Synthesis and Pharmacological Evaluation of Novel Triazole-Pyrimidine Hybrids as Potential Neuroprotective and Anti-neuroinflammatory Agents.

OBJECTIVE: Neuroprotection is a precise target for the treatment of neurodegenerative diseases, ischemic stroke, and traumatic brain injury. Pyrimidine and its derivatives have been proven to use antiviral, anticancer, antioxidant, and antimicrobial activity prompting us to study the neuroprotection and anti-inflammatory activity of the triazole-pyrimidine hybrid on human microglia and neuronal cell model. METHODS: A series of novel triazole-pyrimidine-based compounds were designed, synthesized and characterized by mass spectra, 1HNMR, 13CNMR, and a single X-Ray diffraction analysis. Further, the neuroprotective, anti-neuroinflammatory activity was evaluated by cell viability assay (MTT), Elisa, qRT-PCR, western blotting, and molecular docking. RESULTS: The molecular results revealed that triazole-pyrimidine hybrid compounds have promising neuroprotective and anti-inflammatory properties. Among the 14 synthesized compounds, ZA3-ZA5, ZB2-ZB6, and intermediate S5 showed significant anti-neuroinflammatory properties through inhibition of nitric oxide (NO) and tumor necrosis factor-α (TNF-α) production in LPS-stimulated human microglia cells. From 14 compounds, six (ZA2 to ZA6 and intermediate S5) exhibited promising neuroprotective activity by reduced expression of the endoplasmic reticulum (ER) chaperone, BIP, and apoptosis marker cleaved caspase-3 in human neuronal cells. Also, a molecular docking study showed that lead compounds have favorable interaction with active residues of ATF4 and NF-kB proteins. CONCLUSION: The possible mechanism of action was observed through the inhibition of ER stress, apoptosis, and the NF-kB inflammatory pathway. Thus, our study strongly indicates that the novel scaffolds of triazole-pyrimidine-based compounds can potentially be developed as neuroprotective and anti-neuroinflammatory agents.

Differential role of Pax6 and its interaction with Shh-Gli1-IDH2 axis in regulation of glioma growth and chemoresistance.

Glioma is a major brain tumor, and the associated mortality rate is very high. Contemporary therapies provide a chance of survival for 9-12 months. Therefore, a novel approach is essential to improve the survival rate. Sonic hedgehog (Shh) cell signaling is critical for early development in various tumors. This investigation attempted to explore the potential interaction and regulation of Shh-Gli1 cell signaling in association with paired box 6 (Pax6) and isocitrate dehydrogenase 2 (IDH2). The expression pattern of Shh, Gli1, Pax6, and IDH2 was examined by transcriptome analysis, immunohistochemistry, and confocal images. The results suggest the interaction of Shh-Gli1 cell signaling pathway with Pax6 and IDH2 and potential regulation. Thereafter, we performed protein-protein docking and molecular dynamic simulations (MDS) of Gli1 with Pax6 and IDH2. The results suggest differential dynamic interactions of Gli1-IDH2 and Gli1-Pax6. Gli1 knockdown downregulated the expression of Pax6 and upregulated the expression of IDH2. Moreover, Gli1 knockdown decreased the expression of the drug resistance gene MRP1. The knockdown of Pax6 gene in glioma cells downregulated the expression of Gli1 and IDH2 and promoted cell proliferation. Moreover, the efficacy of the treatment of glioma cells with temozolomide (TMZ) and Gli1 inhibitor GANT61 was higher than that of TMZ alone. MDS results revealed that the interactions of Gli1 with IDH2 were stronger and more stable than those with Pax6. Intriguingly, inhibition of Pax6 promoted glioma growth even in the presence of TMZ. However, the tumor-suppressive nature of Pax6 was altered when Gli1 was inhibited by GANT61, and it showed potential oncogenic character, as observed in other cancers. Therefore, we conclude that Pax6 interacted with IDH2 and Gli1 in glioma. Moreover, the Shh-Gli1-IDH2/Pax6 cell signaling axis provides a new therapeutic approach for inhibiting the progression of the disease and mitigating drug resistance in glioma.

Z-Guggulsterone Is a Potential Lead Molecule of Dawa-ul-Kurkum against Hepatocellular Carcinoma.

An ancient saffron-based polyherbal formulation, Dawa-ul-Kurkum (DuK), has been used to treat liver ailments and other diseases and was recently evaluated for its anticancer potential against hepatocellular carcinoma (HCC) by our research team. To gain further insight into the lead molecule of DuK, we selected ten active constituents belonging to its seven herbal constituents (crocin, crocetin, safranal, jatamansone, isovaleric acid, cinnamaldehyde, coumaric acid, citral, guggulsterone and dehydrocostus lactone). We docked them with 32 prominent proteins that play important roles in the development, progression and suppression of HCC and those involved in endoplasmic reticulum (ER) stress to identify the binding interactions between them. Three reference drugs for HCC (sorafenib, regorafenib, and nivolumab) were also examined for comparison. The in silico studies revealed that, out of the ten compounds, three of them-viz., Z-guggulsterone, dehydrocostus lactone and crocin-showed good binding efficiency with the HCC and ER stress proteins. Comparison of binding affinity with standard drugs was followed by preliminary in vitro screening of these selected compounds in human liver cancer cell lines. The results provided the basis for selecting Z-guggulsterone as the best-acting phytoconstituent amongst the 10 studied. Further validation of the binding efficiency of Z-guggulsterone was undertaking using molecular dynamics (MD) simulation studies. The effects of Z-guggulsterone on clone formation and cell cycle progression were also assessed. The anti-oxidant potential of Z-guggulsterone was analyzed through DPPH and FRAP assays. qRTPCR was utilized to check the results at the in vitro level. These results indicate that Z-guggulsterone should be considered as the main constituent of DuK instead of the crocin in saffron, as previously hypothesized.

Effect of Osmolytes on Conformational Behavior of Intrinsically Disordered Protein α-Synuclein.

α-Synuclein is an intrinsically disordered protein whose function in a healthy brain is poorly understood. It is genetically and neuropathologically linked to Parkinson's disease (PD). PD is manifested after the accumulation of plaques of α-synuclein aggregates in the brain cells. Aggregates of α-synuclein are very toxic and lead to the disruption of cellular homeostasis and neuronal death. α-Synuclein can also contribute to disease propagation as it may exert noxious effects on neighboring cells. Understanding the mechanism of α-synuclein aggregation will facilitate the problem of dealing with neurodegenerative diseases in general and that of PD in particular. Here, we have used molecular dynamics simulations to investigate the behavior of α-synuclein at various temperatures and in different concentrations of urea and trimethyl amine oxide. The residue region from 61 to 95 of α-synuclein is experimentally known as amyloidogenic. In our study, we have identified some other regions, which also have the propensity to form an aggregate besides this known sequence. Urea being a denaturant interacts more with these regions of α-synuclein through hydrogen bond formation and inhibits the ß-sheet formation, whereas trimethyl amine oxide itself does not interact much with the protein and stabilizes the protein by preferentially distributing water molecules on the surface of the protein.

Biophysical and biochemical studies on glycoxidatively modified human low density lipoprotein.

Methylglyoxal (MGO), a reactive dicarbonyl metabolite is a potent arginine directed glycating agent which has implications for diabetes-related complications. Dicarbonyl metabolites are produced endogenously and in a state of misbalance, they contribute to cell and tissue dysfunction through protein and DNA modifications causing dicarbonyl stress. MGO is detoxified by glyoxalase 1 (GLO1) system in the cytoplasm. Reactive oxygen species (ROS) are known to aggravate the glycation process. Both the processes are closely linked, and their combined activity is often referred to as "glycoxidation" process. Glycoxidation of proteins has several consequences such as type 2 diabetes mellitus (T2DM), aging etc. In this study, we have investigated the glycation of low-density lipoprotein (LDL) using different concentrations of MGO for varied incubation time periods. The structural perturbations induced in LDL were analyzed by UV-Vis, fluorescence, circular dichroism spectroscopy, molecular docking studies, polyacrylamide gel electrophoresis, FTIR, thermal denaturation studies, Thioflavin T assay and isothermal titration calorimetry. The ketoamine moieties, carbonyl content and HMF content were quantitated in native and glycated LDL. Simulation studies were also done to see the effect of MGO on the secondary structure of the protein. We report structural perturbations, increased carbonyl content, ketoamine moieties and HMF content in glycated LDL as compared to native analog (native LDL). We report the structural perturbations in LDL upon modification with MGO which could obstruct its normal physiological functions and hence contribute to disease pathogenesis and associated complications.

pH modulates the TGF-ß ligands binding to the receptors: a computational analysis.

In spite of showing high sequence similarity and forming structurally similar ternary complex in vitro, the in vivo role of TGF-ß1 and TGF-ß3 ligands suggests against their functional redundancy and necessitates the importance for the study of the specificity of these ligands. A comparative computational analysis of binary and ternary complexes of these two ligands shows that anchor residues of ligand and receptor at TGF-ß:TßR2 interface are similar in both complexes. However, the potential anchor residues of TGF-ß at TGF-ß:TßR1 interface are different, Tyr50 and Lys51 in TGF-ß3 complex and Lys60 and Tyr6 in TGF-ß1 complex. Pro55 and Asp57 of TßRI may act as anchor residues in complexes of both ligands along with Ile54 for TGF-ß3 complex and Val61 for TGF-ß1 complex. Arg58 of TßR1 acts as a potential hot residue for TGF-ß3 ternary complex but not for TGF-ß1 ternary complex formation whereas Pro55 and Phe60 may act as hot residues for both complexes. The Delphi analysis of the pH dependence of the binding energy indicates that pH has a remarkable effect on the binding energy of TßR2 to the open form of TGF-ß3. Lowering of pH from 7 to 4 favors binding of the open form of TGF-ß3 to TßR2. Now, apart from the residues at pH 7, residues Arg25, Lys31 and Arg94 of TGF-ß3 and Asp118 and Glu119 of TßR2 also contribute significantly to the binding energy. Contrary to the binding energy of TßR2 to TGF-ß3/TGF-ß1, TßR1 shows appreciable pH dependence for its binding in ternary complex of TGF-ß3/TGF-ß1. In TGF-ß3 ternary complex, the TßR1 electrostatic interaction energy disfavors complex formation at pH 7 while it is favored at pH 4.

Computational and physicochemical insight into 4-hydroxy-2-nonenal induced structural and functional perturbations in human low-density lipoprotein.

Lipid peroxidation (LPO) is a biological process that frequently occurs under physiological conditions. Undue oxidative stress increases the level of LPO; which may further contribute to the development of cancer. 4-Hydroxy-2-nonenal (HNE), one of the principal by-products of LPO, is present in high concentrations in oxidatively stressed cells. HNE rapidly reacts with various biological components, including DNA and proteins; however, the extent of protein degradation by lipid electrophiles is not well understood. The influence of HNE on protein structures will likely have a considerable therapeutic value. This research elucidates the potential of HNE, one of the most researched phospholipid peroxidation products, in modifying low-density lipoprotein (LDL). In this study, we tracked the structural alterations in LDL by HNE using various physicochemical techniques. To comprehend the stability, binding mechanism and conformational dynamics of the HNE-LDL complex, computational investigations were carried out. LDL was altered in vitro by HNE, and the secondary and tertiary structural alterations were examined using spectroscopic methods, such as UV-visible, fluorescence, circular dichroism and fourier transform infrared spectroscopy. Carbonyl content, thiobarbituric acid-reactive-substance (TBARS) and nitroblue tetrazolium (NBT) reduction assays were used to examine changes in the oxidation status of LDL. Thioflavin T (ThT), 1-anilinonaphthalene-8-sulfonic (ANS) binding assay and electron microscopy were used to investigate aggregates formation. According to our research, LDL modified by HNE results in changes in structural dynamics, oxidative stress and the formation of LDL aggregates. The current investigation must characterize HNE's interactions with LDL and comprehend how it can change their physiological or pathological functions.Communicated by Ramaswamy H. Sarma.

Exploring the interaction of tepotinib with calf thymus DNA using molecular dynamics simulation and multispectroscopic techniques.

In recent times, there has been a surge in the discovery of drugs that directly interact with DNA, influencing gene expression. As a result, understanding how biomolecules interact with DNA has become a major area of research. One such drug is Tepotinib (TPT), an FDA-approved anti-cancer medication known as a MET tyrosine kinase inhibitor, used in chemotherapy for metastatic non-small cell lung cancer (NSCLC) with MET exon 14 skipping alterations. In our study, we adopted both biophysical and in-silico methods to investigate the binding relationship of TPT and ctDNA. The absorption spectra of ctDNA exhibited a hypochromic effect when titrated with TPT and the binding constant of TPT-ctDNA complex was calculated, Ka = 9.91 × 104 M-1. By computing bimolecular enhancement constant (KB) and thermodynamic enhancement constant (KD) in fluorometric investigations, it was found that the fluorescence enhancement is a result of a static process involving the ctDNA-TPT complex formation in the ground state, as opposed to a dynamic process. The displacement assay results further supported this finding, showing that TPT exhibits a binding preference for minor groove of ct-DNA and was also demonstrated by KI quenching and CD spectroscopy. The molecular docking and molecular dynamic simulations validated TPT's groove binding nature and binding pattern with ctDNA, respectively. Thus, the results of our present investigation offer valuable insights into the interaction between TPT and ctDNA. It is evident that TPT, as an anti-cancer medication, binds to the minor groove of ctDNA.

Discovery of Quinolinone Hybrids as Dual Inhibitors of Acetylcholinesterase and Aß Aggregation for Alzheimer's Disease Therapy.

The development of multitargeted therapeutics has evolved as a promising strategy to identify efficient therapeutics for neurological disorders. We report herein new quinolinone hybrids as dual inhibitors of acetylcholinesterase (AChE) and Aß aggregation that function as multitargeted ligands for Alzheimer's disease. The quinoline hybrids (AM1-AM16) were screened for their ability to inhibit AChE, BACE1, amyloid fibrillation, α-syn aggregation, and tau aggregation. Among the tested compounds, AM5 and AM10 inhibited AChE activity by more than 80% at single-dose screening and possessed a remarkable ability to inhibit the fibrillation of Aß42 oligomers at 10 µM. In addition, dose-dependent screening of AM5 and AM10 was performed, giving half-maximal AChE inhibitory concentration (IC50) values of 1.29 ± 0.13 and 1.72 ± 0.18 µM, respectively. In addition, AM5 and AM10 demonstrated concentration-dependent inhibitory profiles for the aggregation of Aß42 oligomers with estimated IC50 values of 4.93 ± 0.8 and 1.42 ± 0.3 µM, respectively. Moreover, the neuroprotective properties of the lead compounds AM5 and AM10 were determined in SH-SY5Y cells incubated with Aß oligomers. This work would enable future research efforts aiming at the structural optimization of AM5 and AM10 to develop potent dual inhibitors of AChE and amyloid aggregation. Furthermore, the in vivo assay confirmed the antioxidant activity of compounds AM5 and AM10 through increasing GSH, CAT, and SOD activities that are responsible for scavenging the ROS and restoring its normal level. Blood investigation illustrated the protective activity of the two compounds against lead-induced neurotoxicity through retaining hematological and liver enzymes near normal levels. Finally, immunohistochemistry investigation revealed the inhibitory activity of ß-amyloid (Aß) aggregation.

Structural and functional insights into the regulation of Helicobacter pylori arginase activity by an evolutionary nonconserved motif.

Urea producing bimetallic arginases are essential for the synthesis of polyamine, DNA, and RNA. Despite conservation of the signature motifs in all arginases, a nonconserved ¹5³ESEEKAWQKLCSL¹65 motif is found in the Helicobacter pylori enzyme, whose role is yet unknown. Using site-directed mutagenesis, kinetic assays, metal analyses, circular dichroism, heat-induced denaturation, molecular dynamics simulations and truncation studies, we report here the significance of this motif in catalytic function, metal retention, structural integrity, and stability of the protein. The enzyme did not exhibit detectable activity upon deletion of the motif as well as on individual mutation of Glu155 and Trp159 while Cys163Ala displayed significant decrease in the activity. Trp159Ala and Glu155Ala show severe loss of thermostability (14-17°) by a decrease in the α-helical structure. The role of Trp159 in stabilization of the structure with the surrounding aromatic residues is confirmed when Trp159Phe restored the structure and stability substantially compared to Trp159Ala. The simulation studies support the above results and show that the motif, which was previously solvent exposed, displays a loop-cum-small helix structure (Lys161-Cys163) and is located near the active-site through a novel Trp159-Asp126 interaction. This is consistent with the mutational analyses, where Trp159 and Asp126 are individually critical for retaining a bimetallic center and thereby for function. Furthermore, Cys163 of the helix is primarily important for dimerization, which is crucial for stimulation of the activity. Thus, these findings not only provide insights into the role of this motif but also offer a possibility to engineer it in human arginases for therapeutics against a number of carcinomas.

Stability of the Aß42 Peptide in Mixed Solutions of Denaturants and Proline.

Amyloid ß-peptide (Aß) is responsible for the neuronal damage and death of a patient with Alzheimer's disease (AD). Aß42 oligomeric forms are dominant neurotoxins and are related to neurodegeneration. Their different forms are related to various pathological conditions in the brain. We investigated Aß42 peptides in different environments of proline, urea, and GdmCl solutions (in pure and mixed binary forms) through atomistic molecular dynamics simulations. Preferential exclusion from the protein surface and facile formation of a large number of weak molecular interactions are the driving forces for the osmolyte's action. We have focused on these interactions between peptide monomers and pure/mixed osmolytes and denaturants. Urea, as usual, denatures the peptide strongly compared to the GdmCl by accumulation around the peptide. GdmCl shows lesser build-up around protein in contrast to urea but is involved in destabilizing the salt bridge formation of Asp23 and Lys28. Proline as an osmolyte protects the peptide from aggregation when mixed with urea and GdmCl solutions. In mixed solutions of two denaturants and osmolyte plus denaturant, the peptide shows enhanced stability as compared to pure denaturant urea solution. The enhanced stability of peptides in proline may be attributed to its exclusion from the peptide surface and favoring salt bridge formation.

Multicomponent Petasis reaction for the identification of pyrazine based multi-target directed anti-Alzheimer's agents: In-silico design, synthesis, and characterization.

Multi-target directed ligands (MTDLs) have recently attracted significant interest due to their exceptional effectiveness against multi-factorial Alzheimer's disease. The present work described the development of pyrazine-based MTDLs using multicomponent Petasis reaction for the dual inhibition of tau-aggregation and human acetylcholinesterase (hAChE). The molecular structure of synthesized ligands was validated by 1H & 13C NMR and mass spectrometry. The screened compounds were shown to have a strong inhibitory effect at 10 µM concentration against tau-oligomerization and hAChE, but only moderate inhibitory activity against Aß42. Among all the compounds, the half-maximal inhibitory concentration (IC50) for 21 and 24 against hAChE were 0.71 µM and 1.09 µM, respectively, while they displayed half-maximal effective concentrations (EC50) values of 2.21 µM and 2.71 µM for cellular tau-oligomerization, respectively. Additionally, an MTT experiment using tau-expressing SH-SY5Y neuroblastoma cells revealed that 21 was more neuroprotective than the FDA-approved medication donepezil. Furthermore, an MD simulation study was performed to investigate the dynamics and stability of AChE-21 and AChE-24 complexes in an aqueous environment. The MM-PBSA calculations were performed to evaluate the binding of 21 and 24 with AChE, and the relative binding energy was calculated as -870.578 and -875.697 kJ mol-1, respectively. As a result, the study offered insight into the design of new MTDLs and highlighted 21 as a potential roadblock to the development of anti-AD medications.

Identification of new oxospiro chromane quinoline-carboxylate antimalarials that arrest parasite growth at ring stage.

Malaria still threatens half the globe population despite successful Artemisinin-based combination therapy. One of the reasons for our inability to eradicate malaria is the emergence of resistance to current antimalarials. Thus, there is a need to develop new antimalarials targeting Plasmodium proteins. The present study reported the design and synthesis of 4, 6 and 7-substituted quinoline-3-carboxylates 9(a-o) and carboxylic acids 10(a-b) for the inhibition of Plasmodium N-Myristoyltransferases (NMTs) using computational biology tools followed by chemical synthesis and functional analysis. The designed compounds exhibited a glide score of -9.241 to -6.960 kcal/mol for PvNMT and -7.538 kcal/mol for PfNMT model proteins. Development of the synthesized compounds was established via NMR, HRMS and single crystal X-ray diffraction study. The synthesized compounds were evaluated for their in vitro antimalarial efficacy against CQ-sensitive Pf3D7 and CQ-resistant PfINDO lines followed by cell toxicity evaluation. In silico results highlighted the compound ethyl 6-methyl-4-(naphthalen-2-yloxy)quinoline-3-carboxylate (9a) as a promising inhibitor with a glide score of -9.084 kcal/mol for PvNMT and -6.975 kcal/mol for PfNMT with IC50 values of 6.58 µM for Pf3D7 line. Furthermore, compounds 9n and 9o exhibited excellent anti-plasmodial activity (Pf3D7 IC50 = 3.96, 6.71 µM, and PfINDO IC50 = 6.38, 2.8 µM, respectively). The conformational stability of 9a with the active site of the target protein was analyzed through MD simulation and was found concordance with in vitro results. Thus, our study provides scaffolds for the development of potent antimalarials targeting both Plasmodium vivax and Plasmodium falciparum.Communicated by Ramaswamy H. Sarma.

In silico studies of the human IAPP in the presence of osmolytes.

The human islet amyloid polypeptide or amylin is secreted along with insulin by pancreatic islets. Under the drastic environmental conditions, amylin can aggregate to form amyloid fibrils. This amyloid plaque of hIAPP in the pancreatic cells is the cause of type II diabetes. Early stages of amylin aggregates are more cytotoxic than the matured fibrils. Here, we have used the all-atom molecular dynamic simulation to see the effect of water, TMAO, urea and urea/TMAO having ratio 2:1 of different concentrations on the amylin protein. Our study suggest that the amylin protein forms ß-sheets in its monomeric form and may cause the aggregation of protein through the residue 13-17 and the C-terminal region. α-Helical content of protein increases with an increase in TMAO concentration by decreasing the SASA value of protein, increase in intramolecular hydrogen bonds and on making the short-range hydrophobic interactions. Electrostatic potential surfaces show that hydrophobic groups are buried and normalised configurational entropy of backbone, and side-chain atoms is lesser in the presence of TMAO, whereas opposite behaviour is obtained in the case of urea. Counteraction effect of TMAO using Kast model towards urea is also observed in ternary solution of urea/TMAO.

Studying the interaction of scopolamine with calf-thymus DNA: An in-vitro and in-silico approach and genotoxicity.

Scopolamine is used to treat various CNS disorder like urinary incontinence, motion sickness, spasmic movements. Despite its pharmaceutical properties, its interaction with DNA is not yet reported. In this article, the interaction between scopolamine and ct-DNA is reported using a combination of biophysical techniques. UV-visible and steady-state fluorescence spectroscopy were used to study interaction and complex formation. Competitive displacement assays and potassium iodide quenching confirmed the mode of binding between scopolamine and DNA. Structural changes induced in the ct-DNA in the presence of scopolamine were evaluated by CD spectroscopy. The plasmid nicking and NBT assay confirmed the genotoxic effect of scopolamine. In-silico study by molecular docking and molecular dynamics simulation revealed the mode of interaction, major stabilizing forces as well as the nucleotide sequences to which the scopolamine binds.

The inhibitory effect of Sunset Yellow on thermally induced Human Serum Albumin aggregates: Possible role in naturopathy.

Intensive research in the field of protein aggregation confirmed that the deposition of amyloid fibrils of proteins are the major cause for the development of various neurotoxic and neurodegenerative diseases, which could be controlled by ensuring the efficient inhibition of aggregation using anti aggregation strategies. Herein, we elaborated the anti amyloidogenic potential of Sunset Yellow (SY) dye against Human Serum Albumin (HSA) fibrillogenesis utilising different biophysical, computational and microscopic techniques. The inhibitory effect of sunset yellow was confirmed by Rayleigh Light Scattering (RLS) measurements along with different dye binding assays (ANS, ThT and CR) by showing concentration dependent reduction in scattering intensity and fluorescence intensity respectively. Further, destabilization and anti fibrillation activity of HSA aggregates were characterized through spectroscopic techniques like Circular Dichroism (CD) and other microscopic techniques like Transmission Electron Microscopy (TEM) for elucidating the structural properties. The SDS-PAGE was also carried out that render the disaggregation effect of the dye on the protein. Moreover, Molecular Docking studies revealed the binding parameters justifying the stable protein-dye complex. Simulation studies were also performed accordingly. Thus, this dye which is used as food additive can serve as a potential aggregation inhibiting agent that can aid in the prevention of amyloidogenic diseases.

Interaction of memantine with calf thymus DNA: an in-vitro and in-silico approach and cytotoxic effect on the cancerous cell lines.

Memantine belongs to the class of cognition enhancers that functions as NMDA receptor antagonist, used to treat Alzheimer's disease. The interaction of memantine with DNA was not investigated. In the present study, the interaction of memantine with ct-DNA, as well as its cytotoxicity on cancer cells, was evaluated. UV-visible spectroscopy, steady-state fluorescence spectroscopic studies revealed the interaction between memantine and ct-DNA. The quenching studies, chemical denaturation, (CD), and DNA melting studies showed the groove binding mode of memantine with ct-DNA. The thermodynamic parameters revealed that the interaction between memantine and ct-DNA is enthalpically driven, and the stabilizing forces involved were hydrogen bonding and van der Waals interaction. The groove-binding was also observed by molecular docking studies, which corroborated the findings of spectroscopic investigations. Density function theory calculations confirmed the existence of electron donor and recipient groups. The stability of memantine and DNA interaction, as well as the critical residues involved in the interaction, was identified by molecular dynamics simulations. Memantine showed cytotoxicity towards the cancer cells as compared to normal cells, as observed by MTT assay. Inverted compound microscopy analysis of memantine treated cancer cell lines further confirmed the results obtained by MTT assay.Communicated by Ramaswamy H. Sarma.

Biophysical characterization of structural and conformational changes in methylmethane sulfonate modified DNA leading to the frizzled backbone structure and strand breaks in DNA.

Methyl methanesulfonate (MMS) is a highly toxic DNA-alkylating agent that has a potential to damage the structural integrity of DNA. This work employed multiple biophysical and computational methods to report the MMS mediated structural alterations in the DNA (MMS-DNA). Spectroscopic techniques and gel electrophoresis studies revealed MMS induced exposure of chromophoric groups of DNA; methylation mediated anti→syn conformational change, DNA fragmentation and reduced nucleic acid stability. MMS induced single-stranded regions in the DNA were observed in nuclease S1 assay. FT-IR results indicated MMS mediated loss of the assigned peaks for DNA, partial loss of C-O ribose, loss of deoxyribose region, C-O stretching and bending of the C-OH groups of hexose sugar, a progressive shift in the assigned guanine and adenine peaks, loss of thymine peak, base stacking and presence of C-O-H vibrations of glucose and fructose, indicating direct strand breaks in DNA due to backbone loss. Isothermal titration calorimetry showed MMS-DNA interaction as exothermic with moderate affinity. Dynamic light scattering studies pointed towards methylation followed by the generation of single-stranded regions. Electron microscopy pictured the loss of alignment in parallel base pairs and showed the formation of fibrous aggregates in MMS-DNA. Molecular docking found MMS in close contact with the ribose sugar of DNA backbone having non-bonded interactions. Molecular dynamic simulations confirmed that MMS is capable of interacting with DNA at two levels, one at the level of nitrogenous bases and another at the DNA backbone. The study offers insights into the molecular interaction of MMS and DNA.Communicated by Ramaswamy H. Sarma.

The non-native conformations of cytochrome c in sodium dodecyl sulfate and their modulation by ATP.

To understand the interaction of cytochrome c (cyt c) with membranes, a systematic investigation of sodium dodecyl sulfate (SDS)-induced conformational alterations in native horse heart ferricytochrome c (pH 7.0) was carried out using heme absorbance, tryptophan fluorescence and circular dichroism (CD) spectroscopy. ATP interaction with membrane-bound cyt c is known to regulate the process of apoptosis. To understand the effect of nucleotide phosphates on membrane-bound cyt c, we also carried out studies of the interaction of ATP with cyt c in the presence of SDS. Fluorescence and UV-Vis data suggest that SDS induces two different transitions (F to C1, C1 to C2) in cyt c, one in the pre-micellar region and the other in the post-micellar region. The fluorescence data further indicated the increase in distance between Trp 59 and heme in the intermediates in both the regions, suggesting loosening up of cyt c on titration with SDS. The far-UV and near-UV CD data suggest partial loss of secondary and tertiary structure in C1, but complete loss of tertiary structure and no further loss of secondary structure in C2. On titration of C1 and C2 with ATP, the secondary structure is restored. However, the heme ligation pattern and heme exposure change only for C2, but not for C1 on the addition of ATP.

Biophysical insight into the binding mechanism of doxofylline to bovine serum albumin: An in vitro and in silico approach.

Insight into the mechanistic binding of bovine serum albumin (BSA) with doxofylline can layout pivotal enlightenment with relevance to pharmacokinetics and pharmacodynamics properties. Herein, many spectroscopic techniques and computational methods had been employed to interpret the structural and binding dynamics of BSA-doxofylline interaction. Doxofylline quenched the intrinsic fluorescence of BSA by static quenching. The stoichiometry and the binding constant of the BSA-doxofylline complex were 1:1 and in the order of 103 M-1. It was also concluded that the binding process was spontaneous and exothermic, primarily based on the thermodynamic study. Circular dichroism and three-dimensional excitation-emission matrix fluorescence results concluded pronounced conformational and microenvironmental changes in BSA structure on binding with doxofylline. The influence of metal ions and vitamins on the binding affinity of the BSA-doxofylline system were also explored. The in vitro findings were further supported by in silico analysis. With a score value of -6.25 kcal/mol, molecular docking showed strong interactions. Molecular dynamics simulation interpretation also suggested the stable binding with lower deviation in the values of RMSD and RMSF obtained by uninterrupted long simulation run. These studies will propose the optimum potency of distribution of the doxofylline into the bloodstream for asthma treatment.