Potential Neuroprotective Peptide Emerged from Dual

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Potential Neuroprotective Peptide Emerged from Dual Neurotherapeutic Targets: A Fusion Approach for the Development of anti-Alzheimer’s Lead Prasenjit Mondal, Gaurav Das, Juhee Khan, Krishnangsu Pradhan, Rathnam Mallesh, Abhijit Saha, Batakrishna Jana, and Surajit Ghosh ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00115 • Publication Date (Web): 06 Mar 2019 Downloaded from http://pubs.acs.org on March 7, 2019

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ACS Chemical Neuroscience

Potential Neuroprotective Peptide Emerged from Dual Neurotherapeutic Targets: A Fusion Approach for the Development of anti-Alzheimer’s Lead Prasenjit Mondal,1,2 Gaurav Das,1,2 Juhee Khan,1 Krishnangsu Pradhan,1 Rathnam Mallesh,1,3 Abhijit Saha,1 Batakrishna Jana,1 Surajit Ghosh 1,2,3* 1. Organic & Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kol-32, West Bengal, India. Tel: +91-33-2499-5872; Fax: +9133-2473-5197/0284 2. Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical Biology Campus, 4, Raja S. C. Mullick Road, Kol-32, West Bengal, India. 3. National Institute of Pharmaceutical Education and Research, Kolkata, CSIR-Indian Institute of Chemical Biology Campus, 4, Raja S. C. Mullick Road, Jadavpur, Kol-32, West Bengal, India.

ABSTRACT: Amyloid-beta (Aβ) peptide misfolds into fibrillary aggregates (β-sheet) and deposited as amyloid plaques in the cellular environment, which severely damages intraneuronal connections leading to Alzheimer’s disease (AD) pathogenesis. Furthermore, neurons are rich in tubulin/microtubule and the intracellular network of microtubules also gets disrupted by the accumulation of Aβ fiber in brain. Hence, development of new potent molecules, which can simultaneously inhibit Aβ fibrillations and stabilize microtubules, is particularly needed for the efficient therapeutic application in AD. To address these issues, here we introduced an

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innovative fusion strategy to design and develop next generation anti-AD therapeutic leads. This unexplored fusion strategy entails design and development of a potent nonapeptide by taking into account both the hydrophobic core (17-21) of Aβ peptide and the taxol binding region of βtubulin. In vitro results suggest that this newly designed peptide interacts at the taxol binding region of β-tubulin with a moderate binding affinity and promotes microtubule polymerization. It has the ability to bind at the hydrophobic core (17-21) of Aβ, responsible for its aggregation and prevent the amyloid fibril as well as plaques formation. In addition, it interacts at the CAS site (catalytic anionic site) of acetylcholinesterase (AChE) and significantly inhibits AChE induced Aβ fibrillation, stimulates neurite branching, provides stability to intracellular microtubules and extensive protection of neurons against nerve growth factor (NGF) deprived neuron toxicity. Moreover, this newly designed peptide shows good stability in serum obtained from humans and efficiently permeates the blood-brain barrier (BBB) without showing any toxicity towards differentiated PC12 neurons as well as primary rat cortical neurons. This excellent feature of protecting the neurons by stabilizing the microtubules without showing any toxicity towards neurons will make this peptide a potent therapeutic agent of AD in near future. KEYWORDS: Neurodegenerative diseases, amyloid plaques, neuropeptides, microtubule stability, primary cortical neurons 1. INTRODUCTION Alzheimer’s disease (AD) is an advanced neural dementia that worsens over a period of time and grows into a fatal chronic neurodegenerative disorder.1-3 It interferes with the person’s basic ability to carry out the everyday chores by diminishing memory, which reveals the life-


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threatening nature of this disease.4 AD is well-characterized by the existence of two different aggregated proteins inside and outside of the neurons. One is intraneuronal neurofibrillary tangles (NFTs) formed by the aggregation of hyperphosphorylated tau and other one is extracellular amyloid plaques, which results in the assembly of amyloid-β (Aβ) in the interstitial areas of brain.5,6 Moreover, it has been documented before that the production and accumulation of amyloid plaques in brain may be synergizing and accelerating the changes in tau pathology.6 This process starts when Aβ peptide is formed by the chopping of amyloid precursor protein (APP) at specific sites by secretase enzymes, which aggregates and forms senile plaques outside the neurons.7,8 These senile plaques triggers the abnormal function of neurons, which result in severe degeneration of neurons as well as brain tissue.8 Further, this senile plaque increases the activity of kinesin protein inside the neurons, which results in hyperphosphorylation of tau protein.9 Tau proteins are found in abundance in neurons where they help in microtubule polymerization and provides stability to their elaborated lattice. When tau detaches from the microtubule due to hyperphosphorylation, it aggregates to form NFTs.10-13 Although the oligomeric and prefibrillar forms of Aβ are identified to be crucial for AD, but the molecular basis and exact mechanism behind the initiation of amyloid aggregation and the toxic effect of the Aβ peptide aggregation remains unclear.14 The conversion of nontoxic, soluble Aβ protein into toxic, insoluble β-sheet rich fibrillary structure is contemplated to be the critical step of AD pathology.15 Further, in this disease progression, microtubule, one of the key cytoskeleton proteins is disrupted severely and leave an impact on neuron function.16,17 Therefore, inhibition of Aβ fibrillation as well as stabilization of microtubule networks is the vital prerequisite for the development of novel anti-Alzheimer therapeutic agents. In this direction, microtubule stabilizing agents like epothilone D,18 Davunetide (NAP peptide), and Dictyostatin19,20 show



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promising results. Thus, further developments of potent therapeutic agents using unique approaches are essential to protect the neurons as well as to understand the basic pathway of this disease progression. To address this problem, in this work we are introducing an advanced fusion strategy whereby we investigate both the taxol cavity of β-tubulin (as taxol stabilizes microtubules) and hydrophobic region of Aβ to develop a microtubule stabilizing neuroprotective peptide. Microtubule stabilizing anti-cancer drug, Taxol was thought to be a potent anti-AD therapeutic agent, but never verified in AD affected brain due to high toxicity and poor brain penetration ability.21-26 In our previous work, we have also developed a octapeptide from the taxol binding pocket of tubulin using alanine scanning mutagenesis, which shows significant neuroprotection over NGF deprived neurons but couldn’t penenetrate the blood-brain barrier (BBB).27 On the other hand, the hydrophobic core (17-21) of Aβ is chiefly responsible for its fibrillation and aggregation. A molecule that binds to this hydrophobic core of Aβ can act as a potent amyloid inhibitor.28 These facts motivate us to develop this novel peptide based therapeutics for AD, which may execute dual role of action. However, we have to also keep in mind about the careful balance between toxicity and microtubule stability, while designing the microtubule stabilizer.29 Since our whole idea of neuroprotection is based on NGF deprivation,30 which ultimately leads to

amyloidogenesis31

and

eventually

microtubule

destruction

lead

by

tau

hyper-

phosphorylation,32 so microtubule stabilizing molecules will qualify as candidates for neuroprotection also. Hence, if our molecule does provide stability to the microtubules without causing toxicity like the taxols, they will in other words also exhibit neuroprotection. The 17-21 core of Aβ contains several non-polar and hydrophobic amino acids whereas the taxol cavity of β-tubulin consists of polar as well as non-polar amino acids. We designed one


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nonapeptide “GAKEAHDFQ (GAK)” by the counter interaction of amino acids of those pocket by considering the relative frequencies of contacts between amino acids (Figure 1 and Figure S1, ESI).33 Further, we designed a library of 9 peptides by sequentially modifying this designed peptide, and performed molecular docking with both tubulin (PDB ID: 1JFF)34 and Aβ (PDB ID: 1IYT)35 to find out the potent inhibitor peptide. Peptide “NVRDLTEFQ” has the best binding affinity with both tubulin (-6.7 kcal mol-1) and amyloid beta (-5.0 kcal mol-1) respectively, which makes this peptide a powerful candidate amongst the others (Figure 2A). Again, to check the positional impact of each amino acid on this designed peptide, we have scrambled this peptide sequence and constructed a library of additional 10 peptide sequences and performed a similar docking study as described above. We found that nonapeptides “NVRDLTEFQ (NVR)”, “LVQRTDNEF (LVQ)”, and “VRNQDEFTL (VRN)” have very good binding affinity to both sides (Figure 2B). Further, studies reveal that NVR peptide evolves as a potent neuroprotective agent, which moderately binds with the taxol cavity of β-tubulin, promotes tubulin polymerization, and Aβ aggregation inhibitor. Moreover, this newly designed peptide is nontoxic against both differentiated PC12 neurons as well as primary rat cortical neurons. Interestingly, this peptide has good stability in human serum and can efficiently permeate the BBB. This unique approach to develop a neuroprotective peptide with dual targets will facilitate the design of more potent anti-AD therapeutic leads. 2. RESULTS AND DISCUSSION 2.1 Designing of peptides library considering the relative occurrences of contacts between amino acids and their molecular docking with tubulin and Aβ



To design a potent neuroprotective peptide having microtubule stabilizing ability, we considered the taxol binding region of β-tubulin as this is the only site where binding of any molecule results in microtubule stabilization. The binding affinity of taxol molecule is so high that it enforces structural changes in tubulin, which results in long range stability of microtubule and finally shows toxicity. Therefore, the neuroprotective peptide must be designed in such a way that it shouldn’t have very high binding affinity towards tubulin.29 Taxol pocket consists of key most interacting amino acids (Gly 370, Asp 26, Arg 278, Phe 272, Thr 276, His 229), non-charge and hydrophobic amino acids (Ser236, Val23, Ala 233, Leu 275, Ser 277) whereas the hydrophobic region of Aβ contains five key hydrophobic amino acids (LVFFA). We have constructed a nonapeptide sequence “GAKEAHDFQ (GAK)“ considering the aforementioned amino acids of both the pockets and using the respective frequencies of contacts between these amino acids (Figure S1, ESI). Afterthat, we have modified this peptide in a sequential approach and constructed a library of total 9 peptides using the most interacting amino acids contact. Then, the molecular docking analysis of these 9 peptides with both tubulin and Aβ suggest that “NVRDLTEFQ (NVR)“ has the best binding affinity towards both tubulin (-6.7 kcal mol-1) and Aβ (-5.0 kcal mol-1) (Figure 2A). Figure S2,ESI has revealed the chem-draw structure and peptide sequence of this potent NVR peptide. Thereafter, we prepared a library consisting of 11 new peptides by rearranging the NVR peptide sequence to find out whether the change in position of each amino acids has any impact towards the final activity of this peptide. 2.2 Molecular docking experiment to identify the potent peptide for neuroprotection Thereafter, molecular docking analysis with these eleven peptides have been conducted around the taxol binding cavity in tubulin (PDB ID-1JFF)34 and hydrophobic core (17-21) of Aβ in Autodock vina 1.1.2.36 The results of molecular docking experiment show that NVR peptide has


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the best binding towards both the sites (-6.7 kcal mol-1 with tubulin and -5.0 kcal mol-1 with Aβ) whereas two other peptides, “LVQRTDNEF (LVQ)“ (-7.8 kcal mol-1 with tubulin and -4.1 kcal mol-1 with Aβ) and “VRNQDEFTL(VRN)“ (-6.5 kcal mol-1 with tubulin and -4.8 kcal mol-1 with Aβ) have close binding energies (Figure 2B). The interaction partners of NVR in the taxol binding pocket are Asp 26, Glu 27, His 229, Ser 236, Gly 270, Pro 274, Thr 276, Arg 278 (Figure 2C, D). 2.3 Synthesis, characterization and fluorescein dye labeling of designed peptides Synthesis of this NVR peptide, two other shuffled peptides (LVQ, VRN) having close binding energy and its fluorescein conjugation were executed with rink amide resin using a well-known solid phase peptide synthesis (SPPS) protocol. Labeling of peptides with fluorescein dye [5(6)Carboxyfluorescein] was executed at the N-terminal asparagine (Asn) moiety of NVR as it has a free primary amine using coupling reagents HOBT and DIC in DMF. Further, purification and characterization were accomplished by reversed-phase HPLC (>90 % purity) and MALDI-TOF mass analysis respectively (Figure S3-S5, ESI). 2.4 Monitoring the effect of different peptides (NVR, LVQ and VRN) on the polymerization nature of tubulin by performing tubulin turbidity study and microtubule assembly assay We have performed tubulin polymerization assay and microtubule assembly study with purified tubulin in presence of GTP to check the effect of these peptides on tubulin polymerization as these peptides were designed to stabilize the intracellular microtubule. Results suggest that NVR peptide has the ability to enhance the polymerization of tubulin at very low concentration (3.125 and 6.25 μM), which designates that it provides substantial stability to intracellular tubulin (Figure 2E, F). In case of LVQ and VRN peptide, tubulin polymerization was reduced compare



to control experiment, which clearly indicates that these peptides do not provide stability to microtubule rather depolymerization occurs (Figure S6, ESI). Therefore, in our further studies, we mostly look into the detail interaction of NVR peptide. 2.5 Measuring the binding constant (Kb) of NVR towards tubulin Further, the binding constant of NVR with tubulin moiety was evaluated carefully as this peptide is promoting tubulin polymerization. Hence, we executed a commonly used intrinsic tryptophan fluorescence emission quenching study to calculate the interaction of small molecules/peptides with tubulin.37 Generally, the emission intensity value of tubulin’s intrinsic tryptophan gets decreased with addition of different concentration of NVR peptide. Modified Stern-Volmer equation was used to plot the fluorescence quenching values, which shows that NVR peptide has the binding affinity (Kb) of ~ 2.5×104 M-1 with tubulin that specifies substantial binding affinity of NVR peptide towards tubulin (Figure 2G). 2.6 Finding binding location of NVR in tubulin by performing Förster Resonance Energy Transfer (FRET) analysis Initially, we have designed this peptide by keeping in mind the key amino acids of taxol cavity of β-tubulin. Therefore, we were curious to detect the exact binding location of NVR in tubulin. This FRET experiment was previously used to locate the binding position of peptide, small molecule or biologically active protein in interacting protein substances using suitable FRET partners. Previously, it was well documented that tubulin-colchicine complex can be used as a donor and fluorescein dye attached peptide can be used as an acceptor to detect the binding location of peptide in tubulin38 as they possess substantial spectral overlap region between them.


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FRET experiment was performed using fluorescein conjugated NVR (acceptor) and tubulin bound colchicine complex (donor). From Figure 2H, we acquired the efficiency of FRET (FRET) = 0.436 Then, we measured the FRET distance (RDA) between the tub-col donor and fluorescein-NVR acceptor as 30.8 ± 1 Å, that clearly suggests that this newly designed NVR binds at a position which is 30 Å far from the binding region of colchicine in tubulin i.e close to taxol site of βtubulin (Figure 2H). 2.7 Molecular docking experiment to understand the detail interaction of NVR peptide with Aβ peptide As per our designed concept, this NVR peptide is quite well interacts with tubulin and able to stabilize the intracellular microtubule. Now, we were interested to examine whether this peptide can inhibit the amyloid aggregation. We executed a molecular docking experiment of NVR with Aβ (PDB ID-1IYT)35 to identify the binding stretch and detail interaction of NVR with Aβ. Results suggest that this NVR peptide interacts at the hydrophobic stretch (17-21) of Aβ peptide with significant binding energy (B.E: -5.0 kcal mol-1) and interacts with Leu 17 and Ala 21. It is well documented that amyloid inhibitor molecule generally binds at the hydrophobic core (1721) of Aβ.27 Hence, this NVR peptide may also act as a potential amyloid inhibitor (Figure 3A). 2.8 Monitoring the inhibiton of Aβ aggregation by designed peptides using ThT assay and FT-IR analysis Then we checked the amyloid aggregation inhibition ability of NVR by performing ThT assay.39 We have incubated Aβ solution (10 μM) alone, with different concentration of NVR (1-20 μM)



peptide at 37 °C temperature for 10 days and monitored the aggregation of Aβ by recording the fluorescence intensity of ThT (Ex- 435 nm, Em- 485 nm), after mixing it with different incubated solution. Also, in another case different concentration of NVR peptide was mixed with preformed amyloid fibril to check its activity on fibril Aβ. We found that this peptide has significant ability to inhibit oligomeric as well as preformed fibril amyloid aggregation (Figure 3B, C). After 7 days this peptide inhibits almost 45 % of the aggregation and the percentage of amyloid aggregation inhibition increases with increasing concentration of peptide solution (Figure 3D). But, in case of shuffled peptides (LVQ and VRN), they only inhibit 13% (LVQ) and 28% (VRN) of amyloid aggregation (Figure S7, ESI). This aggressive suppression of amyloid aggregation was also supported by FT-IR study, where we didn’t find any characteristic β-sheet peak of amyloid fibrillations (Figure 3E) in presence of NVR peptide. 2.9 Monitoring acetylcholinesterase (AChE) induced Aβ aggregation inhibition by NVR peptide ThT assay and docking study reveals that this NVR can substantially prevent amyloid aggregation. Further, we were interested to find out whether this peptide can act in a similar manner when AChE is present in the solution. In neurodegenerative brain disorder, the concentration of AChE increases rapidly, which enhances the aggregation of Aβ.27,40 Therefore, we explored the effect of NVR in induced amyloid aggregation condition. For this purpose, we incubated different concentration of NVR with Aβ and AChE at 37 OC and kept the final solution in agitation for 12 h. After 12 h of incubation and aggitation, we observed significant inhibition of amyloid aggregation upon increment of NVR peptide concentration (5 µM, 10 µM) compared to control experiment where rapid amyloid aggregation occurs (Figure 3F). This result suggests substantial inhibition activity of NVR in acetylcholinesterase induced extreme amyloid aggregation condition.


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2.10 Evaluation of inhibiton kinetics of AChE enzyme activity by NVR peptide Previous results suggest that NVR peptide acts as a prospective inhibitor for Aβ aggregation in prescence of significant amount of AChE, which further can co-relate with the fact that this peptide might have an inhibitory effect towards AChE. We investigated the detail mechanism of prevention of AChE action by NVR. Therefore, the substrate-velocity curve was measured upon addition of different concentration of NVR (0.5-10 µM) in various acetylthiocholine (substrate) concentration (87.5 - 700 μM) to calculate the binding region of NVR and find out the exact mechanistic pathway of AChE inhibition. We clearly observed the changes of Km value (negative reciprocal of X-intercept) and Vmax value (reciprocal of Y-intercept) with increasing concentration of NVR from the Lineweaver-Burk plot, which suggests its binding at CAS site of AChE in a competitive fashion (Figure 3G). 2.11 Determination the binding interaction of NVR peptide with AChE by molecular docking study AChE has two binding sites of interaction, one is peripherral anionic site (PAS) and other one is catalytic anionic site (CAS). Experimental results show that NVR peptide interacts with AChE, so, we performed molecular docking experiment with NVR to find out the binding location and interaction partners in AChE (PDB ID: 4EY6).41 The binding energy (-8.2 kcal mol-1) of this NVR in CAS site of this enzyme is quite high, which indicates significant binding affinity of NVR towards CAS site of AChE. This interaction of NVR with AChE was stabilised by the interaction of different amino acids S200, Y130, G117, E199, G119, Y121, S122, N85, D72, Y70, R289, S286, W279 of PAS and CAS pockets of AChE (Figure 3H). Hence, the result



suggests that NVR has the ability to interact at the CAS binding region of AChE and inhibit the activity of AChE. 2.12 Transmission Electron Microscopy (TEM) study to monitor Aβ fibrillation inhibition ability of NVR peptide We observed substantial inhibition of Aβ fibrillization in TEM analysis when Aβ solution was mixed with NVR peptide and incubated at 37 °C. TEM images suggest that when Aβ solution (1 μM) is incubated alone for 7 days at 37 °C temperature, it exhibits extensive fibrillar network (Figure S8A, ESI). But after incubating Aβ solution with NVR peptide (1 μM) we observed negligible fibrillar network (Figure S8B, ESI). 2.13 MD simulation study to monitor the interaction of NVR with Aβ After that, we were keen to understand the inhibition of fibril formation through MD simulation study. Therefore, two short peptide sequence “KLVFFAE’’(16-22 region of Aβ42, responsible for its‘ fibrillation) with NVR was prepared and kept in a cubic box for simulation. After 50 ns of simulation, we found that those two short peptides can’t form stable β-sheet structure throughout the simulation (Figure S9A, ESI). This phenomenon was further confirmed by the secondary structure profile image (Figure S9B, ESI). But, they form stable secondary β-sheet structure within 2 ns of simulation (Figure S10, ESI). These findings clearly reveal that NVR binds to the hydrophobic core of Aβ peptide and inhibits its aggregation process. 2.14 Uptake analysis of NVR in neurons through microscopy All our previous studies with the peptide indicates that this newly designed NVR peptide can promotes tubulin polymerisation and inhibits Aβ fibrillation significantly. So we wanted to know


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whether this peptide will replicate similar results in the cell which will be studied through several in vitro assays. Mainly, we want to check the effect of this peptide in the neurons as this peptide was designed to protect the neurons from degeneration. At first, we investigated the uptake efficacy of NVR peptide in differentiated rat pheochromocytoma (PC12) cells. The differentiated neurons were incubated with fluorescein conjugated NVR peptide (10 µM) for 4 h in the incubator. It was followed by fixation of the treated cells with paraformaldehyde and analysis of the cells was performed through microscopy. When observed through 488 nm channel, we found sufficient cellular uptake of NVR peptide showing staining with fluorescein labeled peptides in the dendrites and cell bodies. The merged images of the DIC, DAPI and 488 nm channels clearly support the above statement (Figure 4A-D). 2.15 Cytotoxicity of NVR peptide Next, the toxicity of NVR was evaluated on PC12 derived neurons at various concentrations as it is extremely necessary for a neuroprotective molecule to be non-toxic. We observed that this peptide is non-cytotoxic in differentiated neurons upto 50 µM of peptide concentration (Figure 4E). Further, microscopic DIC images of control and NVR treated (10 µM) differentiated PC12 neurons suggest that in case of NVR treated PC12, neurons are healthier with sufficient neurites out-growth (Figure 4F-G). The average neurite length of the neurons treated with NVR peptide was higher than the untreated control indicating the neuroprotective nature of NVR (Figure 4H) 2.16 Stabilization of intracellular microtubule by NVR peptide Intracellular microtubule network got hampered by both Tau and Aβ aggregates during progression of AD.42 Therefore; microtubule stabilisation is a key requirement for a potent neuroprotective agent. Various in vitro results suggest that NVR peptide promotes microtubule



polymerization and stabilizes microtubule. The stability of microtubule networks by NVR peptide was evaluated through a cellular assay. At first, the neurons were incubated with NVR peptide (10 µM) overnight, thereafter the cells were fixed. The fixed cells were stained with primary antibody targeting α-tubulin and then counterstained by appropriate secondary antibody. Microscopic images clearly revealed that healthier microtubules were observed in NVR treated cells (Figure 4I-L) when comparisons were made with the control cells (Figure S11, ESI). 2.17 Anti-NGF assay to assess the neuroprotective nature of NVR The neuroprotection ability of NVR was further evaluated by performing anti-NGF assay. PC12 cells were differentiated with nerve growth factor (NGF) for 5 days to obtain neurons. NGF deprivation in the differentiated neurons was performed by incubation with anti-NGF which ultimately generates large amounts of Aβ inside the cells. We have used this model to evaluate the degree of neurotoxicity and its reduction on NVR treatment in the cells.27,42 The NGF deprived PC12 neurons were treated with varying doses of NVR (1-10 µM) peptide. The neuroprotection capability of the NVR peptide is further confirmed by looking at the DIC images obtained from this experiment (Figure 5A-C). The NVR peptide starts conferring neuroprotection from 1 µM concentration, which further escalates with increasing concentrations of NVR (Figure 5D). 2.18 Stability assessment of NVR peptide in human serum Results of the aforesaid experiments influenced us for considering this NVR peptide for in vivo studies. So it becomes pertinent to check the stability of this peptide in serum. NVR was incubated at 37 ºC with serum obtained from human and the amount (in %) of remaining peptide was monitored upto 24 h by performing HPLC in every 2 h. The data recorded up to 24 h


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claimed that still 28 % of peptide is intact and hence our peptide shows significant stability in human serum. (Figure 5E).38 2.19 Blood-brain barrier (BBB) permeation ability of NVR Since, NVR has been designed for neuroprotection and therapeutic application in AD, we were intersted to know the BBB permeation ability of NVR. We performed the in vivo mice model experiment with NVR to check its BBB crossing ability. The presence molecular mass peak of NVR with Na+ ions with some cleaved peptides mass in the MALDI mass spectrum of NVR treated brain homogenate (Figure 6A) in comparison with control brain homogenate with no such mass peak clearly reveals that NVR can cross the BBB (Figure S12, ESI). This proves the BBB permeation ability of NVR peptide. 2.20 Morphology study and staining of primary cortical neurons treated with NVR Finally, we have checked whether NVR has any toxicity on primary cortical neurons. For this purpose, we cultured the cortical neurons following a previously reported methodology43,44 and institute‘s animal ethics guidelines. We witnessed that the cortical neurons exhibited good health similar like untreated control (Figure 6B, C). The cortical neurons were further immunostained with MAP2, a marker of mature neurons and again no abnormality in the morphology of the NVR treated neurons were observed (Figure 6D-F) confirming the non-toxic behaviour of NVR towards primary neurons. 3. CONCLUSION In summary, we introduced a state-of-the-art fusion approach for design and development of neurotherapeutics. Through this strategy, we designed a nonapeptide from taxol binding cavity of



β-tubulin and hydrophobic core (17-21) of Aβ to stabilize the intracellular microtubule as well as protect the neurons from degeneration during AD pathogenesis. The high binding affinity of NVR peptide with tubulin and its promotion of tubulin polymerization were studied through various experiments like tubulin polymerization assay, microtubule assembly assay, molecular docking, and quenching of tubulin’s intrinsic tryptophan fluorescence. Further, FRET analysis and molecular docking experiments confirm that this peptide binds near to the taxol binding cavity of tubulin. Also, this peptide inhibits amyloid aggregation by binding at 17-21 region (hydrophobic core) of Aβ. In vitro cell based studies confirm that NVR is not toxic to neurons, stimulates neurite branching in PC12 derived neurons, stabilizes the intracellular microtubules, and confers neuro-protection against the neuro-toxic effects of NGF deprivation. Moreover, NVR is stable in human serum, promotes health of the primary cortical neurons, and crosses the BBB efficiently. Finally, we envision that this new strategy and discovery of new potential antiAD peptide (NVR) that can be a prospective microtubule stabilizing therapeutic agent for further consideration in in vivo system. 4. EXPERIMENTAL SECTION 4.1 Chemicals Fmoc-Rink Amide AM resin, all the required Fmoc protected amino acids, and O-(1HBenzotriazol-1 yl) N, N, N′, N′-Tetramethyluronium hexa fluorophosphate (HBTU) were procured from Novabiochem (Merck). Ethanedithiol (EDT), Phenol, N, N′-Dimethylformamide (DMF), Dichloromethane (DCM), 30 % Hydrogen peroxide solution, Acetone, Dichloromethane (DCM), Trifluoroaceticacid (TFA), and Thioflavin-T (ThT) were bought from Merck. Piperidine, Dimethyl sulphoxide (DMSO), Diethylether (Et2O), 1-Hydroxybenzotriazole


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(HOBT), and Diisopropylethylamine (DIPEA) were obtained from Spectrochem. Methanol was bought from Finar whereas acetonitrile was procured from J. T. Baker. 5(6)-Carboxyfluorescein (fluorescein),

N,

N′-Diisopropylcarbodiimide

(DIC),

4′,6-Diamidine-2′-phenylindole

dihydrochloride (DAPI), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Kanamycin sulfate, and Dulbecco’s Modified Eagle’s Medium (DMEM) medium were procured from Sigma Aldrich. Colorimetric acetylcholinesterase assay kit (ab138871) was bought from Abcam. B27 supplemented Neurobasal media, glutaMAX and pen/strep were procured from Gibco, Life technologies. Human recombinant NGF was bought from Sigma Aldrich. All compounds were used without further purification. 4.2 Construction of neuroprotective peptides library based upon relative occurrences of contacts between different amino acids Molecular docking analysis of Taxol molecule in respective pocket of tubulin suggests some most interacting amino acids (Gly 370, Asp 26, Arg 278, Phe 272, Thr 276, His 229), which stabilize the binding of taxol. Also, some non-charged and hydrophobic amino acids (Ser 236, Val 23, Ala 233, Leu 275, Ser 277) help in this binding, whereas the hydrophobic region (17-21) of Aβ contains 5 hydrophobic amino acids (LVFFA). We have constructed a nonapeptide sequence “GAKEAHDFQ (GAK)“ considering the aforementioned amino acids of both the pockets and using the most preferred contacts of these amino acids. It was described earlier that certain amino acids like Trp (W), Tyr (Y), His (H), and Phe (F) preferred interaction among them except Trp (W) and Tyr (Y); acidic amino acids [Asp (D), Glu (E)] mostly interacts with basic amino acids [Lys (K), Arg (R)]; aliphatic amino acids like to interact with themselves; Also, Thr (T), Asn (N) His (H), Met (M) interact among themselves, whereas Gly (G) preferentially interacts with Asn (N) and Asp (D). Serine doesn’t have any preferences for



interaction. Hence, a short library of 9 peptides has prepared sequentially from GAK peptide using the aforementioned preferred contacts of different amino acids. 4.3 Identifying best neuroprotective peptide from designed library by molecular docking analysis with both tubulin (PDB ID: 1JFF) and Aβ (PDB ID:1IYT) Afterthat, molecular docking of these 9 peptides was performed with both tubulin and Aβ using Autodock Vina 1.1.2.36 A grid box volume of 20×20×16 was set around the taxol area of βtubulin (PDB ID-1JFF)34 and a grid volume of 40×26×54 was set on the receptor Aβ (PDB ID1IYT)35 to perform the molecular docking experiment of NVR peptide with both the protein. Again, screening of neuroprotective peptide from another library of shuffled peptide was performed in similar manner. All the docking images were observed and processed in PyMOL 4.4 Synthesis and characterization of all the unlabeled and labeled (fluorescein) peptides Synthesis of this newly designed NVR peptide, two shuffled peptides (LVQ, VRN), and labeling of NVR with fluorescein dye were performed with Rink amide AM resin using the well known SPPS method. All the peptides were prepared in a microwave based peptide synthesizer in our lab. 20% piperidine solution prepared in DMF was used to deprotect the Fmoc group during peptide synthesis. Resin cleavage of the newly synthesized peptides was acheived by using a well known cleavage solution (cocktail formulation: TFA 91%, Milli Q 3%, EDT 3%, Phenol 3%). Finally, reversed-phase HPLC (Shimadzu) system and MALDI-TOF mass spectrometry were used to purify and characterize the crude peptides. 4.5 Protein biochemistry


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We have followed a well documented protocol for the isolation and purification of tubulin from goat brain.45 This purified tubulin solution was stored with glycerol in liq. nitrogen cryo vials after adjusting the concentration at 200 µM. 4.6 Tubulin turbidity experiment Generally, turbidity experiment executed for monitoring peptide’s effect on the polymerization property of purified tubulin. Absorbance of tubulin increases when turbidity of the solution increases. Therefore, the quantity of microtubule formed by the polymerization of tubulin was recorded by adding different concentrations of NVR peptide with tubulin solution. 20 µM of tubulin with 10% dimethyl sulfoxide, GTP (4 mM) with 3.125 µM of NVR in one vial and with 6.25 µM of NVR in another vial were mixed properly with Brinkley Reassembly Buffer 80 (BRB80) in ice cold condition, taken into a quartz cuvettes having 10 mm path length and recorded the absorbance at 37 °C temperature for 40 min. We have recorded the measurement at 350 nm wavelength in Cary 60 UV-Vis Spectrophotometer. 4.7 Microtubule assembly assay45,47 The effect of NVR on the polymerization rate of tubulin was observed by performing microtubule assembly assay with DAPI molecule. Fluorescence emission of DAPI enhances when it gets bind with microtubule. Hence, the extent of microtubule formed due to tubulin polymerization is quantified by the addition of different NVR peptide concentration into tubulin and DAPI mixture. We prepared the mixtures of DAPI (10 µM) in BRB80 buffer containing tubulin (100 µM), GTP (10 mM), NVR (3.125 µM) in one tube and NVR (6.25 µM) in another tube. The excitation wavelength was 355 nm and the emission was recorded in the range of 400-



600 nm wavelengths at 37 °C for 1 h or till saturation come in an interval of 5 min using PTI QM 40 spectrofluorometer. 4.8 Calculation of binding constant of NVR with tubulin by measuring the quenched emission value of intrinsic tryptophan unit Intrinsic fluorescence of tryptophan residues of tubulin get quenched after it gets bind by small molecules or drugs. Hence, the interaction of peptide or small molecule towards tubulin can be calculated by measuring the emission intensities of intrinsic tryptophan residues. Then we mixed several different concentration of NVR with tubulin solution (10 µM) at 4 ºC and emission intensities were measured using Quanta Master Spectrofluorometer (QM-40). Hence, with this quenched fluorescence values of intrinsic tryptophan we have calculated the binding constant of NVR with the help of modified Stern-Volmer equation.48 The excitation and emission wavelength range was 295 nm 310-450 nm respectively. 4.9 Fluorescence Resonance Energy Transfer (FRET) study to find out the binding location of NVR in tubulin Identification of binding location of NVR peptide in tubulin was measured by performing FRET analysis. In this experiment, a complex of tubulin-colchicine has been used as a donor and fluorescein tagged NVR peptide was used as an acceptor molecule. Colchicine binds at a specific position in tubulin and possess remarkable spectral overlap with fluorescein compounds.38 Recently, this FRET pair was used to measure the distance of fluorescein tagged peptide from colchicine pocket of tubulin. The excitation wavelength for this experiment was 355 nm whereas the emission wavelength range was from 450-650 nm. We measured the fluorescein emission of


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only fluorescein-NVR peptide (A), only tubulin-colchicine complex (D), and complex of both (A-D) (1:1 molar mixture).

IA  ID  I A

The equation to calculate the efficiency of FRET is, FRET =

Where ID and IA are the intensity of acceptor and donor molecules Förster distance (R0) between fluorescein molecule and Tubulin-colchicine complex was documented earlier ~ 29.5 ±1 Å.38

We have measured the distance (RDA) between fluorescein NVR (acceptor) and Tub-col complex (donor) using the following equation. 1

R DA

1   FRET  6   Ro    FRET 

4.10 Preparation of Aβ (1-42) peptide’s stock The Aβ42 peptide’s stock was prepared by adding 1 mg of Aβ42 peptide in 1,1,1,3,3,3Hexafluoro-2-propanol (400 µL). We kept this stock solution at -20 C after preparing small aliquots (10 µL). For experiment purpose, we take out 4 µL of this stock, dried under N2 gas, mixed 1 µL of NH4OH (1%) and adjusted the volume to 30 µL with PBS. By doing this, we prepared an 80 µM of final Aβ42 peptide solution. Further, we diluted this solution as per our requirement with PBS buffer. 4.11 Inhibition of amyloid fibrillation by performing Thioflavin T (ThT) assay 27,39 ThT assay was generally used for monitroing the inhibition of Aβ peptide aggregation. The amount of inhibition of amyloid aggregation was quantified by measuring the fluorescence of



ThT. NVR peptide was mixed with Aβ (10 µM) in different ratio ranging from (0.1:1) upto (1:1) and kept in 37 oC. 2 μL of solution was taken out after every 12 h and added with ThT solution at room temparature and emmission intensity of the solution was measured in spectrofluorimeter (λex- 435 nm and λem-460-650 nm). 4.12 Monitoring acetylcholinesterase (AChE) induced Aβ aggregation inhibition by NVR peptide49 Usually, AChE enzyme enhances the amyloid aggregation process. Therefore, inhibition of amyloid aggregation by NVR peptide in extreme cellular condition (high conc. of AChE) was performed in prescence AChE, 10 M of Aβ, and various concentration of NVR peptide (5 and 10 M).

Control experiment was executed without using peptide. Amount of amyloid

aggregation was monitored for 24 h by measuring the emission intensities of Thioflavin T (ThT) solution. 4.13 Evaluation of inhibiton kinetics of acetylcholinesterase enzyme activity by NVR peptide 49 The effect of NVR peptide on acetylcholinesterase activity was evaluated by Ellman’s assay. First, DTNB (0.01 M) and ATC (0.075 M) were mixed and added with varying concentration of NVR peptide in the acetylcholinesterase enzyme solution. After 1.5 min, the absorption value of the final solution was measured at 412 nm wavelength. Control experiment was executed without using peptide. To regulate the non-enzymatic reaction we have used phosphate buffer saline (PBS) instead of enzyme solution. 0.5 to 10 µM of NVR peptide was mixed with 87.5 - 700 μM concentration of substrate for monitoring the inhibition kinetics AChE activity (substratevelosity curve) by NVR peptide. Lineweaver-Burk plot was prepared by measuring the Km and Vm value of the AChE enzyme inhibition experiment.


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4.14 NVR peptide’s docking with AChE We executed the molecular docking analysis of NVR peptide with AChE in Autodock vina version 1.1.2. to understand the binding location, binding affinity and interaction partner of NVR peptide with AChE enzyme. A grid volume of 32×42×46 was kept in the center of the receptor Aβ peptide (PDB ID-4EY6)41 for a blind docking experiment with NVR peptide. All the docking images were observed and processed in PyMOL. 4.15 Transmission Electron Microscopy (TEM) study to monitor Aβ fibrillation inhibition ability of NVR peptide TEM study was performed by incubating Aβ1-42 peptide solution alone (1 µM) and with NVR peptide (1 µM) at 37 ºC for 7 days to check its effect on amyloid aggregation. After 7 days of incubation, 10 µL of incubated solution was pipette out and placed on a copper grid of 300 mesh. Then, after 2 min, the grids were washed carefully with water for the removal of excess solution. Next, 2% uranylacetate in water solution was used to stain the grids after careful washing. The morphology was studied in an electron microscope operating at 80 kV. 4.16 MD Simulation study MD simulation study was performed to check the inhibitory effect of NVR peptide on Aβ fibrillations using GROMACS version 4.5.5. We have placed two “KLVFFAE” sequence in one cubic box and in another cubic box we placed one NVR peptide with two “KLVFFAE” sequence. For both cases, in all three directions the periodic boundary conditions were applied and GROMOS 96 53a6 force field was used for the simulation with peptides. For electrostatic and Lennard-Jones interactions, we have set the cut-off radii as 0.9 nm and 1.4 nm respectively. Particle-Mesh Ewald (PME) method was used to identify the electrostatics interactions of long-



range. A time step of 2 fs was used to perform this simulation. At first, we have started the simulation for 500 ps in constant volume (NVT) followed by equilibrate the system with 1 ns of constant pressure (NPT) using a well-known Parrinello-Rahman coupling method at 310 K temperature. Relaxation time of 0.1ps and 1ps were used for NVT and NPT respectively. Finally, the simulation run was initiated for 20 and 50 ns for control simulation of two KLVFFAE peptides and with NVR peptide respectively. 4.17 Culture of PC12 cells and its differentiation into neurons The culture of PC12 cells were conducted in Dulbecco’s Modified Eagle’s Medium (DMEM) augmented with fetal bovine serum (FBS) (5%) and horse serum (10%) at a temparature of 37 oC

and in 5 % CO2 environment. For neuronal differentiation of PC12 cells, the cells were

treated with 100 ng/mL of NGF and cultured for 5 days before they are completely converted to neurons. 4.18 Uptake analysis of NVR in differentiated PC12 neurons We checked the cellular entry of fluorescein tagged NVR in differentiated PC12 neurons. 10 µM of fluorescein tagged NVR peptide were incubated with the differentiated neurons for 4 h. Followed by washing the neurons with PBS and thereafter fixation was performed with 4%formaldehyde. Next, the neurons were stained with Hoechst 33258 for nuclear staining. Finally, the cellular uptake of fluorescein-NVR was observed through Olympus microscope (IX83) [equipped with Andor iXon3 897 EMCCD camera] after washing the cells with PBS. 4.19 Cellular viability assay with NVR peptide


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The cells were cultured in 96 well plates overnight and differentiated carefully into neurons. Then with varying doses of NVR (50, 25, 12.5, 6.25, 3.125, 1.5625 μg/mL), the differentiated PC12 neurons were treated for 24 h. After treatment, for all cases MTT solution (5 mg/mL) was prepared in PBS and added to the neurons for 4 h incubation. The formazan formed was solubilized in MeOH-DMSO (1:1) and the absorbance recorded at a wavelength of 550 nm. 4.20 Stabilization of intracellular microtubule by NVR peptide PC12 cells were cultured in glass bottom dishes with cell population of 3000-5000 and grown overnight. After removal of the medium, a solution of 10 µM of NVR peptide was mixed with it. After 16 h of treatment, glass bottom dishes were washed thoroughly with serum free media to remove the culture media. 4% paraformaldehyde solution was used to fix the cells, then blocking and permeabilization through BSA (5%) and triton-X-100 (0.2%) respectively. Then the neurons were incubated for 2 h with primary alpha-tubulin antibody with a dilution of 1:300 overnight and then with secondary antibody with a dilution of 1:600 and incubated for 2 h. Before imaging, Hoechst 33258 (1 μg/mL) solution was mixed and kept at 37 oC for 30 min. We have then captured images 405 and 561 nm channel at various areas of the glass bottom dishes to confirm the microtubule morphology of differentiated PC12 neurons. 4.21 Anti-NGF assay to assess the neuroprotective nature of NVR First the PC12 cells were differentiated with NGF (100 ng/mL) into neurons.The cells were deprived NGF through addition of anti-NGF at a concentration of 2 µg/mL. To this NGF starved cells, variable doses of NVR peptide (1-20 µM) was added for 20 h. Then the cell cytotoxicity is measured following MTT assay as described above.27,38 4.22 Stability of NVR peptide in physiological (serum) condition27,38



Human serum was used to check the stability of NVR peptide in serum condition. A mixture of 50 L of NVR (200 M), 800 L of human serum, and 150 L of milliQ water were taken in centrifuge tube and kept at 37 C for 24 h. After every 2 h of incubation, 100 L of incubated solution was mixed with 100 L of ACN. Then, centrifugation was performed and the above clear portion taken for HPLC. Then, this process was continued up to 24 h. After 24 h, we have plotted the change in intensity profile of NVR’s molecular peak with time to find out whether this peptide is stable under physiological conditions. 4.23 Blood-Brain Barrier crossing experiment Two groups were made with each group containing 3 healthy C57BL/6J female mice. BBB permeability was assessed by giving intraperitoneal injection of 100 µL NVR solution (10 mg/kg body weight of mice).50,51 and in the control group 100 µL saline solution was administered. After 6h of treatment, animals were euthanized by transcardial perfusion method by pumping the blood out of the circulation. The mice brains were isolated and removal of meninges was carefully performed under stereo microscope. Then the brain was crushed with liquid nitrogen using a mortar and pestle. To the crushed brain, water and Acetonitrile were added in equal proportions for dissolving the BBB crossed NVR. After centrifugation, from this mixture the soluble part was taken, detected through HPLC and mass spectrometry for both NVR treated mice as well as control mice. This BBB experiment protocol was previously well studied and documented in detail in our earlier work.49,52 4.24 Consequences after treating the cultured primary rat cortical neurons with NVR peptide The effect of NVR is also studied in cultured primary neurons.51 Briefly, from pregnant sprague dawley rats E18 embryo brain cortices were dissected out, enzymatic digestion performed and


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the isolated cells cultured in glucose rich MEM medium and later maintained in Neurobasal medium with B27 supplementation. These primary neurons are plated on poly-D-lysine coated glass bottom dishes (3-5 x 105/mL) and cultured at 37 0C temperature in 5% CO2. The cells are allowed to grow for a week and then we have treated the cortical neuron cells with 10 µM of NVR peptide. 4.25 Immunostaining of primary rat cortical neurons with MAP2 The obtained cortical neurons, were immunostained by anti-MAP2 following a well-documented protocol established earlier.43,44 In brief, at first the rat cortical neurons were fixed by 4 % formaldehyde and soon after permeabilized with 0.3 % Triton-X. After that, the fixed cells were incubated overnight after treating the cells with primary Mouse anti-MAP2. Next day, the cells were again incubated with a goat anti-Mouse secondary antibody at 37 °C for 2 h. The cells were stained for nucleus by Hoechst 33258 and observed the stained cells under a confocal microscope. 4.26 Result analysis and statistical significance calculation of various experiments Microscopic images were processed and analysed in Image J software and various spectral data were calculated by Origin pro 8.5. One way ANOVA and two tailed student’s t-test were executed to evaluate the statistical significance and their values varies between *p≤0.05 and **p≤0.001 for different studies. ASSOCIATED CONTENT Supporting Information This materials are available free of charge via the internet at http://pubs.acs.org.



Designing of neuroprotective peptide by taking into consideration the relative occurrences of contacts between amino acids, structure of NVR peptide, HPLC and mass spectrum of different peptides, fluorescein conjugation of NVR, tubulin polymerisation and ThT assay of shuffled peptide, MD simulation and TEM study of NVR peptide, and control brain mass analysis. AUTHOR INFORMATION: Corresponding Author Dr. Surajit Ghosh Principal Scientist Department of organic and medicinal chemistry division CSIR-IICB, Jadavpur, Kolkata-700 032, India Tel: +91-33-2499-5872 Fax: +91-33-2473-5197/0284 E-mail: [email protected] ORCID ID Prasenjit Mondal: 0000-0003-0767-449X Gaurav Das: 0000-0002-8432-5384 Surajit Ghosh: 0000-0002-8203-8613 Author contributions P.M. executed various computational analyses for peptide design, identification of potent peptide from the designed library of peptides by molecular docking, synthesized and purified all the peptides and accomplished several in vitro assays like microtubule polymerization assay, tubulin’s intrinsic tryptophan quenching assay, FRET analysis, ThT experiment, FT-IR and binding kinetics determination with AChE. G.D. has accomplished different in vitro cellular assays e.g. cell viability, uptake analysis in cell, neuroprotection analysis, and intracellular microtubule staining experiment. J.K. and G.D. have analyzed the effect of NVR on primary rat


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cortical neurons and blood-brain barrier. K.P. assisted P.M. to perform the inhibiton kinetics of AChE. R.M helped P.M. by synthesizing shuffled peptides. Initially, A.S. and B.J. have helped P.M in some in vitro experiments. Moreover, P.M. performed the MD simulation study with this peptide and assisted S.G. in writing this manuscript. S.G. conceived the project idea, supervised the work and wrote the manuscript. Notes There is no competing financial interest to declare ACKNOWLEDGMENT PM, AS and BJ wish to thank CSIR, GD thanks to ICMR, JK thanks to DST-Inspire, KP thanks to UGC, and RM thanks to NIPER for providing their fellowships. SG would like to acknowledge SERB, India (EMR/2015/002230) for financial support and CSIR-IICB for providing excellent infrastructure.

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Figure 1: Schematic representation for the designing of novel neuroprotective peptide (GAKEAHDFQ) considering the most interacting and hydrophobic amino acids around the taxol binding cavity of β-tubulin and hydrophobic core (17-21) of Aβ by means of relative occurrences of contacts between amino acids.



Figure 2: (A) Binding energies of all the nine designed peptides, where NVR shows best binding energy at both the sites (i.e taxol site of tubulin and hydrophobic region of Aβ). (B) Molecular docking energies (B.E) of various shuffled peptides of NVR peptide with tubulin and Aβ. (C) Molecular docking experiment suggests that NVR interacts close to the taxol site of tubulin. (D) H-bonding interaction of different amino acids with NVR at the taxol cavity of β-tubulin. (E) Tubulin turbidity assay using various concentrations of NVR peptide (F) Microtubule association


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study for the quantification of tubulin polymerization using various concentrations of NVR peptide. (G) Binding constant (Kb) calculation of NVR towards tubulin by recording the quenched emission intensities of intrinsic tryptophan unit of tubulin upon addition of higher concentrated solution NVR. (H) Exact binding location determination of NVR in tubulin by performing FRET analysis by means of tub-col complex as a donor unit and fluorescein conjugated NVR as an acceptor unit.

Figure 3: (A) Molecular docking experiment was performed with NVR and Aβ peptide (PDB1IYT). (B) Evaluating the amyloid aggregation inhibition ability of different concentrated NVR solution by recording the emission intensities of ThT. (C) Inhibition of preformed amyloid aggregates by different concentrations of NVR peptides monitored by ThT assay (D) Bar diagram shows the percent (%) inhibition of amyloid aggregation by NVR peptide. (E) FT-IR spectrum shows no β sheet character when Aβ peptide was incubated with NVR for 7 days at 37



°C. (F) Checking the amyloid aggregation inhibition capability of different concentrated NVR peptide at the time of AChE induced extreme amyloid aggregation condition. Error bar corresponds to standard deviation of the value. (G) Binding kinetics determination of NVR peptide towards AChE activity by plotting Lineweaver-Burk plot by means of different concentrated (10µM, 5µM, 2µM, 1µM, 0.5 μM) solution of NVR with different concentration of substrate (87.5-700µM). (H) Molecular docking analysis of NVR with AChE (PDB ID-4EY6) showing different binding partner of CAS and PAS site (B.E: -8.2 kcal mol-1).

Figure 4: (A-D) Microscopic images of DIC, 405 nm and 488 nm channels with their merged clearly suggests substantial intake of Fluorescein-NVR in differentiated PC12 neurons. Scale bars correspond to 20 µm. (E) Bar diagram reveals the non-toxic nature of NVR in differentiated PC12 neurons. Error bars correspond to standard deviation of the value from mean (*p

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