Quinazolinone-Hydrazine Cyanoacetamide Hybrids as Potent Multitarget-Directed Druggable Therapeutics against Alzheimer's Disease: Design, Synthesis, and Biochemical, In Silico, and Mechanistic Analyses.

The discovery of effective multitarget-directed ligands (MTDLs) against multifactorial Alzheimer's disease (AD) remnants has been focused in an incessant drug discovery pursuit. In this perception, the current study explores the rational design, synthesis, and evaluation of 26 quinazolinone-hydrazine cyanoacetamide hybrids 7(a-j), 8(a-j), and 9(a-f) as MTDLs against AD. These new compounds were synthesized in four-step processes using simple phthalimide as the starting material without any major workup procedures and were characterized by different spectroscopic techniques. In Ellman's assay, the most potent analogues 7i, 8j, and 9d were identified as selective and mixed-type inhibitors of hAChE. Furthermore, biophysical and computational assessments revealed that the analogues 7i, 8j, and 9d were bound to both the catalytic active site and peripheral anionic site of hAChE with high affinity. The molecular dynamics simulation analysis highlighted the conformational changes of hAChE upon binding of 7i, 8j, and 9d and also the stability of resulting biomolecular systems all over 100 ns simulations. In addition to antioxidant activity, the most active congeners were found to protect substantially SK-N-SH cells from oxidative damage. Decisively, the most active analogues 7i, 8j, and 9d were assessed as potent Aß1-42 fibril modulators and protective agents against Aß1-42-induced toxicity in SH-SY5Y cells. Additionally, glioblastoma C6 cell-based assays also demonstrated the use of the most active congeners 7i, 8j, and 9d as protective agents against Aß1-42-induced toxicity. Overall, this multifunctional capacity of quinazolinone-hydrazine cyanoacetamide hybrids demonstrated the noteworthy potential of these hybrids to develop as effectual MTDLs against AD. However, further pharmacokinetics, toxicology, and behavioral studies are warranted.

Deciphering the binding mechanism of gingerol molecules with plasma proteins: implications for drug delivery and therapeutic potential.

Ginger is a highly valued herb, renowned globally for its rich content of phenolic compounds. It has been traditionally used to treat various health conditions such as cardiovascular diseases, digestive issues, migraines, Alzheimer's disease, tumor reduction and chronic inflammation. Despite its potential medicinal applications, the therapeutic effectiveness of ginger is hindered by its limited availability and low plasma concentration levels. In this study, we explored the interaction of ginger's primary phenolic compounds, specifically 6-gingerol (6 G), 8-gingerol (8 G) and 10-gingerol (10 G), with plasma proteins which are human serum albumin (HSA) and α-1-acid glycoprotein (AGP). These two plasma proteins significantly influence drug distribution and disposition as they are key binding sites for most drugs. Fluorescence emission spectra indicated strong binding of 6, 8 and 10 G with HSA, with binding constants of 2.03 ± 0.01 × 104 M-1, 4.20 ± 0.01 × 104 M-1 and 6.03 ± 0.01 × 106 M-1, respectively. However, the binding of gingerols with AGP was found to be negligible. Molecular displacement by site-specific probes and molecular docking analyses revealed that gingerols bind at the IIA domain, with stability provided by hydrogen bonds, van der Waals forces, conventional hydrogen bonds, carbon-hydrogen bonds, alkyl and Pi-alkyl interactions. Further, the partial unfolding of the protein was observed upon binding the gingerol compound with HSA. In addition, molecular dynamic simulations demonstrated that gingerols remained stable in the subdomain IIA over 100 ns. This stability, coupled with Molecular Mechanics Generalized Born Surface Area indicating free energies of -43.765, -57.504 and -66.69 kcal/mol for 6, 8 and 10 G, respectively, reinforces the robust binding potential of these compounds. Circular dichroism studies suggested that the interaction of gingerols leads to the minimal transformation of HSA secondary structure, with the pattern being 10 G > 8 G > 6 G, a finding further substantiated by root mean square deviation and root mean square fluctuation fluctuations. These results propose that HSA has a stronger affinity to gingerols than AGP, which could have significant implications on the therapeutic circulating levels of gingerols.Communicated by Ramaswamy H. Sarma.

Elucidation of binding mechanism of rhodanine derivative P4OC on bovine serum albumin.

Rhodanine is an important scaffold in medicinal chemistry and it act as potent anticancer agent and other pharmacological effects. In pharmacokinetics and pharmacodynamics studies of the drug, the drug binding properties on serum protein is crucial for producing better drug. This study was designed to explore the binding interactions between the Rhodanine derivative (P4OC) on Bovine Serum Albumin (BSA). The interactions between P4OC and BSA were investigated using biophysical approach and molecular docking. The quenching mechanism and binding constants of P4OC on BSA were determined by biophysical approach through fluorescence spectroscopic experiments. Circular dichroism (CD) spectroscopy was used to study the secondary structural changes of BSA upon P4OC binding. The fluorescence experiments of P4OC binding on BSA show good drug binding with static quenching constants using stern Volmer plot and found the quenching constant value KP4OC = 1.12762 × 1013 M-1 with corresponding binding free energy (ΔG) -2.303 kcal/mol. The molecular displacement fluorescence emission on BSA-P4OC complex by site specific markers shows that P4OC binds at I A sub-domain of BSA further confirmed peak shift by synchronous fluorescence of P4OC on BSA with tyrosine, tryptophan and phenylalanine amino acids. Increasing concentration of P4OC on BSA found secondary structural changes, the percentage of α-helix was decreased as well increase percentage of ß-sheet and random coil. The binding of P4OC to BSA was computationally studied by molecular docking methods. Thus, results obtained are in excellent agreement with experimental and theoretical results with respect to the binding mechanism and binding constant of P4OC on BSA. We concluded that, the rhodanine derivative P4OC possesses good drug binding properties on BSA. Further P4OC may be evaluated its potential pharmacological activities on clinical trial.Communicated by Ramaswamy H. Sarma.

Chebulinic and chebulagic acid binding with serum proteins: biophysical and molecular docking approach.

Chebulinic acid (CHN) and chebulagic acid (CHG) have been known for centuries for their anti-cancer, anti-diabetes, HIV and anti-inflammatory properties. In this study, the interaction of these phytochemicals CHN/CHG, with the two major transport proteins for various drugs, human serum albumin (HSA) and α-1-acid glycoprotein (AGP), was unraveled by using several spectroscopic techniques and computational methods. The binding of CHN/CHG quenches the HSA/AGP fluorescence intensities, and also these phytochemicals are bound strongly to HSA/AGP proteins. An apparent decrease in fluorescence intensities of CHN/CHG-HSA and CHN/CHG-AGP complex showed the static mode of fluorescence quenching. Furthermore, the intrinsic fluorescence and using site-specific markers ibuprofen competing with these molecules, thereby replacing it in the binding site of subdomain IIIA. The computational methods substantiated the experimental findings, revealing that CHN interacted with Lys414A, Glu492A, Glu492A and Lys413A residues of subdomain IIIA of HSA and for CHG showed the interaction with Lys545A and Lys413A residues of subdomain IIIA of HSA. Fluorescence and surface plasmon resonance data unveiled a previously unreported binding event between CHN/CHG and HSA; the determined binding affinities of both compounds were slightly higher for HSA than AGP. A change in functionality of protein confirmed the esterase-like activity of HSA in the presence of CHG/CHN upon binding with CHG/CHN. Displacement and circular dichroism (CD) experiments analysis showed that the two CHN/CHG and binding specifically to IIIA subdomain on HSA results in the conformational changes in the HSA. Thus, CD revealed a few conformational changes in HSA due to CHN/CHG. The binding of these two phytochemicals to the plasma proteins would give a path to develop new inspired drug molecules for chronic diseases.Communicated by Ramaswamy H. Sarma.

Comparative binding studies of bacosine with human serum albumin and α-1-acid glycoprotein biophysical evaluation and computational approach.

Bacosine (BAC) is a natural product isolated from a herb and used in the Ayurvedic system of medicine. It is reported to have a wide array of biological activities, which has generated interest in its therapeutic potential. To better understand how BAC may operate as a potential anti-cancer therapeutic, we examined its anti-cancer properties in the human breast cancer cell line, MCF-7. In order to get an idea of how it may behave in vivo, we also evaluated its interaction with human serum albumin (HSA) and α-1-acid glycoprotein (AGP) using fluorescence spectroscopy and in silico molecular modelling. Based on our in vitro studies, we found that BAC inhibited MCF-7 cell growth in a dose-dependent manner with an IC50 value of 9 µM. In addition, the intrinsic fluorescence of HSA and AGP was quenched by BAC, consistent with a static quenching mechanism. Fluorescence emission spectroscopy revealed a binding of 2.97 ± 0.01 × 104 M-1 for HSA-BAC which corresponded to a free energy change of - 6.07 kcal/mol at 25 °C. In addition, we found that BAC had a binding constant of 1.8 ± 0.02 × 103 M-1 to AGP which corresponded to a change in free energy - 4.42 kcal/mol at 25 °C. We also identified the site of BAC binding to the HSA protein using the site-specific marker, phenylbutazone, along with molecular docking studies. Circular dichroism spectra revealed partial changes in the secondary structure of HSA in the presence of BAC suggesting direct interactions. Molecular dynamics simulations demonstrated that the HSA-BAC complex reaches an equilibration state at around 4 ns, suggesting that the HSA-BAC complex is quite stable. Our results provide evidence that serum proteins can act as a carrier protein for BAC, potentially impacting its development as an anti-cancer agent.

Elucidation of binding mechanism of stigmasterol with human serum albumin: a biophysical and molecular dynamics simulation approach.

In the present study, we have analyzed the interaction of a phytochemical, stigmasterol (Stig), with human serum albumin (HSA) under physiological conditions using fluorescence quenching, circular dichroism and molecular modeling methods. Cytotoxic studies with Stig in mouse macrophages (RAW 246.7) and HeLa cell lines showed anti-inflammatory and anti-cancer properties. Further, the intrinsic fluorescence of HSA was quenched by Stig, which was considered a static quenching mechanism. The site-specific marker experiments revealed that Stig binds to the IIIA subdomain of HSA with a binding constant of KStig=1.8 ± 0.03 × 105 M-1 and free energy of -7.26 ± 0.031 Kcal/mol. The secondary structure of HSA was partially unfolded after binding of Stig, which indicates an alteration in the microenvironment of the protein binding site. Molecular docking experiments found that Stig binds strongly with HSA at the IIIA domain of the hydrophobic pocket with one hydrogen bond. The rigidity of the protein-Stig complex and free energies were analyzed by molecular dynamic simulation (MDS) for 100 ns, where the HSA-Stig was stabilized after 40 ns. MDS studies revealed that HSA does not significantly change the secondary structure when it binds with Stig, which is in agreement with the circular dichroism data. Overall, the results obtained gave qualitative and quantitative insight into the binding interaction between HSA and Stig, which is essential in understanding the latter as a therapeutic molecule.Communicated by Ramaswamy H. Sarma.

Deciphering the AChE-binding mechanism with multifunctional tricyclic coumarin anti-Alzheimer's agents using biophysical and bioinformatics approaches and evaluation of their modulating effect on Amyloidogenic peptide assembly.

Investigating the drug-AChE binding mechanism is vital in understanding its cogent use in medical practice against Alzheimer's disease (AD). The production and accumulation of oligomers of ß-amyloid is a central event in the neuropathology of AD. Beside the inhibition of assembly process, modulation of the aggregation process of these proteins towards minimally toxic pathways may be a possible therapeutic strategy for AD. Hence, the present study aims to examine the effect of multifunctional fused tricyclic 7-hydroxy 4-methyl coumarin analogs (HMC1-5) on the self-induced aggregation of ß-amyloid using Thioflavin T (ThT) assay, scanning electron microscopic study, AlamarBlue and immune blotting assays and also the binding mechanism with AChE by fluorescence emission, conformational, molecular docking and molecular dynamic simulation studies under physiological pH 7.4. The ThT assay, FE-SEM study, cell line and western blots establish that the HMC1-5 molecules could irreversibly disrupt preformed Aß42 fibrils, accelerate the aggregates into micro size co-assembled structures, and effectively eliminate the cytotoxicity of Aß1-42. Fluorescence emission studies indicating a strong binding affinity between HMC1-5 and AChE with the binding constants of 1.04 × 105, 3.57 × 104, 1.97 × 104, 3.07 × 104 and 2.95 × 104 M-1, respectively and binding sites number found to be 1. CD studies disclosed a partial unfolding in the secondary structure of AChE upon binding with HMC1-5. Docking analysis inferred that the HMC1-5 were bound through hydrophobic and hydrophilic interactions to the AChE active site. Molecular dynamics simulations emphasized the stability of AChE-HMC1-5 complexes throughout the 100 ns simulations, and the local conformational changes of the residues of AChE validate the stability of complexes. These results provide new and unique complementary approach for modulating the biological effects of the Aß aggregates by coumarin analogs and new insights for further in vivo investigations as novel anti AD agents.

Enhanced Lipid Production in Chlamydomonas reinhardtii Caused by Severe Iron Deficiency.

Microalgae are used as a source of lipids for the production of biofuels. Most algae produce neutral lipids under stress conditions. Here, lipid accumulation by the unicellular alga Chlamydomonas reinhardtii was examined during cultivation under iron-limiting conditions. Severe iron stress caused the cells to accumulate a significant amount of lipid, specifically triacylglycerols (TAGs), by compromising the growth. Semi-quantitative measurements by Fourier transform infrared (FTIR) spectroscopy showed an increase in both carbohydrate and lipid content in iron-stressed C. reinhardtii cells compared to control. Analysis by flow cytometry and thin layer chromatography confirmed that severe iron deficiency-induced TAG accumulation to fourfold higher than in iron-replete control cells. This accumulation of TAGs was mostly degraded from chloroplast lipids accompanied by overexpression of diacylglycerol acyltransferase (DGAT2A) protein. Furthermore, liquid chromatography-mass spectrometry (LC-MS) analysis demonstrated significantly enhanced levels of C16:0, C18:2, and C18:3 fatty acids (FAs). These results indicate that iron stress triggers the rapid accumulation of TAGs in C. reinhardtii cells. The enhanced production of these lipids caused by the iron deficiency may contribute to the efficient production of algal biofuels if we escalate to the photobioreactor's growth conditions.

Improving the inhibition of ß-amyloid aggregation by withanolide and withanoside derivatives.

Here, we have studied the ameliorative effects of Withania somnifera derivatives (Withanolide A, Withanolide B, Withanoside IV, and Withanoside V) on the fibril formation of amyloid-ß 42 for Alzheimer's disease. We analyzed reduction in the aggregation of ß amyloid protein with these Ashwagandha derivatives by Thioflavin T assay in the oligomeric and fibrillar state. We have tested the cytotoxic activity of these compounds against human SK-N-SH cell line for 48 h, and the IC 50 value found to be 28.61 ± 2.91, 14.84 ± 1.45, 18.76 ± 0.76 and 30.14 ± 2.59 µM, respectively. After the treatment of the cells with half the concentration of IC 50 value, there was a remarkable decrease in the number of apoptotic cells stained by TUNEL assay indicating the DNA damage and also observed significant decrease of reactive oxygen species. Also, the binding and molecular stability of these derivatives with amyloid ß was also studied using bioinformatics tools where these molecules were interacted at LVFFA region which is inhibition site of amyloid-ß1 42. These studies revealed that the Withanolides and Withanosides interact with the hydrophobic core of amyloid-ß 1-42 in the oligomeric stage, preventing further interaction with the monomers and diminishing aggregation.

Restoration of photosynthetic activity and supercomplexes from severe iron starvation in Chlamydomonas reinhardtii.

The eukaryotic alga Chlamydomonas (C.) reinhardtii is used as a model organism to study photosynthetic efficiency. We studied the organization and protein profile of thylakoid membranes under severe iron (Fe2+) deficiency condition and iron supplement for their restoration. Chlorophyll (Chl) a fluorescence fast OJIP transients were decreased in the severe Fe2+ deficient cells resulting in the reduction of the photochemical efficiency. The circular dichroism (CD) results from Fe2+ deficient thylakoid membranes show a significant change in pigment-pigment and pigment-protein excitonic interactions. The organization of super-complexes was also affected significantly. Furthermore, super-complexes of photosystem (PS) II and PSI, along with its dimers, were severely reduced. The complexes separated using sucrose gradient centrifugation shows that loss of super-complexes and excitonic pigment-pigment interactions were restored in the severely Fe2+ deficient cells upon Fe supplementation for three generations. Additionally, the immunoblots demonstrated that both PSII, PSI core, and their light-harvesting complex antenna proteins were differentially decreased. However, reduced core proteins were aggregated, which in turn proteins were unfold and destabilized the supercomplexes and its function. Interestingly, the aggregated proteins were insoluble after n-Dodecyl ß-D-maltoside solubilization. Further, they were identified in the pellet form. When Fe2+ was added to the severely deficient cells, the photosynthetic activity, pigment-proteins complexes, and proteins were restored to the level of control after 3rd generation.

Organophosphate hydrolase interacts with ferric-enterobactin and promotes iron uptake in association with TonB-dependent transport system.

Our previous studies have shown the existence of organophosphate hydrolase (OPH) as a part of the inner membrane associated Ton complex (ExbB/ExbD and TonB) of Sphingobium fuliginis. We now show its involvement in iron uptake by establishing direct interactions with ferric-enterobactin. The interactions between OPH and ferric-enterobactin were not affected even when the active site architecture is altered by substituting active site aspartate with either alanine or asparagine. Protein docking studies further substantiated these findings and predicted the existence of ferric-enterobactin binding site that is different from the catalytic site of OPH. A lysine residue (82K) found at the predicted ferric-enterobactin binding site facilitated interactions between OPH and ferric-enterobactin. Substitution of lysine with alanine did not affect triesterase activity, but it abrogated OPH ability to interact with both ferric-enterobactin and ExbD, strengthening further the fact that the catalytic site is not the site for binding of these ligands. In the absence of interactions between OPHK82A and ExbD, OPHK82A failed to target membrane in E. coli cells. The Sphingobium fuliginis TonB-dependent transport (SfTonBDT) system was reconstituted in E. coli GS027 cells generated by deleting the exbD and tonB genes. The E. coli GS030 cells having SfTonBDT system with OPH showed increased iron uptake. Such an increase was not seen in E. coli GS029, cells having SfTonBDT system generated either by omitting OPH or by including its variants, OPHD301A, OPHD301N suggesting a role for OPH in enhanced iron uptake.

Comparative binding of Swertiamarin with human serum albumin and α-1 glycoprotein and its cytotoxicity against neuroblastoma cells.

Communicated by Ramaswamy H. Sarma.

Unraveling the stability of plasma proteins upon interaction of synthesized uridine products: biophysical and molecular dynamics approach.

Most of the drugs binding to human serum albumin (HSA) are transported to various parts of the body. Here, we have studied the molecular interaction between HSA and synthesized uridine derivatives, 1-[(3R, 4S, 5 R)-2-methyl-3, 4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidine-2,4-dion.)(C-MU); [(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxy-4-methyl-tetrahydrofuran-2-yl] methyl methyl phosphochloridate (CM-MU) and [(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-2-methyl-3,4-dihydroxyoxolan-2-yl] methyl dihydrogen phosphate (P-MU). Cytotoxic studies of these synthesized compounds with mouse macrophages (RAW 246.7) and HeLa cells (human cervical cancer cells) and binding mechanism of these uridine derivatives with HSA were performed. Subsequently, fluorescence quenching was observed upon titration of uridine derivatives with HSA via static mode of quenching, and the binding constants (K2-C-MU = 4 ± 0.03 × 104M-1, K5-CM-MU = 1.95 ± 0.03 × 104 M-1 and K5-P-MU =1.56 ± 0.03 × 104 M-1) were found to be in sync with the computational results. Further, molecular displacement and molecular docking data revealed that all the derivatives are binding in the subdomain IIA and IIB regions of HSA. The protein secondary structure of complexes was determined by circular dichroism, indicating partial unfolding of the protein upon addition of the uridine derivatives. Furthermore, atomic force microscopy data reveal the change in topology upon binding of 2-C-MU, 5-CM-MU and 5-P-MU with HSA, indicating change in the microenvironment around tryptophan region. Additionally, cytotoxicity studies on HeLa and Raw Cell lines suggested that these molecules have significant anti-proliferative and anti-inflammatory properties. Hence, the study may be of help for development of new drugs based on uridine derivatives which may be helpful for combating various potential diseases.Communicated by Ramaswamy H. Sarma.

Synthesis and biological evaluation of flavone-8-acrylamide derivatives as potential multi-target-directed anti Alzheimer agents and investigation of binding mechanism with acetylcholinesterase.

In a search for novel multifunctional anti-Alzheimer agents, a congeneric set of seventeen flavone-8-acrylamide derivatives (8a─q) were synthesized and evaluated for their cholinesterase inhibitory, antioxidant, neuroprotective and modulation of Aß aggregation activities. The target compounds showed effective and selective inhibitory activity against the AChE over BuChE. In addition, the target compounds also showed moderate anti-oxidant activity and strong neuroprotective capacities, and accelerated dosage-dependently the Aß aggregation. Also, we presented here a complete study on the interaction of 8a, 8d, 8e, 8h and 8i with AChE. Through fluorescence emission studies, the binding sites number found to be 1, binding constants were calculated as 2.04 × 104, 2.22 × 104, 1.18 × 104, 9.8 × 103 and 3.2 × 104 M-1 and free energy change as -5.83, -5.91, -5.51, -5.41 and -6.12 kcal M-1 at 25 °C which were well agreed with the computational calculations indicating a strong binding affinity of flavones and AChE. Furthermore, the CD studies revealed that the secondary structure of AChE became partly unfolded upon binding with 8a, 8d, 8e, 8h and 8i.

Changes in the photosynthetic apparatus and lipid droplet formation in Chlamydomonas reinhardtii under iron deficiency.

The unicellular photosynthetic alga Chlamydomonas reinhardtii was propagated in iron deficiency medium and patterns of growth, photosynthetic efficiency, lipid accumulation, as well as the expression of lipid biosynthetic and photosynthesis-related proteins were analysed and compared with iron-sufficient growth conditions. As expected, the photosynthetic rate was reduced (maximally after 4 days of growth) as a result of increased non-photochemical quenching (NPQ). Surprisingly, the stress-response protein LHCSR3 was expressed in conditions of iron deficiency that cause NPQ induction. In addition, the protein contents of both the PSI and PSII reaction centres were gradually reduced during growth in iron deficiency medium. Interestingly, the two generations of Fe deficiency cells could be able to recover the photosynthesis but the second generation cells recovered much slower as these cells were severely in shock. Analysis by flow cytometry with fluorescence-activated cell sorting and thin layer chromatography showed that iron deficiency also induced the accumulation of triacylglycerides (TAG), which resulted in the formation of lipid droplets. This was most significant between 48 and 72 h of growth. Dramatic increases in DGAT2A and PDAT1 levels were caused by iron starvation, which indicated that the biosynthesis of TAG had been increased. Analysis using gas chromatography mass spectrometry showed that levels of 16:0, 18:0, 18:2 and 18:3Δ9,12,15 fatty acids were significantly elevated. The results of this study highlight the genes/enzymes of Chlamydomonas that affect lipid synthesis through their influence on photosynthesis, and these represent potential targets of metabolic engineering to develop strains for biofuel production.

Spectroscopic evaluation of synthesized 5ß-dihydrocortisol and 5ß-dihydrocortisol acetate binding mechanism with human serum albumin and their role in anticancer activity.

Our study focus on the biological importance of synthesized 5ß-dihydrocortisol (Dhc) and 5ß-dihydrocortisol acetate (DhcA) molecules, the cytotoxic study was performed on breast cancer cell line (MCF-7) normal human embryonic kidney cell line (HEK293), the IC50 values for MCF-7 cells were 28 and 25 µM, respectively, whereas no toxicity in terms of cell viability was observed with HEK293 cell line. Further experiment proved that Dhc and DhcA induced 35.6 and 37.7% early apoptotic cells and 2.5, 2.9% late apoptotic cells, respectively, morphological observation of cell death through TUNEL assay revealed that Dhc and DhcA induced apoptosis in MCF-7 cells. The complexes of HSA-Dhc and HSA-DhcA were observed as static quenching, and the binding constants (K) was 4.7 ± .03 × 104 M-1 and 3.9 ± .05 × 104 M-1, and their binding free energies were found to be -6.4 and -6.16 kcal/mol, respectively. The displacement studies confirmed that lidocaine 1.4 ± .05 × 104 M-1 replaced Dhc, and phenylbutazone 1.5 ± .05 × 104 M-1 replaced by DhcA, which explains domain I and domain II are the binding sites for Dhc and DhcA. Further, FT-IR, synchronous spectroscopy, and CD results revealed that the secondary structure of HSA was altered in the presence of Dhc and DhcA. Furthermore, the atomic force microscopy and transmission electron microscopy showed that the dimensions like height and molecular size of the HSA-Dhc and HSA-DhcA complex were larger compared to HSA alone. Detailed analysis through molecular dynamics simulations also supported greater stability of HSA-Dhc and HSA-DhcA complexes, and root-mean-square-fluctuation interpreted the binding site of Dhc as domain IB and domain IIA for DhcA. This information is valuable for further development of steroid derivative with improved pharmacological significance as novel anti-cancer drugs.

Elucidating the active interaction mechanism of phytochemicals withanolide and withanoside derivatives with human serum albumin.

Withania somnifera (Ashwagandha) is an efficient medicinal plant known in Ayurveda and Chinese medicine since ancient times, whose extracts are consumed orally as food supplement or as a health tonic owing to its several restorative properties for various CNS disorders, inflammation, tumour, stress, rheumatism etc. In this study, we have analyzed the binding interaction of four derivatives of Withania somnifera (Withanolide A, Withanolide B, Withanoside IV and Withanoside V) with HSA because of their important pharmacological properties. To unravel the binding between derivatives of Withania somnifera and HSA, fluorescence spectroscopy was used. Binding studies were further studied by molecular docking and dynamics and results confirmed greater stability upon binding of derivatives with HSA. Circular dichroism data illustrated change in the secondary structure of protein upon interaction with these derivatives, particularly the helical structure was increased and ß-sheets and random coils were decreased. Furthermore, morphological and topological changes were observed using AFM and TEM upon binding of ligands with HSA indicating that HSA-withnoside/withanolide complexes were formed. All the results cumulatively demonstrate strong binding of withanosides and withanolides derivatives with serum albumin, which should further be explored to study the pharmacokinetics and pharmacodynamics of these derivatives.

New Flavone-Cyanoacetamide Hybrids with a Combination of Cholinergic, Antioxidant, Modulation of ß-Amyloid Aggregation, and Neuroprotection Properties as Innovative Multifunctional Therapeutic Candidates for Alzheimer's Disease and Unraveling Their Mechanism of Action with Acetylcholinesterase.

In line with the modern multi-target-directed ligand paradigm of Alzheimer's disease (AD), a series of 19 compounds composed of flavone and cyanoacetamide groups have been synthesized and evaluated as multifunctional agents against AD. Biological evaluation demonstrated that compounds 7j, 7n, 7o, 7r, and 7s exhibited excellent inhibitory potency (AChE, IC50 of 0.271 ± 0.012 to 1.006 ± 0.075 µM) and good selectivity toward acetylcholinesterase, significant antioxidant activity, good modulation effects on self-induced Aß aggregation, low cytotoxicity, and neuroprotection in human neuroblastoma SK-N-SH cells. Further, an inclusive study on the interaction of 7j, 7n, 7o, 7r, and 7s with AChE at physiological pH 7.2 using fluorescence, circular dichroism, and molecular docking methods suggested that these derivatives bind strongly to the peripheral anionic site of AChE mostly through hydrophobic interactions. Overall, the multifunctional profiles and strong AChE binding affinity highlight these compounds as promising prototypes for further pursuit of innovative multifunctional drugs for AD.

Iron deficiency cause changes in photochemistry, thylakoid organization, and accumulation of photosystem II proteins in Chlamydomonas reinhardtii.

A trace element, iron (Fe) plays a pivotal role in photosynthesis process which in turn mediates the plant growth and productivity. Here, we have focused majorly on the photochemistry of photosystem (PS) II, abundance of proteins, and organization of supercomplexes of thylakoids from Fe-depleted cells in Chlamydomonas reinhardtii. Confocal pictures show that the cell's size has been reduced and formed rosette-shaped palmelloids; however, there is no cell death. Further, the PSII photochemistry was reduced remarkably. Further, the photosynthetic efficiency analyzer data revealed that both donor and acceptor side of PSII were equally damaged. Additionally, the room-temperature emission spectra showed the fluorescence emission maxima increased due to impaired energy transfer from PSII to PSI. Furthermore, the protein data reveal that most of the proteins of reaction center and light-harvesting antenna were reduced in Fe-depleted cells. Additionally, the supercomplexes of PSI and PSII were destabilized from thylakoids under Fe-deficient condition showing that Fe is an important element in photosynthesis mechanism.

Protein stability, conformational change and binding mechanism of human serum albumin upon binding of embelin and its role in disease control.

Here, we present the inclusive binding mode of phytochemical embelin, an anticancer drug with human serum albumin (HSA) established under physiological condition. Also, to understand the pharmacological role of embelin molecule, here, we have studied the anti-cancer activity of embelin on human cervical cancer cell line (HeLa cell line), which revealed that embelin showed dose dependent inhibition in the growth of cancer cells and also induces 26.3% of apoptosis at an IC50 value of 29µM. Further, embelin was titrated with HSA and the fluorescence emission quenching of HSA due to the formation of the HSA-embelin complex was observed. The binding constant of this complex is 5.9±.01×10(4)M(-1) and the number of bound embelin molecules is approximately 1.0. Consequently, molecular displacement and computational docking experiments show that the embelin is binding to subdomain IB to HSA. Further evidence from microTOF-Q mass spectrometry showed an increase in mass from 66,563Da to 66,857Da observed for free HSA and HSA+embelin complex, signifying that there is robust binding of embelin with HSA. In addition, the variations of HSA secondary structural elements in presence of embelin were confirmed by circular dichroism which indicates partial unfolding of protein. Furthermore, the transmission electron micrographs established that complex formation leads to aggregation of HSA plus embelin. Molecular dynamics simulations revealed that the stability of the HSA-embelin complexes and results suggests that at around 3500ps the complex reaches equilibration state which clearly contributes to the understanding of the stability of the HSA-embelin complexes.