interactions. Docking studies provided the atomistic details pertaining to the binding of both forms of chelerythrine and supported the higher binding in favour of the alkanolamine over the iminium. Furthermore, molecular dynamics study provided accurate insights regarding the binding of both chelerythrine forms in accordance with the experimental results obtained. Chelerythrine binding pocket involves the catalytic region and aggregation prone K-peptide region, which are sandwiched between one another. Overall, these results suggest that both the forms of the alkaloid bind to the protein but the neutral form has higher affinity than the cationic form.

Indirect read-out of the promoter DNA by RNA polymerase in the closed complex.

Transcription is initiated when RNA polymerase recognizes the duplex promoter DNA in the closed complex. Due to its transient nature, the closed complex has not been well characterized. How the initial promoter recognition occurs may offer important clues to regulation of transcription initiation. In this article, we have carried out single-base pair substitution experiments on two Escherichia coli promoters belonging to two different classes, the -35 and the extended -10, under conditions which stabilize the closed complex. Single-base pair substitution experiments indicate modest base-specific effects on the stability of the closed complex of both promoters. Mutations of base pairs in the -10 region affect the closed complexes of two promoters differently, suggesting different modes of interaction of the RNA polymerase and the promoter in the two closed complexes. Two residues on σ(70) which have been suggested to play important role in promoter recognition, Q437 and R436, were mutated and found to have different effects on the closed-complex stability. DNA circular dichroism (CD) and FRET suggest that the promoter DNA in the closed complex is distorted. Modeling suggests two different orientations of the recognition helix of the RNA polymerase in the closed complex. We propose that the RNA polymerase recognizes the sequence dependent conformation of the promoter DNA in the closed complex.

Discovery of safe and orally effective 4-aminoquinaldine analogues as apoptotic inducers with activity against experimental visceral leishmaniasis.

Novel antileishmanials are urgently required to overcome emergence of drug resistance, cytotoxic effects, and difficulties in oral delivery. Toward this, we investigated a series of novel 4-aminoquinaldine derivatives, a new class of molecules, as potential antileishmanials. 4-Aminoquinaldine derivatives presented inhibitory effects on L. donovani promastigotes and amastigotes (50% inhibitory concentration range, 0.94 to 127 µM). Of these, PP-9 and PP-10 were the most effective in vitro and demonstrated strong efficacies in vivo through the intraperitoneal route. They were also found to be effective against both sodium antimony gluconate-sensitive and -resistant Leishmania donovani strains in BALB/c mice when treated orally, resulting in more than 95% protection. Investigation of their mode of action revealed that killing by PP-10 involved moderate inhibition of dihydrofolate reductase and elicitation of the apoptotic cascade. Our studies implicate that PP-10 augments reactive oxygen species generation, evidenced from decreased glutathione levels and increased lipid peroxidation. Subsequent disruption of Leishmania promastigote mitochondrial membrane potential and activation of cytosolic proteases initiated the apoptotic pathway, resulting in DNA fragmentation and parasite death. Our results demonstrate that PP-9 and PP-10 are promising lead compounds with the potential for treating visceral leishmaniasis (VL) through the oral route.

Modeling the closed and open state conformations of the GABA(A) ion channel--plausible structural insights for channel gating.

Recent disclosure of high resolution crystal structures of Gloeobacter violaceus (GLIC) in open state and Erwinia chrysanthemii (ELIC) in closed state provides newer avenues to advance our knowledge and understanding of the physiologically and pharmacologically important ionotropic GABA(A) ion channel. The present modeling study envisions understanding the complex molecular transitions involved in ionic conductance, which were not evident in earlier disclosed homology models. In particular, emphasis was put on understanding the structural basis of gating, gating transition from the closed to the open state on an atomic scale. Homology modeling of two different physiological states of GABA(A) was carried out using their respective templates. The ability of induced fit docking in breaking the critical inter residue salt bridge (Glu155ß(2) and Arg207ß(2)) upon endogenous GABA docking reflects the perceived side chain rearrangements that occur at the orthosteric site and consolidate the quality of the model. Biophysical calculations like electrostatic mapping, pore radius calculation, ion solvation profile, and normal-mode analysis (NMA) were undertaken to address pertinent questions like the following: How the change in state of the ion channel alters the electrostatic environment across the lumen; How accessible is the Cl(-) ion in the open state and closed state; What structural changes regulate channel gating. A "Twist to Turn" global motion evinced at the quaternary level accompanied by tilting and rotation of the M2 helices along the membrane normal rationalizes the structural transition involved in gating. This perceived global motion hints toward a conserved gating mechanism among pLGIC. To paraphrase, this modeling study proves to be a reliable framework for understanding the structure function relationship of the hitherto unresolved GABA(A) ion channel. The modeled structures presented herein not only reveal the structurally distinct conformational states of the GABA(A) ion channel but also explain the biophysical difference between the respective states.

Rationalizing lead optimization by consensus 2D- CoMFA CoMSIA GRIND (3D) QSAR guided fragment hopping in search of γ-secretase inhibitors.

γ-Secretase (Gamma Secretase) is a potential drug target in Alzheimer's disease therapeutics. A sequel lead design study was undertaken on a series of bicyclononanes with an aim of identifying potent isofunctional chemotypes. Fragment-based bioisosteric replacement, which considers shape, chemistry, and electrostatics was carried out to mine over four million medicinally relevant fragments of Brood database. The resulting subset, thus, obtained was further mined using consensus QSAR developed from 2D and CoMFA, CoMSIA, GRIND (3D) QSAR predicted endpoints with superior statistical results. The employed consensus prediction and the predicted endpoint values were found to be in good agreement with the experimental values. The predictive ability of the generated model was validated using different statistical metrics, and similarity-based coverage estimation was carried out to define applicability boundaries. Few analogs designed, using the concept of bioisosterism, were found to be promising and could be considered for synthesis and subsequent screening.

Rationalizing fragment based drug discovery for BACE1: insights from FB-QSAR, FB-QSSR, multi objective (MO-QSPR) and MIF studies.

The ability to identify fragments that interact with a biological target is a key step in FBDD. To date, the concept of fragment based drug design (FBDD) is increasingly driven by bio-physical methods. To expand the boundaries of QSAR paradigm, and to rationalize FBDD using In silico approach, we propose a fragment based QSAR methodology referred here in as FB-QSAR. The FB-QSAR methodology was validated on a dataset consisting of 52 Hydroxy ethylamine (HEA) inhibitors, disclosed by GlaxoSmithKline Pharmaceuticals as potential anti-Alzheimer agents. To address the issue of target selectivity, a major confounding factor in the development of selective BACE1 inhibitors, FB-QSSR models were developed using the reported off target activity values. A heat map constructed, based on the activity and selectivity profile of the individual R-group fragments, and was in turn used to identify superior R-group fragments. Further, simultaneous optimization of multiple properties, an issue encountered in real-world drug discovery scenario, and often overlooked in QSAR approaches, was addressed using a Multi Objective (MO-QSPR) method that balances properties, based on the defined objectives. MO-QSPR was implemented using Derringer and Suich desirability algorithm to identify the optimal level of independent variables (X) that could confer a trade-off between selectivity and activity. The results obtained from FB-QSAR were further substantiated using MIF (Molecular Interaction Fields) studies. To exemplify the potentials of FB-QSAR and MO-QSPR in a pragmatic fashion, the insights gleaned from the MO-QSPR study was reverse engineered using Inverse-QSAR in a combinatorial fashion to enumerate some prospective novel, potent and selective BACE1 inhibitors.

Polyamines and its analogue modulates amyloid fibrillation in lysozyme: A comparative investigation.

BACKGROUND: Polyamines can induce protein aggregation that can be related to the physiology of the cellular function. Polyamines have been implicated in protein aggregation which may lead to neuropathic and non neuropathic amyloidosis. SCOPE OF REVIEW: Change in the level of polyamine concentration has been associated with ageing and neurodegeneration such as Parkinson's disease, Alzheimer's disease. Lysozyme aggregation in the presence of polyamines leads to non neuropathic amyloidosis. Polyamine analogues can suppress or inhibit protein aggregation suggesting their efficacy against amyloidogenic protein aggregates. MAJOR CONCLUSIONS: In this study we report the comparative interactions of lysozyme with the polyamine analogue, 1-naphthyl acetyl spermine in comparison with the biogenic polyamines through spectroscopy, calorimetry, imaging and docking techniques. The findings revealed that the affinity of binding varied as spermidine > 1-naphthyl acetyl spermine > spermine. The biogenic polyamines accelerated the rate of fibrillation significantly, whereas the analogue inhibited the rate of fibrillation to a considerable extent. The polyamines bind near the catalytic diad residues viz. Glu35 and Asp52, and in close proximity of Trp62 residue. However, the analogue showed dual nature of interaction where its alkyl amine region bind in same way as the biogenic polyamines bind to the catalytic site, while the naphthyl group makes hydrophobic contacts with Trp62 and Trp63, thereby suggesting its direct influence on fibrillation. GENERAL SIGNIFICANCE: This study, thus, potentiates, the development of a polyamine analogue that can perform as an effective inhibitor targeted towards aggregation of amyloidogenic proteins.

Combinatorial library enumeration and lead hopping using comparative interaction fingerprint analysis and classical 2D QSAR methods for seeking novel GABA(A) alpha(3) modulators.

Selective modulators of GABA(A) alpha(3) (gamma amino butyric acid alpha(3)) receptor are known to alleviate the side effects associated with nonspecific modulators. A follow up study was undertaken on a series of functionally selective phthalazines with an ideological credo of identifying more potent isofunctional chemotypes. A bioisosteric database enumerated using the combichem approach endorsed mining in a lead-like chemical space. Primary screening of the massive library was undertaken using the "Miscreen" toolkit, which uses sophisticated bayesian statistics for calculating bioactivity score. The resulting subset, thus, obtained was mined using a novel proteo-chemometric method that integrates molecular docking and QSAR formalism termed CoIFA (comparative interaction fingerprint analysis). CoIFA encodes protein-ligand interaction terms as propensity values based on a statistical inference to construct categorical QSAR models that assist in decision making during virtual screening. In the absence of an experimentally resolved structure of GABA(A) alpha(3) receptor, standard comparative modeling techniques were employed to construct a homology model of GABA(A) alpha(3) receptor. A typical docking study was then carried out on the modeled structure, and the interaction fingerprints generated based on the docked binding mode were used to derive propensity values for the interacting atom pairs that served as pseudo-energy variables to generate a CoIFA model. The classification accuracy of the CoIFA model was validated using different metrics derived from a confusion matrix. Further predictive lead mining was carried out using a consensus two-dimensional QSAR approach, which offers a better predictive protocol compared to the arbitrary choice of a single QSAR model. The predictive ability of the generated model was validated using different statistical metrics, and similarity-based coverage estimation was carried out to define applicability boundaries. Few analogs designed using the concept of bioisosterism were found to be promising and could be considered for synthesis and subsequent screening.

Cross-talk between allosteric and orthosteric binding sites of γ-amino butyric acid type A receptors (GABAA-Rs): A computational study revealing the structural basis of selectivity.

The γ-amino butyric acid type A receptors (GABAA-Rs) are GABA-gated chloride ion channels that mediate fast inhibitory neurotransmissions. Due to their essential role in normal brain function, neuromodulatory therapies are targeted at them for restoring GABA-mediated inhibition. The receptor modulation by benzodiazepine (BZD) shows therapeutically useful actions. The mechanisms, by which BZD-site performs selective transduction while modulating GABAA-Rs, and its correlation with the occurrence of sedation is not fully known. In pursuance, we performed a computational study starting from modeling of α2-subtype GABAA-R, docking of α1/2-selective ligands followed by molecular dynamics simulations of the obtained complexes. The results show that during early stages of activation, a) allosteric binding initiate structural changes through BZD-site for GABA-elicited activation; b) selective BZD-binders positively modulate orthosteric GABA-bound site with fin-like C- and F-loop movements, which supports twisting of inner and outer ß-barrel; c) modulation by α1/2-selective ligands was only evident at site 1, mimicking mandatory doubly bound state; d) strength of allosteric communication was prominent for α2-modulators, however, the basic nature of allosteric-orthosteric site cross-talk remains same for both α1/2-modulators; and e) ratio of hydrophobic:hydrophilic ligand contact surface decides α2-selectivity, less value of ratio favors it. These insights would enable us to design better α2-selective modulator/s. Altogether our computational study reveals early stages of allosteric modulation, highlighting subtype selective activation and pathways recommending GABA binding sites during selective modulation. Communicated by Ramaswamy H. Sarma.

Fragment-based virtual screening approach and molecular dynamics simulation studies for identification of BACE1 inhibitor leads.

Traditional structure-based virtual screening method to identify drug-like small molecules for BACE1 is so far unsuccessful. Location of BACE1, poor Blood Brain Barrier permeability and P-glycoprotein (Pgp) susceptibility of the inhibitors make it even more difficult. Fragment-based drug design method is suitable for efficient optimization of initial hit molecules for target like BACE1. We have developed a fragment-based virtual screening approach to identify/optimize the fragment molecules as a starting point. This method combines the shape, electrostatic, and pharmacophoric features of known fragment molecules, bound to protein conjugate crystal structure, and aims to identify both chemically and energetically feasible small fragment ligands that bind to BACE1 active site. The two top-ranked fragment hits were subjected for a 53 ns MD simulation. Principle component analysis and free energy landscape analysis reveal that the new ligands show the characteristic features of established BACE1 inhibitors. The potent method employed in this study may serve for the development of potential lead molecules for BACE1-directed Alzheimer's disease therapeutics.

Computational analysis of BACE1-ligand complex crystal structures and linear discriminant analysis for identification of BACE1 inhibitors with anti P-glycoprotein binding property.

More than 100 years of research on Alzheimer's disease didn't yield a potential cure for this dreadful disease. Poor Blood Brain Barrier (BBB) permeability and P-glycoprotein binding of BACE1 inhibitors are the major causes for the failure of these molecules during clinical trials. The design of BACE1 inhibitors with a balance of sufficient affinity to the binding site and little or no interaction with P-glycoproteins is indispensable. Identification and understanding of protein-ligand interactions are essential for ligand optimization process. Structure-based drug design (SBDD) efforts led to a steady accumulation of BACE1-ligand crystal complexes in the PDB. This study focuses on analyses of 153 BACE1-ligand complexes for the direct contacts (hydrogen bonds and weak interactions) observed between protein and ligand and indirect contacts (water-mediated hydrogen bonds), observed in BACE1-ligand complex crystal structures. Intraligand hydrogen bonds were analyzed, with focus on ligand P-glycoprotein efflux. The interactions are dissected specific to subsites in the active site and discussed. The observed protein-ligand and intraligand interactions were used to develop the linear discriminant model for the identification of BACE1 inhibitors with less or no P-glycoprotein binding property. Excellent statistical results and model's ability to correctly predict a new data-set with an accuracy of 92% is achieved. The results are retrospectively analyzed to give input for the design of potential BACE1 inhibitors.

Identification of novel hits as highly prospective dual agonists for mu and kappa opioid receptors: an integrated in silico approach.

Opioid agonists are used clinically for the treatment of acute and chronic pain, however, their clinical use is limited due to the presence of undesired side effects. Dual agonists, simultaneously targeting mu and kappa opioid receptors, show fewer side effects than that of selective agonists. In the present work, 2D- and 3D- Quantitative Structure Activity Relationship studies were performed on a series of aminomorphinan derivatives as dual agonists, using a wide range of descriptors. The aim of the study was to identify the structural requirements for the activity of these compounds towards mu and kappa opioid receptors and using the models, with best external predictability, for predicting the activities of new hits obtained from shape based virtual screening of drug like compounds from ZINC database. Genetic algorithm-based GFA and G/PLS techniques were used to derive the 2D-QSAR models. Common feature-based pharmacophore was used for aligning the compounds for 3D-QSAR. All the models were validated both internally and externally using statistical metrics. The coverage estimation of the models was carried out with applicability domain calculation. Six enriched hits were identified as novel prospective dual agonist based on good Blood Brain Barrier permeability and their activities towards mu and kappa opioid receptors, predicted by the best QSAR models. The known potent dual agonist, cyclorphan, and two highly prospective dual agonists were docked to both the receptors and binding free energies were calculated using MMGBSA. Molecular dynamics studies were performed on the docked complexes with both the receptors to establish stability of the complexes.

Aristolochic acid and its derivatives as inhibitors of snake venom L-amino acid oxidase.

Snake venom L-amino acid oxidase (LAAO) exerts toxicity by inducing hemorrhage, pneumorrhagia, pulmonary edema, cardiac edema, liver cell necrosis etc. Being well conserved, inhibitors of the enzyme may be synthesized using the template of the substrate, substrate binding site and features of the catalytic site of the enzyme. Previous findings showed that aristolochic acid (AA), a major constituent of Aristolochia indica, inhibits Russell's viper venom LAAO enzyme activity since, AA interacts with DNA and causes genotoxicity, derivatives of this compound were synthesized by replacing the nitro group to reduce toxicity while retaining the inhibitory potency. The interactions of AA and its derivatives with LAAO were followed by inhibition kinetics and surface plasmon resonance. Similar interactions with DNA were followed by absorption spectroscopy and atomic force microscopy. LAAO-induced cytotoxicity was evaluated by generation of reactive oxygen species (ROS), cell viability assays, confocal and epifluorescence microscopy. The hydroxyl (AA-OH) and chloro (AA-Cl) derivatives acted as inhibitors of LAAO but did not interact with DNA. The derivatives significantly reduced LAAO-induced ROS generation and cytotoxicity in human embryonic kidney (HEK 293) and hepatoma (HepG2) cell lines. Confocal images indicated that AA, AA-OH and AA-Cl interfered with the binding of LAAO to the cell membrane. AA-OH and AA-Cl significantly inhibited LAAO activity and reduced LAAO-induced cytotoxicity.

REMOVED: Novel aminoquinonoid analogues of diospyrin: Synthesis, antitumour activity and preliminary QSAR studies.

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Integration of ligand and structure based approaches for identification of novel MbtI inhibitors in Mycobacterium tuberculosis and molecular dynamics simulation studies.

Mycobacterium tuberculosis is an obligate pathogen of mammals and is responsible for more than two million deaths annually. The ability to acquire iron from the extracellular environment is a key determinant of pathogenicity in mycobacteria. M. tuberculosis acquires iron exclusively through the siderophores. Several lines of evidence suggest that siderophores have a critical role in bacterial growth and virulence. Hence, in the present study, we have used a combined ligand and structure-based drug design approach for identification of novel inhibitors against salicylate synthase MbtI, a unique and essential enzyme for the biosynthesis of siderophores in M. tuberculosis. We have generated the ligand based and structure based pharmacophores and validated exhaustively. From the validation results it was found that GH (Goodness of Hit) scores for the selected ligand based and structure based pharmacophore models were 0.89 and 0.97, respectively, which indicate that the quality of the pharmacophore models are acceptable as GH value is >0.7. The validated pharmacophores were used for screening the ZINC database. A total of 73 hits, obtained through various insilico screening techniques, were further enriched to 17 hits using docking studies. Molecular dynamics simulations were carried out to compare the binding mode and stability of complexes of MbtI bound with substrate, known inhibitors, and three top ranked hits. The results obtained in this study gave assurance about the identified hits as prospective inhibitors of MbtI.

Molecular Shape Analysis-Guided Virtual Screening Platform for Adenosine Kinase Inhibitors.

We propose a new application of molecular shape descriptors in hierarchical selection during virtual screening (VS). Here, a structure-based pharmacophore and docking-guided VS protocol have been evolved to identify inhibitors against adenosine kinase (AK). The knowledge gained on the shape requirements has been extrapolated in classifying active and inactive molecules against this target. This classification enabled us to pick the appropriate ligand conformation in the binding site. We have suggested a set of hierarchical filters for VS, from a simple molecular shape analysis (MSA) descriptor-based recursive models to docking scores. This approach permits a systematic study to understand the importance of spatial requirements and limitations for inhibitors against AK. Finally, the guidelines on how to select compounds for AK to achieve success have been highlighted. The utility of this approach has been suggested by giving an example of database screening for plausible active compounds.

Capturing state-dependent dynamic events of GABAA-receptors: a microscopic look into the structural and functional insights.

The γ-amino butyric acid type A receptors (GABAA-Rs) are the key players in the mammalian brain that meditate fast inhibitory neurotransmission events. The structural integrity of these ligand-gated ion channel controls chloride ion permeability, which in turn monitors important pharmacological functions. Despite ample studies on GABAA-Rs, there was a need for a study on full-length receptor structures, devoted to track structure-function correlations based on their dynamic behavior consideration. We have employed molecular dynamics simulations accompanied by other biophysical methods to shed light on sequential and unaddressed questions like How GABAA-R structure facilitates the entry of GABA molecules at its two orthosteric binding sites? After entry, what structural features and changes monitor site-wise GABA binding differences? In the same context, what are the roles and responsibilities of loops such as C and F? On physiologically relevant time scales, how open to close state transition occurs? How salt bridges such as E155-R207 and E153-R207 maintain state-dependent C-loop structures? In an attempt, our simulation study unravels the complete course of GABA binding-unbinding pathway. This provides us with the relevant understanding of state-dependent dynamic events of GABAA-Rs.

Structure-based designing of sordarin derivative as potential fungicide with pan-fungal activity.

Fungal infections have become a significant problem for immunosuppressed patients. Sordarin, a promising fungicidal agent, inhibits fungal protein synthesis by impairing elongation factor-2 (eEF2) function. Intriguingly, despite high sequence similarity among eEF2s from different species, sordarin has been shown to inhibit translation specifically in certain fungi while unable to do so in some other fungal species (e.g. Candida parapsilosis and Candida lusitaniae). The sordarin binding site on eEF2 as well as its mechanism of action is known. In a previous study, we have detailed the interactions between sordarin and eEF2 cavities from different fungal species at the molecular level and predicted the probable cause of sordarin sensitivity. Guided by our previous analysis, we aimed for computer-aided designing of sordarin derivatives as potential fungicidal agents that still remain ineffective against human eEF2. We have performed structural knowledge-based designing of several sordarin derivatives and evaluated predicted interactions of those derivatives with the sordarin-binding cavities of different eEF2s, against which sordarin shows no inhibitory action. Our analyses identify an amino-pyrrole derivative as a good template for further designing of promising broad-spectrum antifungal agents. The drug likeness and ADMET prediction on this derivative also supports its suitability as a drug candidate.

Structural and thermodynamic analysis of the binding of tRNA(phe) by the putative anticancer alkaloid chelerythrine: Spectroscopy, calorimetry and molecular docking studies.

The interaction of the putative anticancer alkaloid chelerythrine with tRNA(phe) was characterized by spectroscopy, calorimetry and molecular docking studies. The charged iminium form of chelerythrine binds with tRNA(phe) in a cooperative mode with a binding affinity value of (4.06±0.01)×10(5)M(-1). The neutral alkanolamine form does not bind to tRNA(phe) but in the presence of high concentration of tRNA(phe) this form gets converted to the iminium form and then binds with tRNA(phe). The partial intercalative mode of binding of chelerythrine to the tRNA(phe) was characterized from the steady state anisotropy, iodide ion-induced fluorescence quenching and viscosity measurements. Chelerythrine binding induced conformational perturbations in tRNA(phe) as observed from the circular dichroism spectroscopy. The strong binding was also supported by the ethidium bromide displacement assay. The binding was favoured by both enthalpy and entropy contributions. Although the binding was dependent on the [Na(+)], non-electrostatic forces contributed predominantly to the Gibbs energy change. The negative value of the heat capacity change proposed the involvement of hydrophobic forces in the binding. Molecular docking study was carried out to decipher the details of the recognition of tRNA(phe) by chelerythrine. The study provided insights about the chelerythrine binding pockets on tRNA(phe) and marked the necessary interactions for binding of chelerythrine molecule. Partially intercalative mode of the alkaloid binding was supported by docking studies. In total, docking studies corroborated well with our experiential observations. The structural and thermodynamic results of chelerythrine binding to tRNA(phe) may be helpful to develop new RNA therapeutic agents.