Crafting of Neuroprotective Octapeptide from Taxol ... - ACS Publications

Subscriber access provided by READING UNIV

Article

Crafting of Neuroprotective Octapeptide from Taxol-binding Pocket of #-Tubulin Prasenjit Mondal, Gaurav Das, Juhee Khan, Krishnangsu Pradhan, and Surajit Ghosh ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00457 • Publication Date (Web): 20 Nov 2017 Downloaded from http://pubs.acs.org on November 22, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Neuroscience is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

Crafting of Neuroprotective Octapeptide from Taxol-binding Pocket of β-Tubulin Prasenjit Mondal,1,2 Gaurav Das,1,2 Juhee Khan,1,2 Krishnangsu Pradhan,1 Surajit Ghosh 1,2*

1. Organic & Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, West Bengal, India. Fax: +91-33-24735197/0284; Tel: +91-33-2499-5872 2. Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical Biology Campus, 4 Raja S. C. Mullick Road, Kolkata 700032, India.

ABSTRACT: Microtubules play crucial role in maintaining the shape and function of neurons. During progression of Alzheimer’s disease (AD), severe destabilization of microtubule occurs, which leads to the permanent disruption of signal transduction process and memory loss. Thus, microtubule stabilization is one of the key requirements for the treatment of AD. Taxol, a microtubule stabilizing anti-cancer drug has been considered as potential anti-AD drug but never tested in AD patients, likely because of its’ toxic nature and poor brain exposure. However, other microtubule-targeting agents such as epothilone D (BMS-241027) and TPI-287 (abeotaxane) and NAP peptide (davunetide) have entered in AD clinical programme. Therefore, taxol binding pocket of tubulin could be a potential site for designing of mild and non-cytotoxic microtubule stabilizing molecules. Here, we adopted an innovative strategy for the development of peptide based microtubule stabilizer, considering the taxol binding pocket of β-tubulin and by using alanine scanning mutagenesis technique. This approach lead us to a potential octapeptide, which

ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

strongly binds with taxol pocket of β-tubulin, serves as an excellent microtubule stabilizer, increases the expression of acetylated tubulin, acts as an Aβ aggregation inhibitor and neuroprotective agent. Further, results revealed that this peptide is non-toxic against both PC12 derived neurons as well as primary cortical neurons. We believe that our strategy and discovery of peptide-based microtubule stabilizer will open the door for the development of potential antiAD therapeutics in near future. KEYWORDS: Alzheimer’s disease (AD), Amyloid beta (1-42) fibrillation, Neuroprotective peptide, Microtubule stabilization, Primary cortical neurons, Alanine scanning mutagenesis. 1. INTRODUCTION Microtubules are the key cytoskeleton filaments in neuron that play an important role in axonal growth, transportation, migration, signal transduction, polarity and maintaining the embellished shape for morphogenesis of brain.1-13 It is interesting to note that, assembly, orientation, stability and spacing of microtubules in neurons are maintained by Tau, a microtubule associated protein (MAP).14-17 During Alzheimer’s disease (AD) progression, Tau protein is hyperphosphorylated and detached from the microtubule lattice and forms insoluble aggregates known as neurofibrillary tangles, which causes axonal dysfunction and memory loss.18,19 Thus, microtubules destabilizes during AD that causes severe disconnection in signal processing resulting in permanent damage to the brain function. In addition to neurofibrillary tangles, aggregation of Amyloid beta (Aβ) is the major constituent of AD pathology formed by the proteolytic cleavage of transmembrane amyloid precursor protein (APP) during regular cell metabolism.20 It has been shown that Aβ and its oligomeric form also destabilizes microtubule


Page 2 of 42

Page 3 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


structure, which causes severe damage in the signal transduction process.21,22 Therefore, microtubule stabilization is the important prerequisite along with reduction of Tau hyperphosphorylation and inhibition of Aβ aggregation. In this direction, various attempts have been made to explore whether microtubule stabilizing molecules are effective as therapeutic agents for AD or not.23-36 Fascinatingly, paclitaxel a microtubule stabilizing anti-cancer drug shows promising result for further consideration in AD therapeutics.37-42 However, it was not tested in AD patient due to poor brain exposure and toxic nature.43 Although, some of the microtubule targeting agent such as epothilone D (BMS-241027)44, TPI-287 (abeotaxane) (http://www.alzforum.org/therapeutics/tpi-287), Dictyostatin45 and NAP peptide (davunetide)46 have able to entered into the clinical programs. Thus, development of non-toxic microtubule stabilizer is essential for the treatment of AD. Here, we applied an interesting strategy for the development of microtubule stabilizing neuroprotective molecule. We have chosen taxol binding region of β-tubulin, because when taxol binds with β-tubulin it enforces a structural change in βtubulin through interaction with the key amino acids of that pocket, causing a long range structural stability of microtubules.47 We considered the ̴4.5 Å cut-off distance around the taxol binding pocket of β-tubulin and applied alanine scanning mutagenesis technique (using ABSscan web server)48 for characterization of the entire ligand binding site, where each residue in a given cut off distance is mutated with the alanine and evaluated for its effect on the function. Using this strategy, we designed a peptide sequence “YDVWIKDY (Primary Sequence: PS1)” from the taxol binding region. Next, we constructed a small peptide library using the concept of most interacting amino acid partners.49 We first constructed an octapeptide “MRAFVRNF” (PS2) upon replacement of all the amino acids of PS1. Then, we made octapeptide “NEVFLDTQ” (PS3) from PS2 following the same strategy. Similarly, following this strategy



we constructed additional three octapeptides such as TEVFLEHQ (PS4), MKAFADGQ (PS5) and YEVFVEHY (PS6). These six octapeptides have been docked in the taxol binding pocket and their binding energy has been calculated. We found that PS3 shows maximum binding energy among all the octapeptides. Further, we made sequence shuffling approach to construct a library of ten octapeptides from PS3 to understand whether positional change of amino acids in PS3 can make any differences in the binding energy or not. Docking results reveal that PS3 remains the best binder in the taxol binding pocket. Next, we evaluated the potential role of PS3 as a microtubule stabilizer, Aβ fibrillation inhibitor and neuroprotective agent by using various in vitro assays. Remarkably, we found that PS3 strongly binds with tubulin, promotes microtubule polymerization, increases the expression of acetylated tubulin, inhibits Aβ aggregation, inhibits AChE induced Aβ aggregation by interacting at the PAS binding site of AChE, enters into the PC12 derived neurons, promotes neurite growth, provides microtubule stabilization and shows significant neuroprotection against NGF deprived neurons. Finally, we found that it is non-toxic to both PC12 derived neurons as well as primary cortical neurons. 2. RESULTS AND DISCUSSION 2.1 In-sillico alanine scanning mutagenesis for finding the most interacting amino acids at taxol binding pocket of β-tubulin Result of alanine scanning mutagenesis in the taxol binding cavity of β-tubulin with a cut-off distance of ~4.5 Å revealed presence of seventeen different important amino acid residues, which has a very high autodock score value (Figure S1, S2, ESI). The importance of a residue was calculated by comparing the intermolecular energies of a wild type variant with a mutant one through ∆∆G values. The full list of conserved amino acids (based on ∆∆G value) of taxol


Page 4 of 42

Page 5 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


binding pocket of β-tubulin are listed in Figure S3. We have constructed a peptide sequence having eight amino acids residue “YDVWIKDY” which is the counter amino acids partner of the most active amino acids49 (Figure S4) of taxol binding cavity having a high autodock score and ∆∆G value. We named this peptide as primary sequence (PS1). 2.2 Designing of neuroprotective peptides from relative frequencies of most interacting amino acids of β-tubulin We constructed an octapeptide sequence PS1 from alanine scanning mutagenesis. But, to develop the potential microtubule stabilizer peptide from this designed sequence in a rational way, further designing of peptides was essential. For this purpose, we designed and constructed a short peptide library of six octapeptides from PS1 using an approach described before49,50. In brief, here we used concept of most interacting amino acid contacts. Using sequential approach, we constructed PS2 from PS1 then PS3 from PS2 and following same method we constructed PS4, PS5 and PS6 peptides (Figure 1). 2.3 Molecular docking study to find out the best neuroprotective peptide Next, we have performed molecular docking of the six octapeptides in the taxol binding pocket of β-tubulin (PDB ID-1JFF)51 in Autodock vina 1.1.2.52 Among them, PS3 is showing the maximum binding with a minimum binding energy (B.E.: -7.2 KcalM-1), in taxol binding pocket of β-tubulin (Figure 1, Figure S5, ESI). We found that interaction partners in the taxol binding pocket are Ser 277, Thr 276, Pro 274, Gly 370, Pro 360, His 229, Glu 22 (Figure 2a,b). 2.4 Screening of best peptide binders as taxol binding pocket of β-tubulin



Although PS3 among the six designed octapeptides strongly interacts with taxol pocket of βtubulin but, it is difficult to conclude that this octapeptide is the best binder. Thus, we constructed another library of ten octapeptides through positional alteration of amino acids of PS3. Then we performed molecular docking of these designed octapeptides at taxol pocket of βtubulin. To our surprise, we found that among all these octapeptides, PS3 peptide is showing the best binding energy (Figure S6, ESI). This result, confirmed that PS3 is the best binder in the taxol pocket and in this manuscript we studied the effect of PS3 peptide as a microtubule stabilizer as well as neuroprotecting agent. The molecular structure and sequence of this peptide is shown in Figure S7, ESI. 2.5 Synthesis, purification and characterization of PS3, SCR1, SCR2 and NP peptides as well as fluorescein conjugation with the PS3 peptide Synthesis of this PS3 peptide, its fluorescein conjugation, two scrambled peptides (SCR1 and SCR2) and one tubulin non-interacting peptide NP (IIGLMVGGVVI) were performed in solid phase peptide synthesis (SPPS) method using rink amide resin in CEM Liberty 1 peptide synthesizer. These peptides were purified by reversse phase HPLC equipped with C18 column and characterized by MALDI-ToF mass spectrometry (Figure S8-S12, ESI). 2.6 Effect of PS3 and NP peptide on tubulin polymerization monitored by microtubule assembly assay and tubulin turbidity assay. Since our designed peptide PS3 shows strong interaction at taxol pocket of β-tubulin from computational study and it has been described before that taxol promotes microtubule polymerization and stabilization,53, 54 we were curious to know whether PS3 can exert similar effects or not? For this purpose, we performed an assay, which is microtubule polymerization


Page 6 of 42

Page 7 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


assay using DAPI as fluorescence marker. We found that upon addition of PS3, polymerization of microtubules enhances, which clearly indicates that PS3 promotes microtubule polymerization (Figure 2c). For control experiment, we have checked the effect of synthesized NP peptide on tubulin by turbidity assay and found that this peptide doesn’t have any effect on tubulin polymerization (Figure S13, ESI). 2.7 Affinity of PS3 peptide with tubulin Next, we were eager to know the binding strength of PS3 with tubulin. For this purpose, we performed the tryptophan quenching experiment, which is routinely used for monitoring the interaction of small molecules with tubulin.55 Data of fluorescence quenching was plotted using modified Stern-Volmer equation, which shows the affinity of PS3 is Kb ~ 2.65 X 104 M-1 with tubulin indicating the significant binding of PS3 with tubulin (Figure 2d, Figure S14, ESI). Also, the binding affinity of two scrambled peptides (SCR1 and SCR2) with tubulin was determined to check the reliance of peptide selection on molecular modeling. The binding energies of SCR1 and SCR2 were 1.1 X 104 M-1 and 2.2 X 104 M-1 respectively (Figure S15, S16, ESI), which were almost 2.4 times and 1.2 times lower than PS3 peptide’s binding energy. 2.8 Surface Plasmon Resonance (SPR) experiment for the binding analysis of PS3 with tubulin Surface plasmon resonance (SPR) experiment was performed to find the binding analysis of PS3 peptide with tubulin. After analyzing the kinetics of binding of PS3 peptide by plotting the curve with a local fitting, we found the association constant (ka) is ~1.52 X 104 M-1s-1 and dissociation constant is (kd)~0.0857±0.008 s-1(Figure S17, ESI). The observed association constant is quite low compared to taxol (3.6 X 106 M-1 at 37 °C and 7.6 X 108 M-1 at 4 °C) which is almost 236 times lower in 37 °C and 50000 times lower in 4 °C.56 Also, the dissociation constant is 4 times



higher than taxol molecule’s first step dissociation constant (0.022 ±0.001 s-1) whereas it is 20 times slower compared to the second step dissociation constant of taxol (1.63 ±0.18 s-1)57. Thus, we can presume that the lower binding of PS3 and relatively fast dissociation compare to taxol has an effect towards microtubule stabilization without showing any toxicity. 2.9 Experimental confirmation of PS3 binding at taxol binding pocket of β-tubulin using Förster Resonance Energy Transfer (FRET) experiment Next, we performed Förster Resonance Energy Transfer (FRET) experiment to understand whether PS3 binds at taxol binding pocket or not. It has been described before that tubulin bound colchicine complex (donor) and fluorescein conjugated peptide (acceptor) was used to determine the binding location of peptide in tubulin58 due to their possession of significant spectral overlap region. Therefore, we have performed FRET experiment using tubulin bound colchicine complex as donor and fluorescein conjugated PS3 peptide as acceptor.

γ

+

IA

Efficiency of FRET, εFRET =

IA ID

Where IA and ID are the intensity of donor and acceptor molecules Here, in this case as obtained from Figure. 2d, εFRET = 0.48 Also, the reported Forster distance (R0) between Tub-col complex and fluorescence was ~ 29.5 ±1 Å. 58 Now, the distance (RDA) between Tub-col complex (donor) and fluorescein PS3 (acceptor) was calculated using the following equation.

FRET FRET

DA

 −ε = Ro   ε

  

1111 6666

1111

RRRR

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 8 of 42

Page 9 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The calculated FRET distance between fluorescein-tagged PS3 peptide and tubulin bound colchicine is ~33.4 ±1 Å , which represents taxol site in β-tubulin, indicating PS3 peptide binds close to the taxol binding pocket of β-tubulin (Figure. 2e). This result shows that PS3 indeed binds near to the taxol pocket. 2.10 Inhibiton of amyloid beta aggregation monitored by ThT assay Next, we have explored whether PS3 has ability to inhibit the aggregation of Aβ or not. For this purpose we have used ThT assay. It has been described before that ThT assay is routinely used for screening of Aβ aggregation inhibitor.59 PS3 peptide was incubated at various concentration (1-10µM) with Aβ peptide (10 µM) at 37 ºC. The inhibition of Aβ aggregation was monitored by mixing ThT with Aβ and PS3 solution followed by recording of ThT fluorescence (Ex- 435 nm, Em- 485nm). From Figure 3a, we found that with increasing concentration of PS3 peptide Aβ aggregation was decreased. Interestingly, we observed that Aβ aggregation was reduced upto ~30% when 1:1 molar ratio of PS3 and Aβ peptide was used. For control study, we have performed this experiment with SCR1 and SCR2 peptide and found that there are almost 13% inhibition of Aβ aggregation (Figure S18, ESI) for both the cases, which is quite low compare to PS3 activity. Along with this, we have performed another experiment where we have measured the ThT fluorescence of pre-formed Aβ with or without PS3 peptide to check that this inhibition is due to our compound not forThT (Figure S19, ESI). 2.11 Understanding interaction of PS3 with Aβ peptide using molecular docking Now, the question is how PS3 inhibits the Aβ peptide aggregation? It has been observed that inhibitor of Aβ generally interacts with 17-21 hydrophobic stretch of Aβ.33 Considering this fact



we performed molecular docking of PS3 with Aβ at 17-21 hydrophobic region of Aβ. Interestingly, we found that PS3 peptide binds at 17-21 region of Aβ peptide (PDB ID-1IYT)60 through interacting with Val 18 and Glu 22 with significant binding energy (B.E: -4.8 KcalM-1), which indicates that this peptide may have potential as amyloid inhibitor (Figure 3b, Figure S20, ESI). 2.12 Inhibition of Acetylcholinesterase (AChE) induced Amyloid-β aggregation by PS3 Molecular docking result of PS3 with Aβ peptide motivated us to investigate further whether PS3 can be a potential amyloid aggregation inhibitor or not. Here, we investigated role of PS3 in Acetylcholinesterase (AChE) induced Aβ aggregation. It has been documented before that Acetylcholinesterase (AChE) concentration increases during amyloid aggregation in extreme cellular condition in brain.61 Thus, we have incubated PS3 peptide with Aβ in AChE enzyme and agitated this mixture for 24 h. From figure 3c, we have observed that amyloid aggregation was decreased significantly with increasing concentration of PS3 peptide (5 µM, 10 µM) after 24 h of incubation whereas in control study amyloid aggregation increased rapidly. This result clearly indicates that PS3 has significant inhibitory effect against Acetylcholinesterase (AChE) induced Aβ aggregation. 2.13 Understanding the inhibition of Acetylcholinesterase activity by PS3 peptide Subsequently, we have investigated the effect of PS3 in the acetylcholinestrase activity as our PS3 emerges as potential amyloid aggregation inhibitor in prescence of high amount of AChE. This phenonmenon can co-relate with the fact that this peptide might inhibit AChE activity. Therefore, we are interested to investigate the mode and mechanistic aspects of inhibition of AChE activity by PS3 peptide. The substrate-velocity curve was calculated with varrying


Page 10 of 42

Page 11 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


concentration of PS3 (0.25 µM - 2 µM) with different substrate (acetylthiocholine) concentration (87.5 - 700 µM) to analyze the mechanism of inhibition. From the Lineweaver-Burk plot (Figure 3d) we have found that the Vmax value (reciprocal of Y-intercept) was decresing whereas Km value (negetive reciprocal of X-intercept) was increasing with increasing concentration of PS3. Therefore, we can conclude that this peptide was binding to the AChE enzyme in a non competitive manner. 2.14 Molecular docking study of PS3 peptide in the CAS and PAS binding site of AChE Experimental evidence shows that PS3 interacts with AChE. Now the question is, in which site does PS3 binds at AChE?. As we know that AChE has two binding site Catalytic anionic site (CAS) and Peripherral anionic site (PAS). For this purpose, we have performed molecular docking study of PS3 peptide in the CAS and PAS sites of AChE enzyme. The binding energy (8.2 KcalM-1) of this PS3 is quite high at PAS site, which indicates high affinity of binding of PS3 peptide in PAS site of AChE enzyme. The binding was stabilised by the H-bonding interaction of amino acids Y130, E199, S122, Y70, Y121, W279, S286, G335 of PAS binding site of AChE enzyme (Figure 3e,f). Thus, we can conclude that PS3 peptide is mainly interacting at the PAS binding site of the enzyme and inhibits its activity. 2.15 TEM study TEM study reveals inhibition of amyloid fibrillization when co-incubated with PS3 peptide. From Figure S21a, we have seen that Aβ forms fibrillar network when incubated alone at 37 °C for 7 days. Aβ co-incubated with PS3 peptide at 37 °C for 7 days shows insignificant aggregation morphology (Figure S21b, ESI). 2.16 Cellular uptake of PS3 peptide



So far, various in vitro results and computational study reveals that PS3 is an efficient microtubule stabilizer and potential Aβ aggregation inhibitor. Now, we are interested to evaluate the effect of this peptide using in vitro cell based experiments. First, we were interested to investigate the effect of this peptide in the neurons. We checked the cellular uptake of PS3 peptide using PC12 (Rat pheochromocytoma cells) derived neurons. Differentiated PC12 cells were treated with 10 µM of fluorescein conjugated PS3 peptide for 4 h. Then PC12 derived neurons were fixed and observed under microscope. Their cell bodies and dendrites were found to stain green when observed under 488 channel, revealing sufficient cellular uptake of PS3 peptide. The merged images of the 488 channel along with the DIC and DAPI channel, showed the healthy morphology of neurons stained with fluorescein conjugated PS3 in the cell bodies and dendrites surrounding the nucleus (Figure 4a-d). 2.17 Cell viability of PS3 peptide Next, we checked the toxicity of PS3 in differentiated PC12 cells at various concentrations. Figure 4e shows that the peptide doesn’t have any toxic affect towards PC12 derived neurons upto 100 µM i.e. the peptide is non-toxic to PC12 cells upto 100 µM. Further, DIC images of 10 µM PS3 treated PC12 derived neurons show healthy morphology and prominent neurite outgrowth (Figure 4f-g). This significant neurite growth indicates the neuroprotective behaviour of PS3. 2.18 Microtubule stabilization study During AD progression both Tau and Aβ aggregates disrupts the intracellular microtubule network.62 In vitro result shows that PS3 is a good microtubule stabilizer. Therefore, we assessed the microtubule stabilization of PS3 peptide using in vitro cell based assay. For this purpose, we


Page 12 of 42

Page 13 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


treated the PC12 cells with 10 µM of PS3 and immunostained it with tubulin specific primary antibody followed by fluorescent tagged secondary antibody and imaged it under fluorescence microscope. Figure 4h-k showed that microtubules were healthier in presence of PS3 peptide as compared to the control cells (Figure S22, ESI). 2.19 Immunoblotting Experiment 63 For the quantification of the above observation i.e. the stabilization of microtubules we performed western blot experiment with acetylated tubulin as a marker. We observed that there was a significant increase in the expression of acetylated tubulin in the sample treated with PS3 as compared to control but in case of the SCR1 or NP (a microtubule non-interacting peptide), the expression of acetylated tubulin was considerably lower (Figure 5a,b). Thus, our observations clearly indicate that our peptide has microtubule stabilizing capability and lends credibility to our earlier performed immunofluorescence studies. 2.20 Evaluation of neuroprotective effect using Anti-NGF assay Next, we have investigated the neuro-protection ability of PS3 peptide against anti-NGF toxicity. We treated PC12 cells with NGF for 5 days for differentiation of the cells and then we treated with anti-NGF which led to over production of Aβ1-42. This NGF deprivation model is now frequently used to understand the kinetics of neuronal death due to NGF deprivation.33 Thus, we used this model for neuro-protection assay. We have performed the anti-NGF assay with various concentrations (6.25-50 µM) of PS3 peptide. Result suggests that PS3 peptide is able to show protection to the NGF deprived cells starting from 6.25 µM concentration, which further improves with higher concentrations (Figure 6a). Microscopic DIC images of this experiment were shown in Figure S23, ESI.



2.21 Serum stability of PS3 peptide upto 24 h Above results motivated us to consider the PS3 in in vivo system. Thus, we checked whether this peptide is stable in human serum or not. Figure 6b reveals that 28% of PS3 remains stable after 24 h of incubation in human serum at 37 ºC

60

. This result indicates that PS3 has good serum

stability and suitable for in vivo validation. 2.22 Effect of PS3 peptide in the primary cortical neuronal Next, we have evaluated the effect of PS3 peptide in the primary cortical neuron.64,65 This experiment has been performed following institute animal ethics guidelines. It was observed that the PS3 treated primary cortical neurons maintained healthy morphology just like the control primary neurons (Figure 6c, d). This result clearly indicates that PS3 is non-toxic against primary neurons which encourage us for further exploration of PS3 in in vivo mice model.

3. CONCLUSION In summary, we have designed and developed an octapeptide through an innovative strategy for stabilizing microtubule during AD progression. This peptide has been evaluated using various in vitro assays such as microtubule assembly assay, intrinsic tryptophan fluorescence quenching assay and molecular docking, which clearly demonstrates that PS3 binds to tubulin with a significant binding affinity and enhances the polymerization rate of tubulin. FRET and molecular docking experiments clearly reveals that PS3 binds at taxol binding pocket of tubulin. We also observed that PS3 inhibits Aβ aggregation through binding at 17-21 region of Aβ and inhibits AChE induced Aβ aggregation through interacting at PAS binding site. In vitro cell based assays


Page 14 of 42

Page 15 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


reveal that PS3 is non-cytotoxic, stabilizes the intracellular microtubules, increases the expression of acetylated tubulin, promotes neurite out-growth in PC12 derived neurons and provides significant neuroprotection against NGF deprived neurons. Further, PS3 shows good serum stability and maintains healthy morphology of the primary cortical neurons. Although there are some limitations of using peptide based therapeutic as they have less central nervous system (CNS) bioavailability and poor metabolic stability. However, recently NAP peptide entered in advance stage clinical trial as microtubule stabilizer for AD. Therefore, PS3 peptide could be a potential candidate for further exploration in in vivo system for the development of microtubule stabilizing anti-AD therapeutics. 4. EXPERIMENTAL SECTION 4.1 Chemicals All the Fmoc protected amino acids, Fmoc-Rink Amide AM resin and O-(1H-Benzotriazol-1 yl) N, N, N′, N′-Tetramethyluronium hexa fluorophosphate (HBTU) were purchased from Novabiochem (Merck). Phenol, Ethanedithiol (EDT), Dichloromethane (DCM), Thioflavin-T (ThT), Trifluoroaceticacid (TFA), Hydrogen peroxide (30% solution), Dichloromethane (DCM), N, N′-Dimethylformamide (DMF) and Acetone were purchased from Merck. Diethylether (Et2O), Dimethyl sulphoxide (DMSO), Diisopropylethylamine (DIPEA), Piperidine and 1Hydroxybenzotriazole (HOBT) were purchased from Spectrochem. Acetonitrile was purchased from J. T. Baker. Methanol was purchased from Finar. N, N′-Diisopropylcarbodiimide (DIC), 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 5(6)-Carboxy Fluorescein (fluorescein), 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI), Kanamycin sulfate, Dulbecco’s Modified Eagle’s Medium (DMEM) medium, were purchased from Sigma Aldrich.



Acetylcholinesterase assay kit (Colorimetric) (ab138871) was bought from Abcam. Human recombinant NGF was purchased from Sigma (St. Louis,MO, USA). Neurobasal media supplemented with B27, glutaMAX and pen/strep were bought from Gibco, Life technologies. All compounds were used without further purification. 4.2 In-Sillico alanine scanning mutagenesis for finding the most interacting amino acids at taxol binding pocket of β-tubulin The alanine scanning mutagenesis (ASM) was performed considering taxol bound tubulin molecule (PDB ID-1JFF)51 in ABS-Scan web server48 This web server was used regularly for finding most interacting partners of a receptor with ligand in a ligand binding pocket of receptor. We have uploaded taxol-tubulin complex (1JFF.pdb) to this server and defined a standard cut off distance (4.5 Å) around taxol pocket of β-tubulin. Then we have selected the ligand as taxol (TA1:B:601) and performed alanine scanning. Site-specific mutagenesis was performed using Modeller package on all selected residues that coupled with energy minimization steps. In addition, we have performed the conserved analysis around taxol pocket of β-tubulin to find the most conserved sequence of that pocket using this webserver. In this study, the protein sequence is compared against NCBI NR database where this protein is aligned following clustalO alignment and conservation is calculated from this alignment using Al2co program. 4.3 Designing of neuroprotective peptides from relative frequencies of most interacting amino acids of β-tubulin. After finding the most interacting amino acids at taxol binding pocket by ASM we have prepared a linear peptide sequence. Now, we conceived interesting concept described by Faure et al. where they showed that some amino acids preferentially interact with certain counter amino


Page 16 of 42

Page 17 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


acids like Phe (F), Tyr (Y), Trp (W), His (H) interacts with themselves except Tyr (Y) and Trp (W); aliphatic amino acid interacts among themselves; Glu (E), Asp (D) interacts with Arg (R), Lys (K); Met (M), Thr (T), His (H), Asn (N) interacts with themselves and Gly (G) preferentially interacts with Asp (D) and Asn (N). Hence, we have prepared a short library of six peptides using these relative frequencies of this amino acid contacts.49 4.4 Most potent peptide screening by molecular docking study Screening of these six designed peptides were performed using molecular docking study in autodock vina version 1.1.2 software52 Since, these peptides were originated from taxol binding pocket of β-tubulin we made a grid box having volume 20x20x16 around the taxol binding cavity of tubulin considering its Protein Data Bank (PDB) structure (PDB ID-1JFF).51 Next, we constructed another library of 10 peptides by shuffling the amino acids sequence of this most promising peptide sequence to understand whether positional change of amino acids can make any differences in the binding energy or not. Hence, we docked those peptides in the taxol binding pocket using the above described parameters to find the best binder among them. 4.5 Synthesis, purification, characterization and fluorescein conjugation with PS3 peptide Sythesis of the peptide NEVFLDTQ (PS3) and its conjugation with fluorescein, two scrambled peptides (SCR1 and SCR2) and one tubulin non-interacting peptide NP (IIGLMVGGVVI) were performed following solid phase peptide synthesis (SPPS) method using microwave peptide synthesizer (Liberty 1 from CEM). This peptide was synthesized using RINK Amide AM resin. Fmoc-deprotection was performed using 20% piperidine in DMF. Standard cleavage cocktail (TFA 91%, EDT 3%, Milli Q 3%, Phenol 3%) solution was used to cleave the peptide from the



resin. Finally, the crude peptide was purified by reverse phase HPLC (Shimadzu) and characterized by MALDI-TOF Mass Spectroscopy. 4.6 Protein biochemistry Isolation and purification of tubulin from goat brain was performed in our laboratory following previously described method.66 The concentration of this purified tubulin was maintained to 200 µM and stored with glycerol in liquid N2 cryo chamber. 4.7 Microtubule assembly assay and tubulin turbidity assay53,54 Microtubule assembly assay was performed for monitoring the effect of our peptide on the polymerization rate of tubulin in presence of DAPI. We have mixed 100 µM tubulin, 10 mM GTP, 10 µM DAPI and various concentration of PS3 peptide in Brinkley Reassembly Buffer 80 (BRB80) (pH 6.9 and contains 80 mM PIPES, 1 mM MgCl2, 1 mM EGTA). The fluorescence was monitored for 50 min at 37 ºC with an excitation wavelength of 355 nm and emission is ranging from 460-650 nm in a PTI Quanta Master Spectrofluorometer (QM-40). Fluorescence intensity of DAPI increases as it binds with microtubules due to its restricted degree of freedom. Therefore, the amount of microtubule formation was quantified by addition of various concentration of PS3 peptide into the tubulin and DAPI mixture. The binding of NP peptide with tubulin was monitored by tubulin turbidity assay following previous work54 using 10 mM GTP and 10 µL of DMSO to initiate the polymerization. The data was analyzed in origin Pro 8.5 software. 4.8 Binding affinity (Kb) of peptide with tubulin was monitored by quenching of intrinsic tryptophan fluorescence of tubulin


Page 18 of 42

Page 19 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Intrinsic tryptophan residues fluorescence is quenched when a small molecule or drug binds to the tubulin. Therefore, the binding of small molecule or peptide towards tubulin is monitored by recording the fluorescence value of intrinsic tryptophan residue. Various concentration of PS3, SCR1 and SCR2 peptide were mixed with 10 µM of tubulin in BRB80 buffer at 4 ºC and fluorescence was recorded using Quanta Master Spectrofluorometer (QM-40) equipped with Peltier to control the temperature. So, from the quenched value of intrinsic tryptophan, the binding constant of PS3, SCR1 and SCR2 peptide towards tubulin was calculated using a modified Stern-Volmer equation.55 The excitation wavelength was 295 nm and the emission wavelength was ranging from 310-450 nm. 4.9 Surface plasmon resonance (SPR) experiment for the binding analysis of PS3 with tubulin Surface plasmon resonance (SPR) experiment was performed to understand the binding analysis of PS3 peptide with tubulin. We have used NTA biosensor chip surface to perform this analysis. First, we immobilized Ni+2 on the chip by flowing 500 µM NiCl2 solution over the surface. Then we flowed Streptavidin-His6 solution and immobilized it onto the surface. After washing the surface with BRB80 buffer, we flowed 20 µg/mL biotin-tubulin and incubated for 15 min. Next, the surface was washed with BRB80 buffer with a flow rate of 100 µL/min. Then we prepared a series of PS3 peptide solutions (10 µM to 1 mM) as analyte and flowed over the tubulin immobilized NTA surface with a flow rate of 30 µL/min. Then PS3 binding kinetics was recorded and analyzed by plotting the curve with a local fitting. 4.10 Identification of binding pocket of PS3 into the tubulin using Fluorescence Resonance Energy Transfer (FRET)



FRET was performed to identify the binding region of PS3 in tubulin. Recently, tubulincolchicine complex was used to find out the distance of fluorescein attached small molecule/peptide binds to tubulin. Colchicine has a specific binding site in tubulin and posseses significant overlap region with fluorescein compound.58 The excitation wavelength was 355 nm and the emission ranging from 450-650 nm. We have recorded the emission of only tubulincolchicine complex, only fluorescein-conjugated peptide and complex (mixed in 1:1 molar ratio). 4.11 Amyloid-Beta (1-42) peptide’s stock solution preparation Amyloid Beta (1-42) (Aβ42) peptide solution was prepared by mixing 1 mg of Aβ42 peptide in 400 µL of 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP). This solution was then aliquots in 10 µL volume tube and stored at -20 °C. Then at the time of experiment 4 µL of this stock solution was used, dried under nitrogen gas, 1 µL of 1% NH4OH added into it and the volume was then adjusted to 30 µL by using phosphate buffer saline (PBS buffer). The final concentration of the Aβ42 peptide solution was 80 µM. Then we further diluted this solution with PBS to adjust the concentration as per our experiment. 4.12 Thioflavin T (ThT) asay for monitoring inhibition of Amyloid β (Aβ) peptide aggregation 59 Inhibition of Aβ peptide aggregation was monitored using ThT fluorescence intensity. PS3, peptide and Aβ42 peptide were mixed with in different ratio ranging from (0.1:1) upto (1:1). For control study, SCR1 and SCR2 was mixed with 10 µM of Aβ in 1:1 ratio for 48 h. ThT was added into the 48 h incubated solution at room temparature and fluorescence was recorded in PTI QM-40 spectrofluorimeter at 435 nm as excitation wavelength and at 460-650 nm as emission wavelength range. 4.13 Molecular docking of PS3 peptide with Aβ peptide


Page 20 of 42

Page 21 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Molecular docking of PS3 peptide with Aβ peptide was performed to understand whether this peptide binds with Aβ peptide or not using Autodock vina version 1.1.2. We have performed a blind docking experiment with grid volume 40x26x54 and centered on the receptor Aβ peptide (PDB ID-1IYT).60 4.14 Inhibition of Acetylcholinesterase induced Aβ peptide aggregation by PS3 peptide61 Inhibition of Aβ peptide aggregation by PS3 peptide was monitored in prescence of AChE enzyme as it accelerates the aggregation process. Various dose of PS3 peptide (5 and 10 µM) was mixed with 10 µM of Aβ for monitoring the role of this peptide in neuroprotection in extreme Aβ aggregation condition. Control study was performed in absence of peptide. Fluorescence of Thioflavin T (ThT) was recorded (λex- 435 nm and λem- 460-650 nm) for monitoring the aggregation of Aβ peptide using PTI QM-40 spectrofluorimeter for 24 h. 4.15 Inhibition of Acetylcholinesterase enzyme activity using various concentration of PS3 peptide61 Ellman’s assay was performed using various concentration of the PS3 peptide to understand whether this peptide can inhibit the AChE enzyme activity or not. Mixture of 0.01 M DTNB and 0.075 M ATC were added into the incubated solution of the acetylcholinesterase enzyme with various concentration of PS3 peptide. Absorption of the mixture was recorded at 412 nm sharply after 1.5 min. Control experiment was performed in absence of peptide. Phosphate buffer saline (PBS) was used in place of enzyme solution for regulation of the non-enzymatic reaction. We have mixed various concentration of PS3 peptide (0.5 to 10 µM) with various concentration of substrate (87.5 to 700 µM) to monitor the kinetics (substrate-velosity curve) of AChE inhibition. Vm and Km value of the inhibition were measured from Lineweaver-Burk plot of this experiment.



4.16 Transmission Electron Microscopy (TEM) study of PS3 peptide with Aβ Aβ peptide alone (having concentration of 1 µM) and Aβ42 peptide with PS3 peptide (final concentration of 1 µM) were incubated at 37 ºC for 7 days. Then, 10 µL of each incubated solution was placed on 300 mesh copper grids from ProSciTech. After 2 min, excess solution was removed and grids were washed properly with water followed by staining with 2% uranylacetate in water. These TEM grids were seen in a TECNAI G2 SPIRITBIOTWIN CZECH REPUBLIC 120 kV electron microscope operating at 80 kV. 4.17 Cell culture We got a kind gift of rat pheochromocytoma (PC12) cell line from Dr. Suvendra Nath Bhattacharyya, Principal Scientist, CSIR-IICB. PC12 cells are cultured in DMEM media that contains 5% fetal bovine serum (FBS) and 10% horse serum at 37 oC temparature and 5 % CO2 atmosphere in our lab. 4.18 Understanding the cellular entry of PS3 peptide into the PC12 cells using fluorescein conjugated PS3 peptide Cellular uptake of PS3 peptide was performed in PC12 cells. Cells were plated in confocal dish and adhered cells were differentiated using 100 ng/mL of NGF for 5 days. Next, PC12 derived neurons were treated with 10 µM of fluorescein conjugated PS3 for 4 h in serum free media. PS3 treated cells were washed with PBS and fixed with 4% formaldehyde for 1 h. Subsequently, the cells were washed with PBS and stained with Hoechst 33258 for 30 min. Finally, the cells were washed with PBS and observed under Olympus (IX83) microscope equipped with Andor iXon3 897 EMCCD camera.


Page 22 of 42

Page 23 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4.19 Cell viability of PS3 was performed by MTT assay using differentiated PC12 cells Cells were cultured in 96 well plates for 24 h and treated with serum free DMEM supplemented with 1% horse serum and 100 ng/mL NGF and incubated for 5 days for differentiation. Differentiated cells were treated with various concentration of PS3 peptide (100, 50, 25, 12.5, 6.25 µg/mL) and incubated for 24 h at 37 ˚C under 5 % CO2 environment. Then cells were treated with MTT solution (5 mg/mL) prepared in cell culture medium. The culture media was then replaced with DMSO-MeOH (1:1) solution to solubilize the yellow formazan before recording the absorbance. The absorbance was recorded in a Microplate ELISA reader (MultiscanTM GO Microplate Spectrophotometer) at 550 nm wavelength. Further, differentiated PC12 cells after treatment of peptide was imaged using Olympus (IX83) microscope equipped with Andor iXon3 897 EMCCD camera in DIC mode. 4.20 Effect of PS3 peptide on neuronal microtubule monitored by fluorescence microscope PC12 cells were cultured in a confocal dish with cell density of 3000-5000 and harvested overnight. The culture media was replaced with 25 µM of PS3 peptide containing treatment solution. After 16 h, serum free media was used to wash the confocal dishes and for complete removal of culture media. Cells were fixed using 4% paraformaldehyde and incubated with 5% BSA and 0.2% triton-X in PBS for 1 h. Cells were then incubated with polyclonal anti-α-tubulin IgG antibody with dilution 1:300 after a single wash with 1X PBS for 2 h. Secondary antibody (Cy3.5 pre-absorbed goat anti-rabbit IgG) was mixed with this solution having dilution 1:600 after washing the cells with PBS and kept for 2 h. Prior to imaging, cells were washed with 1X PBS and incubated with Hoechst 33258 (1 µg/mL) for 30 min. Images were captured in a confocal microscope having a 40× objective (Olympus) and an Andor iXon3 897 EMCCD



camera in 405 and 561 nm wavelength laser lights. The microtubule morphology of differentiated PC12 cells was captured at various areas of culture dish. 4.21 Immunoblotting experiment 63 PC12 differentiated neurons plated in 6 well plates with 10 µM of PS3 peptide, its scrambled analogue SCR1 (NLDFVTEQ) and NP (a microtubule non-interacting peptide) for 12 h at 37 ºC. After 12 h of treatment, cells were lysed on ice using RIPA cell lysis buffer supplemented with 1% protease inhibitor cocktail and then centrifuged at 12000xg at 4 °C for 20 min. The protein concentrations in the supernatants were quantified using Bradford reagent. The cell lysates were subjected to 12% SDS-polyacrylamide gel electrophoresis, then transferred onto PVDF membranes. The membranes were then probed with appropriate dilution of Anti-alpha Tubulin (acetyl K40) primary antibody overnight at 4 ºC. Membranes were then washed thrice with 1X TBS buffer and incubated with Anti-Mouse HRP-conjugated secondary antibody for 2 h at room temperature. All proteins were detected using LuminataTM Forte chemiluminescence reagent and quantified using densitometry adjusted against loading control alpha-Tubulin. 4.22 Monitoring neuroprotective effect of PS3 peptide in NGF deprived PC12 derived neurons 100 ng/mL of NGF in serum free DMEM media containing 1% horse serum was treated to the differentiated PC12 neuron and incubated at 37 ˚C in a 5 % CO2 environment in our lab for 5 days. Next, differentiated PC12 neurons were treated with anti-NGF (2 µg/mL) alone and with varying concentrations of PS3 peptide for upto 20 h. The cells containing serum free media was treated with MTT solution (5 mg/mL) and incubated for 4 h at 37 ˚C. The incubation media was replaced by 1:1 MeOH:DMSO solution in each well and scanned using a microplate ELISA reader (Thermo; MultiscanTM GO Microplate Spectrophotometer) at 550 nm33


Page 24 of 42

Page 25 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4.23 Serum stability of PS3 peptide 58 Serum stability of the PS3 peptide was monitored in human serum. A solution of 50 µL of peptide (200 µM) in 800 µL of human serum and 150 µL of milliQ water were incubated at 37 °C. Next, 100 µL of mixture was separately taken out and mixed with 100 µL of acetonitrile. The mixture was centrifuged and the filtrate part was then used for HPLC. Then, the same step was repeated in every 2 h interval upto 24 h. Intensity of PS3 peptide’s molecular peak in HPLC was plotted with time up to 24 h. 4.24 Effect of PS3 peptide on primary cortical neuron culture In our lab, primary cortical neurons were cultured by following the previously described method.64,65 In brief, we have taken timed-pregnant Sprague Dawley rat and isolated brain from E18 embryos. Brain cortices were then micro-dissected, digested, dissolved, filtered and then suspended in MEM medium that contained 10% horse serum and glucose (0.6% wt/vol). The suspended cells (3-5 x 105/mL) were taken and cultured on confocal dishes coated with poly-Dlysine at 37 0C with 5% CO2 environment. The culture medium was changed with Neurobasal media supplemented with B27, Pen/Strep and GlutaMAX after 4 h of incubation. We have treated the cells with 10 µM of PS3 peptide after continuing the cell culture for another 4 days. 4.25 Data Analysis Image J software was used to analyse the microscopic images whereas Origin 8.5 pro was used to study the spectroscopic and statistical data. For statistical analysis, two tailed student’s t-test and one-way ANOVA was performed. Statistical values was varied between *P≤0.05, *P≤0.01, **P≤0.001 in various experiment.



ASSOCIATED CONTENT Supporting Information Figures S1-S23. This materials are available free of charge via the internet at http://pubs.acs.org. 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 Surajit Ghosh: 0000-0002-8203-8613 Author contributions P.M. performed alanine scanning mutagenesis, screening of lead peptide from peptide library by molecular docking, synthesized and purified all the peptides and performed various in vitro microtubule assays e.g. microtubule assembly assay, tryptophan quenching experiment, FRET experiment and ThT assay. G.D. performed various cell based assays like cell viability, cellular uptake, neuroprotection, intracellular microtubule staining assay and immunoblotting experiment. J.K. performed the primary cortical neurons experiment. K.P. helped P.M. for AChE enzyme inhibiton assay. Also, P.M. executed various molecular docking studies, MD simulation


Page 26 of 42

Page 27 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


experiment with this peptide and helped S.G. in writing manuscript. S.G. conceived the idea, supervised the project and wrote the manuscript. Notes The authors declare no competing financial interest ACKNOWLEDGMENT PM wish to thank CSIR, GD thanks to ICMR, JK thanks to DST-Inspire and KP thanks to UGC for awarding their fellowships. SG kindly acknowledges SERB, India (EMR/2015/002230) for financial assistance and CSIR-IICB for infrastructure.

Reference: 1. Reiner, O., and Sapir, T. (2009) Polarity regulation in migrating neurons in the cortex, Mol. Neurobiol. 40, 1–14. 2. Barnes, A. P., and Polleux, F. (2009) Establishment of axondendrite polarity in developing neurons, Annu. Rev. Neurosci. 32, 347–381. 3. Conde, C., and Caćeres, A. (2009) Microtubule assembly, organization and dynamics in axons and dendrites, Nat. Rev. Neurosci. 10, 319–332. 4. Marıń, O., Valiente, M., Ge ,X., and Tsai, L. H. (2010) Guiding neuronal cell migrations, Cold Spring Harb. Perspect. Biol. 2, a001834. 5. Kuijpers, M., and Hoogenraad, C. C. (2011) Centrosomes, microtubules and neuronal development, Mol. Cell. Neurosci. 48, 349–358. 6. Stiess, M., and Bradke, F. (2011) Neuronal polarization: the cytoskeleton leads the way, Dev. Neurobiol. 71, 430–444. 7. Rolls, M. M. (2011) Neuronal polarity in Drosophila: sorting out axons and dendrites, Dev. Neurobiol. 71, 419–429.



8. Baas, P. W., and Lin, S. (2011) Hooks and comets: the story of microtubule polarity orientation in the neuron, Dev. Neurobiol. 71, 403–418. 9. Yu, W., Schwei, M.J., and Baas, P.W. (1996) Microtubule transport and assembly during axon growth, J. Cell Biol. 133, 151–157. 10. Slaughter, T.S., Wang, J., and Black, M. M. (1997) Transport of microtubules from the cell body into the axons of cultured neurons, J. Neurosci. 17, 5807–5819. 11. Tanaka, E., Ho, T., and Kirschner, M. W. (1995) The role of microtubule dynamics in growth cone motility and axonal growth, J. Cell Biol. 128, 139–155. 12. Baas, P. W., Black, M. M., and Banker, G. A. (1989) Changes in micromicrotubule polarity orientation during the development of hippocampal neurons in culture, J. Cell Biol. 109, 3085–3094. 13. Baas, P. W., Slaughter, T., Brown, A., and Black, M. M. (1991) Microtubule dynamics in axons and dendrites, J. Neurosci. Res. 30, 134–153. 14. Brandt, R., and Lee, G. (1994) Orientation, assembly and stability both of microtubule bundles induced by a fragment of tau protein, Cell Motil. Cytoskel. 28, 143–154. 15. Baas, P. W., Pienkowski, T. P., Cimbalnik, K. A., Toyama, K., Bakalis, S., Ahmad, F. J., and Kosik, K. S. (1994) Tau confers drug-stability and but not cold-stability to microtubules in living cells, J. Cell Sci. 107, 135-143. 16. Black, M. M. (1987) Comparison of MAP-2 and tau on the packing of assembled microtubules, Proc. Natl. Acad. Sci. USA. 84, 7783–7787. 17. Chen, J., Kanai, Y., Cowan, N. J., and Hirokawa, N. (1992) Projection of MAP-2 and tau determine spacings between microtubules in dendrites and axons, Nature 360, 674–677.


Page 28 of 42

Page 29 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


18. Mandelkow, E. M., Stamer, K., Vogel, R., Thies, E., and Mandelkow, E. (2003) Clogging of axons by tau, inhibition of axonal traffic and starvation of synapses, Neurobiol Aging 24, 1079–1085. 19. Tolnay, M., and Probst, A. (1999) Review: tau protein pathology in Alzheimer’s disease and related disorders, Neuropathol Appl Neurobiol 25, 171–187. 20. LaFerla, F. M., Green, K. N., and Oddo, S. (2007) Intracellular amyloid-beta in Alzheimer's disease, Nat Rev Neurosci. 8, 499-509. 21. Zempel, H., Luedtke, J., Kumar, Y., Biernat, J., Dawson, H., Mandelkow, E., and Mandelkow, E. M. (2013) Amyloid-β oligomers induce synaptic damage via Taudependent microtubule severing by TTLL6 and spastin, EMBO J. 32(22), 2920-2937. 22. Saha, A., Mohapatra, S., Kurkute, P., Jana, B., Mondal, P., Bhunia, D., Ghosh, S., and Ghosh, S. (2015) Interaction of Aβ peptide with tubulin causes an inhibition of tubulin polymerization and the apoptotic death of cancer cells, Chem Commun (Camb). 51(12), 2249-2252. 23. Michaelis, M. L., Chen, Y., Hill, S., Reiff, E., Georg, G., Rice, A., and Audus, K. (2002) Amyloid peptide toxicity and microtubule-stabilizing drugs, J Mol Neurosci. 19(1-2), 101-105. 24. Michaelis, M. L., Ansar, S., Chen, Y., Reiff, E. R., Seyb, K. I., Himes, R. H., Audus, K. L., and Georg, G. I. (2005) {beta}-Amyloid-induced neurodegeneration and protection by structurally diverse microtubule-stabilizing agents, J Pharmacol Exp Ther. 312(2), 659-668. 25. Nelson, R. (2005) Microtubule-stabilising drugs may be therapeutic in AD, Lancet Neurol. 4(2), 83-84.



26. Trojanowski, J. Q., Smith, A. B., Huryn, D., and Lee, V. M. (2005) Microtubulestabilising drugs for therapy of Alzheimer's disease and other neurodegenerative disorders with axonal transport impairments, Expert Opin Pharmacother. 6(5), 683-686. 27. Doraiswamy, P. M., and Xiong, G. L. (2006) Pharmacological strategies for the prevention of Alzheimer's disease, Expert Opin Pharmacother. 7(1), 1-10. 28. Lee, V. M., and Trojanowski, J. Q. (2006) Progress from Alzheimer's tangles to pathological tau points towards more effective therapies now, J Alzheimers Dis. 9, 257262. 29. Butler, D., Bendiske, J., Michaelis, M. L., Karanian, D. A., and Bahr, B. A. (2007) Microtubule-stabilizing agent prevents protein accumulation-induced loss of synaptic markers, Eur J Pharmacol. 562(1-2), 20-27. 30. Brunden, K. R., Zhang, B., Carroll, J., Yao, Y., Potuzak, J. S., Hogan, A. M., Iba, M., James, M. J., Xie, S. X., Ballatore, C., Smith, A. B 3rd., Lee, V. M., and Trojanowski, J. Q. (2010) Epothilone D improves microtubule density, axonal integrity, and cognition in a transgenic mouse model of tauopathy, J Neurosci. 30(41), 13861-13866. 31. Brunden, K. R., Yao, Y., Potuzak, J. S., Ferrer, N. I., Ballatore, C., James, M. J., Hogan, A. M., Trojanowski, J. Q., Smith, A. B 3rd., and Lee, V. M. (2011) The characterization of microtubule-stabilizing drugs as possible therapeutic agents for Alzheimer's disease and related tauopathies, Pharmacol Res. 63(4), 341-351. 32. Brunden, K. R., Trojanowski, J. Q., Smith, A. B 3rd., Lee, V. M., and Ballatore, C. (2014) Microtubule-stabilizing agents as potential therapeutics for neurodegenerative disease, Bioorg Med Chem. 22(18), 5040-5049.


Page 30 of 42

Page 31 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


33. Biswas, A., Kurkute, P., Saleem, S., Jana, B., Mohapatra, S., Mondal, P., Adak, A., Ghosh, S., Saha, A., Bhunia, D., Biswas, S. C., and Ghosh, S. (2015) Novel hexapeptide interacts with tubulin and microtubules, inhibits Aβ fibrillation, and shows significant neuroprotection, ACS Chem Neurosci. 6(8), 1309-1316. 34. Biswas, A., Kurkute, P., Jana, B., Laskar, A., and Ghosh, S. (2014) An amyloid inhibitor octapeptide forms amyloid type fibrous aggregates and affects microtubule motility, Chem Commun (Camb). 50(20), 2604-2607. 35. Magen, I., and Gozes, I. (2013) Microtubule-stabilizing peptides and small molecules protecting axonal transport and brain function: focus on davunetide (NAP), Neuropeptides. 47(6), 489-495. 36. Shprung, T., and Gozes, I. (2009) A novel method for analyzing mitochondrial movement: inhibition by paclitaxel in a pheochromocytoma cell model, J Mol Neurosci. 37(3), 254-262. 37. Burke, W. J., Raghu, G., and Strong, R. (1994) Taxol protects against calcium-mediated death of differentiated rat pheochromocytoma cells, Life Sci. 55(16), 313-319. 38. Michaelis, M. L., Ranciat, N., Chen, Y., Bechtel, M., Ragan, R., Hepperle, M., Liu, Y., and Georg, G. (1998) Protection against beta-amyloid toxicity in primary neurons by paclitaxel (Taxol), J Neurochem. 70(4), 1623-1627. 39. Li, G., Faibushevich, A., Turunen, B. J., Yoon, S. O., Georg, G., Michaelis, M. L., and Dobrowsky, R. T. (2003) Stabilization of the cyclin-dependent kinase 5 activator, p35, by paclitaxel decreases beta-amyloid toxicity in cortical neurons, J Neurochem. 84(2), 347362. 40. Holzgrabe, U. (2005) Paclitaxel for Alzheimer treatment, Pharm Unserer Zeit. 34(2), 96.



41. Shemesh, O. A., and Spira, M. E. (2011) Rescue of neurons from undergoing hallmark tau-induced Alzheimer's disease cell pathologies by the antimitotic drug paclitaxel, Neurobiol Dis., 43(1), 163-175. 42. Pittman, S. K., Gracias, N. G., Vasko, M. R., and Fehrenbacher, J. C. (2014) Paclitaxel alters the evoked release of calcitonin gene-related peptide from rat sensory neurons in culture, Exp Neurol. 253, 146-153. 43. Gornstein, E. L., and Schwarz, T. L. (2017) Neurotoxic mechanisms of paclitaxel are local to the distal axon and independent of transport defects, Exp Neurol. 288, 153-166. 44. Zhang, B., Carroll, J., Trojanowski, J. Q., Yao, Y., Iba, M., Potuzak, J. S., Hogan, A. M., Xie, S. X., Ballatore, C., Smith, A. B, Lee, V. M., and Brunden. K. R., (2012) The microtubule-stabilizing agent, epothilone D, reduces axonal dysfunction, neurotoxicity, cognitive deficits, and Alzheimer-like pathology in an interventional study with aged tau transgenic mice. J Neurosci., 32, 3601-3611. 45. Brunden, K. R., Gardner, N. M., James, M. J., Yao, Y., Trojanowski, J. Q., Lee, V. M., Paterson, I., Ballatore, C., and Smith, A. B.,.(2013) MT-Stabilizer, Dictyostatin, Exhibits Prolonged Brain Retention and Activity: Potential Therapeutic Implications. ACS Med Chem Lett.; 4, 886-889. 46. Gozes, I. (2011) NAP (davunetide) provides functional and structural neuroprotection, Curr Pharm Des., 17, 1040-1044. 47. Xiao, H., Verdier-Pinard, P., Fernandez-Fuentes, N., Burd, B., Angeletti, R., Fiser, A., Horwitz, S. B., and Orr, G. A. (2006) Insights into the mechanism of microtubule stabilization by Taxol, Proc Natl Acad Sci U S A. 103(27), 10166-10173.


Page 32 of 42

Page 33 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


48. Anand, P., Nagarajan, D., Mukherjee, S., and Chandra, N. (2014) ABS–Scan: In silico alanine scanning mutagenesis for binding site residues in protein–ligand complex, F1000Research. 3, 214 (doi: 10.12688/f1000research.5165.2) 49. Faure, G., Bornot, A., and de Brevern, A. G. (2008) Protein Contacts, Inter-residue Interactions and Side-chain Modelling, Biochimie. 90 (4), 626-639. 50. Bhunia, D., Mohapatra, S., Kurkute, P., Ghosh, S., Jana, B., Mondal, P., Saha, A., Das, G. & Ghosh, S. (2016) .Novel Tubulin-targeted Cell Penetrating Antimitotic Octapeptide, Chem. Commun. 52, 12657-12660. 51. Lowe, J., Li, H., Downing, K. H., and Nogales, E. (2001) Refined Structure of Alpha Beta-tubulin at 3.5 A Resolution, J. Mol. Biol. 313 (5), 1045-1057. 52. Trott, O., and Olson, A. J. (2010) AutoDock Vina: Improving the Speed and Accuracy of Docking with a New Scoring Function, Efficient Optimization, and Multithreading, J. Comput. Chem. 31, 455-461. 53. Bonne, D., Heusele, C., Simon, C., and Pantaloni, D. (1985) 4',6-Diamidino-2phenylindole, a Fluorescent Probe for Tubulin and Microtubules, J. Biol. Chem. 260 (5), 2819-2825. 54. Ghosh, J. G., Houck, S. A., and Clark, J. I. (2007) Interactive Domains in the Molecular Chaperone Human αB Crystallin Modulate Microtubule Assembly and Disassembly, PLoS ONE. 2 (6), e498. 55. Gupta, K., and Panda, D. (2002) Perturbation of Microtubule Polymerization by Quercetin through Tubulin Binding: a Novel Mechanism of its Antiproliferative Activity, Biochemistry 41 (43), 13029-13038.



56. Dıáz, J. F., Barasoain, I., and Andreu, J. M. (2003) Fast Kinetics of Taxol Binding to Microtubules, J. Biol. Chem. 278(10), 8407-8419. 57. Matesanz, R., Barasoain, I., Yang, C., Wang, L., Li, X., Ine´ s, C., Coderch, C., Gago, F., Barbero, J., Andreu, J. M., Fang, W., and Dıáz, J. F. (2008) Optimization of taxane binding to microtubules: binding affinity dissection and incremental construction of a high-affinity analog of paclitaxel, Chemistry & Biology, 15, 573-585. 58. Jana, B., Mondal, P., Saha, A., Adak, A., Das, G., Mohapatra, S., Kurkute, P., and Ghosh, S. (2017) Designed Tetrapeptide Interacts with Tubulin and Microtubule, Langmuir, Article ASAP DOI: 10.1021/acs.langmuir.7b01326 59. Paul, A., Nadimpally, K. C., Mondal, T., Thalluri, K., and Mandal, B. (2015) Inhibition of Alzheimer's amyloid-β peptide aggregation and its disruption by a conformationally restricted α/β hybrid peptide, Chem. Commun. 51, 2245-2248. 60. Crescenzi, O., Tomaselli, S., Guerrini, R., Salvadori, S., D'Ursi, A.M., Temussi, P.A., and Picone, D. (2002) Solution structure of the Alzheimer's disease amyloid betapeptide (1-42), Eur J Biochem. 269(22), 5642-5648. 61. Chen, X., Wehle, S., Kuzmanovic, N., Merget, B., Holzgrabe, U., Kö nig, B., Sotriffer, C. A., and Decker, M. (2014) Acetylcholinesterase Inhibitors with Photoswitchable Inhibition of β-Amyloid Aggregation, ACS Chem. Neurosci. 5, 377-389. 62. Adak, A., Das, G., Barman, S., Mohapatra, S., Bhunia, D., and Ghosh, S. (2017) Biodegradable Neuro-Compatible Peptide Hydrogel Promotes Neurite Outgrowth, Shows Significant Neuroprotection, and Delivers Anti-Alzheimer Drug, ACS Appl Mater Interfaces., 9, 5067- 5076.


Page 34 of 42

Page 35 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


63. Portran, D., Schaedel, L., Xu, Z., Théry, M. & Nachury M.V. (2017).Tubulin acetylation protects long-lived microtubules against mechanical ageing. Nat Cell Biol.19, 391-398. 64. Xu, S. Y., Wu, Y. M., Ji, Z., Gao, X. Y., & Pan, S. Y. A (2012) Modified Technique for Culturing Primary Fetal Rat Cortical Neurons, Journal of Biomedicine and Biotechnology.1–7. 65. Beaudoin III, G. M. J., Lee, S. H., Singh, D., Yuan, Y., Ng, Y. G., Reichardt, L. F., and Arikkath, J. (2012) Nature Protocols, 7, 1741. 66. Hyman, A., Drechsel, D., Kellogg, D., Salser, S., Sawin, K., Steffen, P., Wordeman, L., and Mitchison, T. (1991) Preparation of Modified Tubulins, Methods in Enzymology, 196, 478-485.



Figure 1: Design of PS3 peptide and its analogues from taxol binding pocket of β-tubulin by InSillico alanine scanning mutagenesis in ABS Scan web server.


Page 36 of 42

Page 37 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2: (a) Molecular docking study of PS3 peptide at the taxol site of β-tubulin (PDB ID1JFF) (b) Interacting amino acids at the taxol pocket of β-tubulin interacts with PS3 peptide. (c) Microtubule polymerization assay using DAPI and tubulin upon addition of various concentration of PS3 peptide. (d) Binding affinity of PS3 peptide (Kb) with tubulin was quantified by measuring the intrinsic tryptophan fluorescence quenching with increasing concentration of PS3 peptide. (e) Determination of binding site of PS3 peptide in tubulin by FRET experiment using Tubulin-colchicine complex (donor) and fluorescein-PS3 (acceptor).



Figure 3: (a) Inhibition of Aβ aggregation upon addition of PS3 peptide was monitored by thioflavin T (ThT) assay. Error bar corresponds to standard deviation of the value (*p

Crafting of Neuroprotective Octapeptide from Taxol ... - ACS Publications

Recommend Documents