Screening of BACE1 inhibitors with antiamyloidogenic activity: A study of flavonoids and flavonoid derivatives.

Aggregates of ß-amyloid peptide are found to occur in brains of AD patients and are formed upon sequential cleavage of the amyloid precursor protein by BACE1 and γ-secretase. Strategies inhibiting either peptide aggregation or the rate limiting enzyme BACE1 have been in demand for its implication in AD therapeutics. The present study is undertaken to mine compounds with dual ability. In this context, some natural compounds that were already predicted as BACE1 inhibitors by our group, were further tested for their activity as aggregation inhibitors. A pharmacophore model built with known antiamyloidogenic compounds was then applied for screening the natural compounds previously predicted as BACE1 inhibitors. Subsequently experimental validation by Thioflavin-T and Aß-GFP assay filtered four compounds genistein, syringetin, tamarixetin and ZINC53276039. Out of them, ZINC53276039 showed promising antiamyloidogenic activity to act as a potent inhibitor of aggregation. Interestingly, our previous study revealed syringetin and ZINC53276039 to be good BACE1 inhibitors while tamarixetin to be a moderate BACE1 inhibitor. These good to moderate BACE1 inhibitors with moderate to reasonable antiamyloidogenic activity might show potency in reducing the amyloid load of AD brains.

Structural and functional implications of leucine-rich repeats in toll-like receptor1 subfamily.

Leucine-rich repeats (LRRs) - the protein-protein and protein-ligand interaction motif of proteins participating in a plethora of functions in plants, vertebrates, invertebrates, and prokaryotes - are a fascinating piece of conserved yet versatile structural motif. In toll-like receptors (TLRs), this domain forms the extracellular part that is preceded by an intracellular toll/interleukin-1 receptor (TIR) domain. The extracellular part is crucial for recognizing a structurally diverse set of viral, bacterial, fungal, and parasite-derived components, while the TIR domain is recruited for activation of downstream signaling following recognition. The distinct ability of the paralogs TLR1 and TLR6 to dimerize with TLR2 and recognize different ligands intrigued and motivated us to exchange the dimerizing and ligand-binding residues between TLR1/6 and note the effect on dimer formation and ligand binding. The appreciable sequence modification brought about no significant alteration in the native scaffold of the motif, as revealed from the comparison of simulations with wild-type dimers. Moreover, docking of the exchanged ligands to the variant proteins supported favorable binding. Thus, the structural stability and the functional plasticity offered by the motif might be the reason for its extensive use across cellular functions and life forms, a feature crucial for coevolution and the knowledge essential for therapeutics.

Apoptotic and autophagic death union by Thuja occidentalis homeopathic drug in cervical cancer cells with thujone as the bioactive principle.

OBJECTIVE: "Multi-targeting" drugs can prove fruitful to combat drug-resistance of multifactorial disease-cervical cancer. This study envisioned to reveal if Thuja homeopathic mother tincture (MT) and its bioactive component could combat human papillomavirus (HPV)-16-infected SiHa cervical cancer cells since it is globally acclaimed for HPV-mediated warts. METHODS: Thuja MT was studied for its antiproliferative and antimigratory properties in SiHa cells followed by microscopic determination of reactive oxygen species (ROS) generation by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining and loss in mitochondrial membrane potential (MtMP) by rhodamine 123 (Rh123) staining. Apoptosis and autophagy inductions were studied by acridine orange/ethidium bromide (AO/EB) staining and immunoblot analyses of marker proteins. The bioactive component of Thuja MT detected by gas chromatography-mass spectrometry was studied for antiproliferative and antimigratory properties along with in silico prediction of its cellular targets by molecular docking and oral drug forming competency. RESULTS: Thuja MT showed significant antiproliferative and antimigratory potential in SiHa cells at a 50% inhibitory concentration (IC50) of 17.3 µL/mL. An increase in DCFDA fluorescence and loss in Rh123 fluorescence prove that Thuja MT acted through the burst of ROS and loss in MtMP respectively. AO/EB-stained cells under the microscope and immunoblot analyses supported Thuja-induced cellular demise via dual pathways-apoptosis and autophagy. Immunoblots showed cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) along with upregulation of Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B)-II, and p62 proteins. Hence, the apoptotic cascade followed a caspase-3-dependent pathway supported by PARP-1 cleavage, while autophagic death was Beclin-1-dependent and mediated by accumulation of LC3BII and p62 proteins. Thujone, detected as the bioactive principle of Thuja MT, showed greater anti-proliferative and anti-migratory potential at an IC50 of 77 µg/mL, along with excellent oral drug competency with the ability for gastrointestinal absorption and blood-brain-barrier permeation with nil toxicity. Molecular docking depicted thujone with the strongest affinity for mammalian target of rapamycin, phosphoinositide 3-kinase, and protein kinase B followed by B-cell lymphoma 2, murine double minute 2 and adenosine monophosphate-activated protein kinase, which might act as upstream triggers of apoptotic-autophagic crosstalk. CONCLUSION: Robust "multi-targeting" anticancer potential of Thuja drug and thujone for HPV-infected cervical cancer ascertained its therapeutic efficacy for HPV infections.

Flavonoids: The Innocuous Agents Offering Protection against Alzheimer's Disease Through Modulation of Proinflammatory and Apoptotic Pathways.

BACKGROUND: Beginning from mild cognitive impairment in patients suffering from Alzheimer's disease (AD), dementia sets in with the progress of the disease. The pathological changes in the brain begin fifteen to twenty years before AD related dementia develops. Presence of senile plaques and neurofibrillary tangles are considered the hallmarks of AD brain. Chronic inflammation resulting from the disruption of the equilibrium between anti-inflammatory and pro-inflammatory signalling emerges as another important feature of AD and also other neurodegenerative diseases. Substantial studies demonstrate that this sustained immune response in the brain is associated with neuronal loss, along with facilitation and aggravation of Aß and NFT pathologies. Although it is well accepted that neuroinflammation and oxidative stress have both detrimental and beneficial influences on the brain tissues, the involvement of microglia and astrocytes in the onset and progress of the neurodegenerative process in AD is becoming increasingly recognized. Although the cause of neuronal loss is known to be apoptosis, the mechanism of promotion of neuronal death remains undisclosed. OBJECTIVE: Controlling the activation of the resident immune cells and/or the excessive production of pro-inflammatory and pro-oxidant factors could be effective as therapeutics. Among the phytonutrients, the neuroprotective role of flavonoids is beyond doubt. This review is an exploration of the literature on the role of flavonoids in these aspects. CONCLUSION: Flavonoids are not only effective in ameliorating the adverse consequences of oxidative stress but also impede the development of late onset Alzheimer's disease by modulating affected signalling pathways and boosting signalling crosstalk.

Estimation of the reducing power and electrochemical behavior of few flavonoids and polyhydroxybenzophenones substantiated by bond dissociation energy: a comparative analysis.

Oxidative stress that damages cellular components affects various organs including the brain. It is thus believed to play an active role in neurodegenerative diseases, wherein the intrinsic antioxidant enzymes metabolize toxic intermediates. For therapeutic purpose, instead of antioxidant enzymes, small organic compounds as antioxidants may be more effective. Here, reducing power and electrochemical behavior of some flavanols, flavanonols, flavones, flavonols and O-methylated flavonols have been estimated and confirmed by the calculated bond dissociation energy. Compared to other classes, flavonols exhibited increased reducing power that decreased with methylation of the oxygen atom in the B-ring. Gossypetin emerged as the most effective of these flavonols. Generally, compounds with two hydroxyl groups in two consecutive positions of the phenyl ring and an enolic group in the C-ring with more preference for the hydroxyl group in the ortho position with respect to each other in the catechol moiety showed major activity. 5 position of the A-ring showed the least effect on the activity. The present understanding therefore may be applied for identifying compounds to be used as scaffold for designing potent antioxidants.

Insights into the structure and function of the rate-limiting enzyme of chlorophyll degradation through analysis of a bacterial Mg-dechelatase homolog.

The Mg-dechelatase enzyme encoded by the Stay-Green (SGR) gene catalyzes Mg2+ dechelation from chlorophyll a. This reaction is the first committed step of chlorophyll degradation pathway in plants and is thus indispensable for the process of leaf senescence. There is no structural information available for this or its related enzymes. This study aims to provide insights into the structure and reaction mechanism of the enzyme through biochemical and computational analysis of an SGR homolog from the Chloroflexi Anaerolineae (AbSGR-h). Recombinant AbSGR-h with its intact sequence and those with mutations were overexpressed in Escherichia coli and their Mg-dechelatase activity were compared. Two aspartates - D34 and D62 were found to be essential for catalysis, while R26, Y28, T29 and D114 were responsible for structural maintenance. Gel filtration analysis of the recombinant AbSGR-h indicates that it forms a homo-oligomer. The three-dimensional structure of AbSGR-h was predicted by a deep learning-based method, which was evaluated by protein structure quality evaluation programs while structural stability of wild-type and mutant forms were investigated through molecular dynamics simulations. Furthermore, in concordance with the results of enzyme assay, molecular docking concluded the significance of D34 in ligand interaction. By combining biochemical analysis and computational prediction, this study unveils the detailed structural characteristics of the enzyme, including the probable pocket of interaction and the residues of structural and functional importance. It also serves as a basis for further studies on Mg-dechelatase such as elucidation of its reaction mechanism or inhibitor screening.

Multi-target inhibition ability of neohesperidin dictates its neuroprotective activity: Implication in Alzheimer's disease therapeutics.

The polygenic nature of Alzheimer's disease (AD) and cross-talk between several signaling cascades make it harder to decode the disease pathogenesis. ß-secretase (BACE1) works upstream in the amyloidogenic processing of amyloid precursor protein (APP) to generate Aß that rapidly aggregates to form fibrils, the most abundant component of plaques observed in AD brains. Here, we report dual inhibition of BACE1 and Aß aggregation by neohesperidin, a flavonoid glycoconjugate, using multi-spectroscopic approaches, force microscopy, molecular modeling, and validated the potency in SH-SY5Y neuroblastoma cell lines. Steady-state and time-resolved fluorescence reveal that neohesperidin binds close to the catalytic aspartate dyad. This binding conformationally restricts the protein in closed form which possibly precludes APP recognition and thereby inhibits BACE1 activity. Neohesperidin also dose-dependently inhibits the amyloid fibril formation, as evident from ANS, ThT assay, and AFM. Neohesperidin ameliorates aggregated Aß25-35 induced ROS generation and mitochondrial dysfunction in the SH-SY5Y cell line. As a result, the amyloid induced apoptosis is significantly prohibited and normal neuronal morphology is rescued. These findings suggest neohesperidin as an inhibitor of the pathogenic conversion of Aß to fibrillar amyloid assembly. Neohesperidin thus emerges as a non-toxic multi-potent scaffold for the development of AD therapeutics.

Theaflavin-Containing Black Tea Extract: A Potential DNA Methyltransferase Inhibitor in Human Colon Cancer Cells and Ehrlich Ascites Carcinoma-Induced Solid Tumors in Mice.

Tea is the most popularly consumed beverage in the world. Theaflavin and thearubigins are the key bioactive compounds of black tea that have anticarcinogenic properties as reported in several studies. However, the epigenetic potential of these compounds has not yet been explored. DNA methyltransferase (DNMT) enzymes induce methylation of DNA at cytosine residues and play a significant role in epigenetic regulation and cancer therapy. The present study has explored the role of black tea as a DNMT inhibitor in the prevention of cancer. Herein, the effect of theaflavin has been studied in colon cancer cell line (HCT-116) and EAC-induced solid tumors in mice. It was found that theaflavin prevented cell proliferation and inhibited tumor progression as well. In silico study showed that theaflavin interacted with DNMT1 and DNMT3a enzymes and blocked their activity. Theaflavin also decreased DNMT activity In Vitro and In Vivo as evident from the DNMT activity assay. Results of immunohistochemistry revealed that theaflavin reduced DNMT expression in the tumors of mice. Taken together, our findings showed that theaflavin has a potential role as a DNMT inhibitor in HCT-116 cell line and EAC induced solid tumors in mice.

Identifying the key residues instrumental in imparting stability to amyloid beta protofibrils - a comparative study using MD simulations of 17-42 residues.

Extracellular plaques, the hallmark of Alzheimer's disease brains, consist of insoluble amyloid fibrils that result from the aggregation of amyloid beta peptides. None of the few therapeutic options currently adopted, address the cause of the disease. Instead, they reduce symptom of the disease. Inhibition of aggregation or destabilization of aggregates therefore, emerges as a preferable therapeutic approach. Designing inhibitors or destabilizers demands comprehensive knowledge of the residues of amyloid beta responsible for the phenomenal structural stability of the aggregate. For the purpose, we have compared the effect on structural destabilization of 13 in silico mutations (single and double) with the wild type counterpart of beta-strand-turn-beta-strand motif of the amyloid beta protofibrils by molecular dynamics simulation. Besides the already known salt bridge interaction between K28 and D23, our analyses expose more significant role of K28 as the only positive charge present in the vicinity. Amongst the two consecutive aromatic residues, F19 is involved in stacking interaction; although effect of F20 mutation is more pronounced. Face to face arrangement of A21 and V36 acts as a pillar maintaining the necessary optimum distance between consecutive chains to promote stabilizing interactions. In addition to providing stability to the first beta-strand, large sized negatively charged E22 facilitates salt bridge formation by ensuring fixed relative position of D23 and in turn K28. Likewise, the hydrophobic residues I32 and L34 pack the protofibril core, once again fostering salt bridge interaction. Prospectively, these findings may be compiled for efficient identification or design of scaffolds accountable for protofibril destabilization.Communicated by Ramaswamy H. Sarma.

Flavonoids as BACE1 inhibitors: QSAR modelling, screening and in vitro evaluation.

Alzheimer's disease (AD) is marked by the presence of amyloid plaques, neurofibrillary tangles, oxidatively damaged neuronal macromolecules and redox sensitive ions. Reduction of amyloid plaques and oxidative stress emerge as a convincing treatment strategy. Plaque reduction is achieved by inhibition of BACE1, the rate limiting enzyme generating the prime constituent of plaques, Aß, through proteolysis of the amyloid precursor protein. Here, we report a QSAR model with five descriptors, developed to screen natural compounds as potent BACE1 inhibitors. Seven compounds out of which five flavonols namely isorhamnetin, syringetin, galangin, tamarixetin, rhamnetin and two flavanonols namely dihydromyricetin, taxifolin were screened. The ability of these compounds were validated using the BACE1 activity assay. The antioxidant property were estimated by the DPPH and ABTS assay. Although inhibition assay implied syringetin to be a promising BACE1 inhibitor, its poor antioxidant activity leaves it less effective as a multitarget ligand. Exhibiting moderate dual ability, isorhamnetin and taxifolin qualified as multi-target scaffolds for AD therapeutics. Our study reveals the importance of 4'-OH in the B ring of flavonols and the lack of any effect of 5'-OH in flavanonols for BACE1 inhibition. In case of antioxidant activity favourable association of 3'-O-methylation derivatives was observed in flavonols.

Hybrid approach to sieve out natural compounds against dual targets in Alzheimer's Disease.

Excess Aß production by the key protease BACE1, results in Aß aggregation, forming amyloid plaques, all of which contribute to the pathogenesis of Alzheimer's disease. Besides the multi-factorial nature of the disease, the diversity in the size and shape of known ligands that bind to the active site of BACE1, that is the flexibility of the enzyme, pose a serious challenge for the identification of drug candidates. To address the issue of receptor flexibility we have carried out ensemble docking with multiple receptor conformations. Therein, two representative structures each from closed and semi-open BACE1 conformations were selected for virtual screening to identify compounds that bind to the active site of both the conformations. These outperformed compounds were ranked using pharmacophore models generated by a ligand-based approach, for the identification of BACE1 inhibitors. The inhibitors were further predicted for anti-amyloidogenic activity using a QSAR model already established by our group thus enlisting compounds with dual potency. BACE1 inhibitory and anti-amyloidogenic activity for the commercially available compounds were validated using in vitro studies. Thus, incorporation of receptor flexibility in BACE1 through ensemble docking in conjunction with structure and ligand-based approach for screening might act as an effective protocol for obtaining promising scaffolds against AD.

Insights into the evolution of extracellular leucine-rich repeats in metazoans with special reference to Toll-like receptor 4.

The importance of the widely spread leucine-rich repeat (LRR) motif was studied considering TLRs, the LRR-containing protein involved in animal immune response. The protein connects intracellular signalling with a chain of molecular interactions through the presence of LRRs in the ectodomain and TIR in the endodomain. Domain analyses with human TLR1-9 reported ectodomain with tandem repeats, transmembrane domain and TIR domain. The repeat number varied across members of TLR and remained characteristic to a particular member. Analysis of gene structure revealed absence of codon interruption with TLR3 and TLR4 as exceptions. Extensive study with TLR4 from metazoans confirmed the presence of 23 LRRs in tandem. Distinct clade formation using coding and amino acid sequence of individual repeats illustrated independent evolution. Although ectodomain and endodomain exhibited differential selection pressure, within the ectodomain, however, the individual repeats displayed positive, negative and neutral selection pressure depending on their structural and functional significance.

Multi-targeting Strategies for Alzheimer's Disease Therapeutics: Pros and Cons.

Alzheimer's Disease (AD) is a single major cause of dementia in middle to old age individuals involving several different etiopathological mechanisms that are yet to be properly characterized. Major invariant and characteristic features consist of the progressive cerebral deposition of the Amyloid ß-protein (Aß) and the neurofibrillary degeneration through Neurofibrillary Tangles (NFT) formed by hyperphosphorylation of the tau proteins in the regions of the brain that deal with memory and cognition. There are at least five subgroups of AD that can be identified by determining Cerebrospinal Fluid (CSF) levels of Aß1-42, tau and ubiquitin. This multifactorial nature of the disease thus demands promising approaches for the development of rational disease-modifying drugs. A large number of agents have been discovered against individual targets but the success rate is very low and the number of compounds progressing to regulatory review is among the lowest found in any therapeutic area. A very promising modern approach solicits the design of Multi-Target-Directed Ligands (MTDLs) based on the "one molecule multiple targets" paradigm that has been specifically adopted for the treatment of disorders with complex pathological mechanisms. AD is one such disorder in which MTDL has found applicability. This review aims at providing an overview of the research carried out in discovering more efficient treatment against AD using MTDL, with a goal to ascertain safer drugs.

Multi-functional activities of citrus flavonoid narirutin in Alzheimer's disease therapeutics: An integrated screening approach and in vitro validation.

Alzheimer's disease (AD) is a complex disorder and the disease mechanism is yet to be properly characterized. Over the years, "amyloid cascade" emerges as principal pathogenic event in AD. ß-Secretase 1 (BACE1) controls the rate-limiting step in amyloid beta (Aß) generation and Aß rapidly aggregates to form neurotoxic amyloid fibrils. Oxidative stress is one of the principal mediators of the observed neurotoxicity of amyloid fibril. The disease pathogenesis involves induction of multiple signaling cascades and the cross-talk therein. Thus inhibiting multiple targets could be an attractive therapeutic strategy. Here, a multi-target virtual screening protocol has been devised and used to screen an in house developed phytochemical library. Narirutin comes out as a multi-potent phytochemical. Steady-state and time-resolved fluorescence along with molecular modelling studies demonstrate its binding to the BACE1 active site that induces a conformational transition of the protein from open to closed form which precludes substrate recognition. Narirutin possesses strong Aß aggregation inhibitory potential, evident from ANS and Thioflavin-T binding assay and confirmed by AFM study. ABTS·+ scavenging assay shows moderate antioxidant activity for narirutin. The applicability of a multi-target screening strategy in AD therapeutics is thus demonstrated and the identified hit, narirutin, shows strong multi-potent activity.

Dual inhibition of BACE1 and Aß aggregation by ß-ecdysone: Application of a phytoecdysteroid scaffold in Alzheimer's disease therapeutics.

Current medications for the complex neurological disorder, Alzheimer's disease (AD), can neither stop disease progression nor revert back disease pathogenesis. The present study demonstrates the applicability of a phytoecdysteroid, ß-ecdysone, as a multi-potent agent in AD therapeutics. ß-ecdysone strongly binds to the active site cavity of BACE1 with calculated dissociation constant of 1.75±0.1µM. Steady-state and time-resolved fluorescence spectroscopy reveal that binding of ß-ecdysone induces conformational transition of the protein from open to closed form thereby blocking substrate binding. Even 500nM of the compound completely blocks the enzyme activity. Furthermore, ß-ecdysone strongly inhibits Aß aggregation, evident from ANS and ThT binding assay. Co-incubation of equimolar peptide and ß-ecdysone completely inhibits Aß fibril formation which is further complemented by the AFM study. Low systemic toxicity of ß-ecdysone further extends the applicability of the compound as functional food and dietary supplement for disease management.

Split-ubiquitin yeast two-hybrid interaction reveals a novel interaction between a natural resistance associated macrophage protein and a membrane bound thioredoxin in Brassica juncea.

Natural resistance associated macrophage proteins (NRAMPs) are evolutionarily conserved metal transporters involved in the transport of essential and nonessential metals in plants. Fifty protein interactors of a Brassica juncea NRAMP protein was identified by a Split-Ubiquitin Yeast-Two-Hybrid screen. The interactors were predicted to function as components of stress response, signaling, development, RNA binding and processing. BjNRAMP4.1 interactors were particularly enriched in proteins taking part in photosynthetic or light regulated processes, or proteins predicted to be localized in plastid/chloroplast. Further, many interactors also had a suggested role in cellular redox regulation. Among these, the interaction of a photosynthesis-related thioredoxin, homologous to Arabidopsis HCF164 (High-chlorophyll fluorescence164) was studied in detail. Homology modeling of BjNRAMP4.1 suggested that it could be redox regulated by BjHCF164. In yeast, the interaction between the two proteins was found to increase in response to metal deficiency; Mn excess and exogenous thiol. Excess Mn also increased the interaction in planta and led to greater accumulation of the complex at the root apoplast. Network analysis of Arabidopsis homologs of BjNRAMP4.1 interactors showed enrichment of many protein components, central to chloroplastic/cellular ROS signaling. BjNRAMP4.1 interacted with BjHCF164 at the root membrane and also in the chloroplast in accordance with its proposed function related to photosynthesis, indicating that this interaction occurred at different sub-cellular locations depending on the tissue. This may serve as a link between metal homeostasis and chloroplastic/cellular ROS through protein-protein interaction.

Multi-target screening mines hesperidin as a multi-potent inhibitor: Implication in Alzheimer's disease therapeutics.

Alzheimer's disease (AD) is the most frequent form of neurodegenerative disorder in elderly people. Involvement of several pathogenic events and their interconnections make this disease a complex disorder. Therefore, designing compounds that can inhibit multiple toxic pathways is the most attractive therapeutic strategy in complex disorders like AD. Here, we have designed a multi-tier screening protocol combining ensemble docking to mine BACE1 inhibitor, as well as 2-D QSAR models for anti-amyloidogenic and antioxidant activities. An in house developed phytochemical library of 200 phytochemicals has been screened through this multi-target procedure which mine hesperidin, a flavanone glycoside commonly found in citrus food items, as a multi-potent phytochemical in AD therapeutics. Steady-state and time-resolved fluorescence spectroscopy reveal that binding of hesperidin to the active site of BACE1 induces a conformational transition of the protein from open to closed form. Hesperidin docks close to the catalytic aspartate residues and orients itself in a way that blocks the cavity opening thereby precluding substrate binding. Hesperidin is a high affinity BACE1 inhibitor and only 500 nM of the compound shows complete inhibition of the enzyme activity. Furthermore, ANS and Thioflavin-T binding assay show that hesperidin completely inhibits the amyloid fibril formation which is further supported by atomic force microscopy. Hesperidin exhibits moderate ABTS(+) radical scavenging assay but strong hydroxyl radical scavenging ability, as evident from DNA nicking assay. Present study demonstrates the applicability of a novel multi-target screening procedure to mine multi-potent agents from natural origin for AD therapeutics.

Structural insight into the mechanism of amyloid precursor protein recognition by ß-secretase 1: A molecular dynamics study.

ß-secretase 1 (BACE1) initiates the proteolysis of amyloid precursor protein (APP) to generate Aß, aggregation of which has been considered to be the main histopathological feature of Alzheimer's Disease. Here, we have explored the conformational switching of BACE1 during APP recognition using molecular dynamics simulation thereby suggesting the recognition to be a conformational selection process. Free BACE1 is highly flexible and exists as an ensemble of conformations. The ß-hairpin flap that covers the active site of BACE1 visits numerous conformations during the simulation. Essential dynamics reveal that concerted movements in several loops including the flap region lead to a conformational switching from open to closed form. During the simulation, free BACE1 visits both the open and closed forms multiple times. Binding of APP to the BACE1 cavity shifts the equilibrium towards a stable complex stabilized by strong electrostatic surface complementarity along with several van der Waals and hydrogen bonding interactions.

Probing the Conformational Dynamics of the Bioactive Peptide TLQP-21 in Solution: A Molecular Dynamics Study.

VGF-derived peptide, TLQP-21, is a physiologically active neuropeptide exhibiting important roles in energy expenditure and balance, gastric contractility, reproduction, pain modulation, and stress. Although the physiological functions of the peptide constitute a research area of considerable interest, structural information is clearly lacking. Here, using extensive 550 nanoseconds molecular dynamics simulation in explicit water model, we have explored the folding energy landscape of the peptide. Principal component analysis and cluster analysis have been used to identify highly populated conformational states of the peptide in solution. The most populated structure of the peptide adopts a highly compact globular form stabilized by several hydrogen-bonding interactions and π-cationic interactions. Strong surface complementarity of hydrophobic residues allows tighter spatial fit of the residues within the core region of the peptide. Our simulation also predicts that the peptide is highly flexible in solution and that the region A7 -R9 and three C-terminal residues, P19 -R21 , possess strong helical propensity.

Fragment-based designing for the generation of novel leads against BACE1.

BACE1, the aspartate protease that generates amyloid-ß peptide (Aß) in the brain of AD (Alzheimer's disease) patients, has emerged as a pharmaceutically relevant target. Here, a fragment-based in silico approach has been adopted to design novel compounds with increased ligand efficiency for BACE1, before screening for brain permeability and toxicity. Fragments docked to the active site of BACE1 and sorted into two groups using binding energy cut-off, were joined to create novel ligands with binding energy lying in the range between -11.36 kcal/mol and -8.56 kcal/mol. Interestingly, QIN, a known inhibitor of BACE1 with an IC50 of 11nM, when docked to BACE1, shows a binding energy (-9.43 kcal/mol) lying within the range of the novel ligand-BACE1 complexes. The present strategy thus enabled the design of four novel inhibitors of BACE1 with favourable binding energy, brain permeability and no toxicity that might show promise as leads in future.