Discovery of Neuro-regenerative Peptoid from Amphibian

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Discovery of Neuro-regenerative Peptoid from Amphibian Neuropeptide Inhibits A# Toxicity and Crossed Blood-Brain Barrier Krishnangsu Pradhan, Gaurav Das, Varsha Gupta, Prasenjit Mondal, Surajit Barman, Juhee Khan, and Surajit Ghosh ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00427 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 12, 2018

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ACS Chemical Neuroscience

Discovery of Neuro-regenerative Peptoid from Amphibian Neuropeptide Inhibits Aβ Toxicity and Crossed Blood-Brain Barrier Krishnangsu Pradhan,1 Gaurav Das,1,2 Varsha Gupta,1 Prasenjit Mondal, 1,2 Surajit Barman,1 Juhee Khan1,2 and Surajit Ghosh1,2* 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: Development of potential therapeutics for Alzheimer’s disease (AD) required multifaceted strategy considering the high level of complexities of human brain and its mode of function. Here, we adopted an advanced strategy targeting two key pathological hallmarks of AD such as senile plaque and neurofibrillary tangles. We derived a lead short tetrapeptide Ser-Leu-Lys-Pro (SLKP) from dodeca-neuropeptide of amphibian (frog) brain. Results suggest that SLKP peptide has superior effect compared to the dodecapeptide in neuroprotection. This result encourages us to adopt peptidomimetic approach to synthesize SLKP peptoid. Remarkably, we found that SLKP peptoid is more potent than its peptide analogue, which significantly inhibits Aβ fibrillization, moderately binds with tubulin and promotes tubulin polymerization as well as stabilization of microtubule networks. Further, we found that SLKP peptoid is stable in serum, showed significant neuroprotection against Aβ mediated toxicity, promotes significant neurite outgrowth, maintains healthy morphology of rat primary cortical neurons and crosses the Blood-Brain Barrier (BBB). To the best of our knowledge, our SLKP peptoid is the first shortest peptoid showed significant neuroprotection, neuro-regeneration against Aβ toxicity as well as crossed the BBB offering a potential lead for AD therapeutics.

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Keywords: Neuropeptide,

Peptoid,

Aβ

aggregation,

Microtubule,

Alzheimer’s

disease

(AD),

neuroprotection, Neuro-regeneration.

1. Introduction Neurodegenerative diseases are characterized by progressive impairment of neuronal function and death in the brain. Alzheimer’s disease (AD) is a neurodegenerative disease, associated with progressive dementia.1.The two major histological hallmarks that define AD are amyloid plaques and neurofibrillary tangles (NFT). Amyloid plaques are clumps of beta-amyloids found in the interstitial spaces of the brain and are extremely toxic to neurons. According to the amyloid hypothesis, Beta amyloids are formed through sequential enzymatic cleavage of amyloid precursor protein (APP) by -secretase and later by -secretase resulting in the formation of insoluble A which causes toxicity and damage to the neuronal networks.2,3 In healthy brain, pico-molar concentrations of A is always present and it takes active part in synapse formation, neural plasticity and homeostasis.4 In normal human brain, 80% of A contains A40, but in AD brain excess A42 is produced and accumulated as amyloid plaques, which deleteriously affect a number of important intracellular pathways finally leading to neuronal death.5 The toxicity of A originates from the oligomeric and fibrillar aggregates of A species, this pathway is known as amyloid cascade hypothesis. The formation of toxic fibrillar aggregates through oligomeric species generated from the A monomers depends on the monomeric concentration, known as the primary nucleation step. After formation of fibrillar structure through primary nucleation, the fibril catalyzes the formation of oligomers, known as secondary nucleation.6 Recently, it has been also observed that oligomeric aggregate of Aβ is more toxic as compared to fibrillary aggregates.7 Another feature of AD are the NFTs which are intracellular filaments of the hyperphosphorylated tau


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protein.1,8 Tau is a protein known to stabilize neuronal intracellular microtubules which is responsible for cargo transport from the cell body to the synaptic end of neurons. In AD, Tau becomes hyperphosphorylated and disrupts microtubule function thus leading to the propagation of the disease.9-13 A number of hypothesis has been proposed for the oligomerization and aggregation of A, tau hyper phosphorylation, metal mediated toxicity, oxidative stress, apoE4 impairment, A clearance, hydrolysis of acetylcholine by acetyl cholinesterase, activation of the inflammatory system, and dysregulation of autophagy in AD disease.4 How exactly all these factors contrive to impair the neuronal functions as well as survival still remains to be determined.5 In order to tackle AD, a number of multifaceted approaches for the development of effective agents for the treatment of AD are very important. In this regard, designing of multifaceted molecules capable of dissolving the Aβ oligomers and fibrils,14 disruption of the redox metal ion from Aβ-metal ion complex,15 preventing ROS generation as well as cell membrane disruption, showing antioxidant effect are some of the very promising approaches. Several peptides and peptoids that act as an inhibitor of Aβ aggregation and microtubule stabilizer have already been reported in the literature by inhibition of tau hyper phosphorylation. Turner et al. have shown that a peptoid mimicking KLVFF peptide modulates Aβ40 aggregation as well as decreases lag time of βsheet aggregation.16 Luo et al. have explored IAM1 and its dimeric form (IAM1)2 with a higher affinity for Aβ42 which ultimately translates to inhibition of aggregation of Aβ42.17 Moreover, (IAM1)2 has the ability to protect the primary hippocampal neurons from the Aβinduced toxicity in vitro.17 Zhao et al. discovered that AIP1 has a binding affinity with Aβ42, inhibition of Aβ42 oligomerization and rescued Aβ42-induced cytotoxicity. AIP1 also has the ability to cross the blood brain barrier.18 Gozes et al. have discovered NAP peptide (NAPVSIPQ), as a potent neuroprotector.19 NAP peptide inhibited the hyper phosphorylation of microtubule associated tau protein to stabilize the microtubule.19 The above reasons have



prompted the development of anti-A therapeutics, markers for serum Aβ42 and microtubule stabilizing agents for targeting AD.20-35 In spite of all the above efforts, the clinical success of the above studies have been severely limited chiefly due to the poor blood-brain barrier (BBB) permeability, severe adverse effects, no selectivity and low affinity of these compounds for Aβ42.36,37 In the wake of the above problems, several small molecules and peptides that inhibit Aβ aggregation have been designed but like all others they also suffer from low bioavailability and rapid degradation in vivo.38-53 Therefore, further extensive efforts are necessary for the development of potential therapeutics of AD. In this direction, ideal approach would be development of poly-N-substituted glycines (peptoids) which are resistant to protease degradation.54,55 Peptoids belongs to a class of peptidomimetics where they bear structural similarity to the peptides except for the fact that the side chains in peptoids are appended to the amide group rather than the α-carbon. Due to this change in backbone structure, the peptoids are proteolytically stable and hold promise for improved anti-AD therapeutics. In this manuscript, we adopted a top down approach to design a short peptoid56 from a long neuropeptide (SLKPAANLPLRF) of frog brain (Rana RFamide, RRFa)57 which on blast shows 100% identity with sequences from Rana catesbeiana (American bullfrog) with accession numbers PIO32748.1 and Q8JIM3.1. This peptoid was designed from a small peptide, which was a fission part of the R-RFa.57 Our strategy involved truncation of the dodecapeptide into three small tetrapeptides (Figure 1). Further, screening of these three tetrapeptides has been performed using standard ThioflavinT (ThT) assay for inhibition of Aβ aggregation. Results suggest that among three tetrapeptides, Ser-Leu-LysPro (SLKP) was the most effective one. This result further motivated us to design a peptoid based on this tetrapeptide, which showed strong interaction with Aβ peptide (B.E. -4.1 Kcal/mol) at 17-21 hydrophobic pocket. This led to detailed study of this peptoid, it’s potential to inhibit of Aβ fibril formation and its ability of neuronal regeneration using


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various in vitro assays. We found that SLKP peptoid significantly inhibits Aβ fibrillization and moderately binds with tubulin. It promotes tubulin polymerization as well as provides stabilization of microtubule networks. Further, it showed stability in serum, promoted neurite outgrowth and conferred significant neuroprotection in PC12 derived neurons, maintained healthy neuronal architecture of rat primary cortical neurons and successfully crossed the blood brain barrier.

2. Results and Discussion 2.1 Synthesis, purification, characterisation of peptides R-RFa, SLKP, AANL and PRLF peptides were synthesized following the solid phase method using rink amide resin in our laboratory. These peptides were purified through reverse phase high performance liquid chromatography and characterized by ESI mass spectrometry (Figure. S1-S8, ESI†). All the peptides were stored in -20 0C freezer. 2.2 Synthesis, purification, characterisation of peptoid and 5(6)-carboxyfluorescein attached peptoid SLKP peptoid was synthesized using rink amide resin following the solid phase method42 (Figure. S9. ESI†). The peptoid was purified and characterised by reverse phase high performance liquid chromatography, mass spectroscopy respectively (Figure. S10, S11, ESI†). Then, 5(6)-carboxyfluorescein was attached with the peptoid using N,N′Diisopropylcarbodiimide (DIC) as a coupling reagent using solid phase method. 5(6)carboxyfluorescein attached peptoid was purified by RP-HPLC (Figure. S12, ESI†) and characterised by MALDI mass spectroscopy (Figure. S13, ESI†). 2.3 Screening of potential lead peptide from dodecapeptide R-RFa In brief, the R-RFa dodecapeptide of frog brain was truncated into the three tetra peptides such as SLKP, AANL and PLRF. ThT assay was performed with R-RFa and three tetra



peptides for identification of most effective inhibitor of Aβ fibrilization. ThT assay revealed that R-RFa and SLKP peptide showed better inhibition of Aβ fibrillization compared to the other two tetra peptides (Figure. 2A). Since R-RFa is a neuropeptide, we were curious to know whether this peptide has potential in neuroregeneration or not. Therefore, the cell viability of R-RFa and three tetra peptides was checked in PC12 derived neurons, which suggested that R-RFa, SLKP, AANL were not cytotoxic but PRLF was slight toxic in higher concentration (Figure. S14, ESI†). Then, we performed neurite outgrowth assay of PC12 derived neurons with R-RFa, SLKP peptide. Interestingly, images of PC12 derived neurons reveal that upon treatment with SLKP peptide neurite outgrowth and length is significantly better compared to the R-RFa dodecapeptide (Figure. 2B-D), whereas other two tetra peptides (AANL, PLRF) are ineffective in promotion of neurite outgrowth (Figure. S15, ESI†). Potential of SLKP peptide in neurite outgrowth was further evaluated using immunocytochemistry assay of intracellular microtubule networks, which clearly supports that SLKP tetrapeptide has superior effect compared to the R-RFa peptide (Figure. 2E, 2F) (Figure. S16, ESI†). This result further motivated us to design peptoid (poly-N-substituted glycines), of SLKP peptide as peptides often suffer from proteolytic degradation (Figure 3A).54,56 2.4 FT-IR experiment for inhibition of amyloid fibrillation In AD brain, one of the major constituents is senile plaque composed of A peptide, originated from proteolysis of the amyloid precursor protein (APP). Hydrophobic interactions of A monomers lead to the generation of insoluble A oligomers as well as fibril structures, which generates large fibrillary aggregated plaque (Senile plaque).58 Since our main focus is to develop inhibitors of A aggregation, at first we performed the FTIR Spectroscopy of Aβ42 with SLKP peptoid. FTIR spectra revealed that A42 peptide formed -sheet structure (1636 cm-1) after 7 days of incubation, but in presence of SLKP peptoid there was no sign of


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-sheet (1651 cm-1) structure. Moreover, the conformation of the SLKP peptoid was also checked. FTIR spectra showed that SLKP peptoid even after 7 days of incubation did not show any -sheet (1679 cm-1) peak indicating it was unable to self-assemble (Figure. S17, ESI†). Thus, above result clearly indicates that SLKP peptoid is able to interact with Aβ and inhibit its β-sheet structure formation. 2.5 Inhibition Aβ42 oligomeric Aggregation by Thioflavin T (ThT) assay Preliminary observation from FTIR data encouraged us to further investigate the inhibitory role of SLKP peptoid against Aβ aggregation. It is known that A42 has a number of polymorphic forms such as monomer, dimer, oligomer and fibrillar aggregates.59 Among all these forms, oligomeric aggregates is considered as the most toxic form A. Amyloid oligomers induce synaptic dysfunction, membrane disruption and finally to neuronal death in the AD brain.60 Therefore, design of an inhibitor against oligomeric aggregates is crucial for the inhibition of amyloid toxicity. Thus, we have performed amyloid aggregation inhibition with the SLKP peptoid through Thioflavin T (ThT) assay. Generally, ThT fluorescence intensity is the indicator of the amount of aggregates present in the solution and it was described before that the fluorescence intensity of ThT was increased after binding with A and the quantity of aggregation is directly proportion with the ThT intensity.61,62 ThT assay revealed that in presence of SLKP peptoid the fluorescence intensity of the ThT was decreased as compared to SLKP peptide (Figure. 3B). Interestingly, the fluorescence intensity was decreased up to 55% using 20 M of SLKP peptoid, which also supports that the SLKP peptoid has the ability to inhibit the self-assembly of A peptide. This data clearly supports that the design of peptoid from the peptide is important. 2.6 Dot blot experiment for inhibition of amyloid aggregation with SLKP peptide and peptoid From the above ThT assay, it was evident that SLKP peptoid has better amyloid fibrillar aggregates inhibition affect than the SLKP peptide. For further confirmation, dot blot



experiment was performed with SLKP peptide and peptoid following previously established method.63 In this experiment, Aβ42 monomer (10 μM) was incubated with SLKP peptide and peptoid. Then, the incubated samples (2 L) were spotted on a nitrocellulose membrane. The membrane was treated with the A11 antibody to detect Aβ42 aggregates and the 6E10 antibody (control) to detect all forms of Aβ species. The intensity of the spots were measured and quantified in comparison to the control experiment. The data revealed that SLKP peptide (40 M) inhibited Aβ42 fibrillar aggregates 45% whereas at same concentration the SLKP peptoid inhibitated Aβ42 aggregates around 87% (Figure. S18 ESI†). The dot blot experiment also supports that SLKP peptoid has better efficiency than its counter peptide for Aβ42 aggregates inhibition and aggregation is decreased in a concentration dependent manner (Figure.3C, S19A ESI†). This experiment also supports design of peptoid from the peptide analouge is crucial aspect in terms of designing therapeutics against AD. 2.7 Inhibition of preformed Aβ42 fibrillation by Thioflavin T (ThT)assay Although, Aβ42 is known to be a disordered peptide, it folds to form an ordered β-sheet structure. Further it self-assembles to amyloid fibrillar aggregates which are toxic in nature. These fibrillar aggregates initiates the progression of AD.64 Therefore, inhibiting the formation of amyloid fibrillar aggregates is known to be considered as one of the most important technique for developing the therapeutic agents for curing or inhibiting AD. With the disease progression, the amount of amyloid fibril increases. Therefore, the effect of SLKP peptoid on the preformed fibril of A was also examined to check whether the SLKP peptoid could dissociate the pre-existing fibrils of Aβ through ThT assay. For this experiment, the complete fibrillation of Aβ was formed and then the peptoid was added. Result revealed that concentration dependent gradual decrease of fluorescence intensity, which is due to the consistent loss of amyloid fibrils (Figure. 3D). The fluorescence intensity of the ThT was


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decreased up to 47% at 20 µM. This result clearly suggests that SLKP peptoid has potential to dissociate the preformed fibril of A. 2.8 Inhibition Aβ42 fibrillation by dot blot assay To further confirm Aβ42 fibrillar aggregation inhibition by SLKP peptoid, dot blot experiment was performed.63 For this experiment, we have used OC antibody, which detects Aβ42 fibrillar aggregates and 6E10 antibody, which binds to all forms of Aβ species. The intensities of the spot on nitrocellulose membrane were measured and quantified. The quantification data showed that SLKP peptoid (40 M) prevents the fibril formation 78% (Figure.3E, S19B ESI†). This data clearly supports that SLKP peptoid inhibited amyloid aggregation as well as amyloid fibrillation to reduce the amyloid toxicity. 2.9 Visulisation of A42 fibril inhibition using TEM study It was well established that in AD, the fibrilar structure of Aβ is one of the key characteristic features. To further consolidate, the effect of SLKP peptoid for the inhibition of A42 fibril structure, we were keen to visualize the fibril inhibition of Aβ by SLKP peptoid. For that purpose TEM study was performed. TEM images revealed that after 7 days of incubation, Aβ formed fibrillar structure but in the same condition SLKP peptoid inhibited fibril formation of A peptide (Figure. S20, ESI†). This result is clearly support that SLKP peptoid inhibits the fibril formation. This TEM data is in agreement with the conclusion drawn from the ThT and dot blot assay. 2.10 Molecular docking for revealing the molecular interactions of peptoid, peptide responsible for amyloid fibril inhibiton So far various experimental results reveal that SLKP peptoid inhibits the Aβ peptide fibrillization. Now, the question is how this peptoid inhibits the fibril formation and what is the mechanism involved, which leads to this inhibition. To understand this event, we performed molecular docking experiment. One of the main characteristics of AD is the



formation of fibril structure of A42 peptide. Fibril structure contains high amount antiparallel -sheet structure which is stabilised by hydrophobic interaction with A42 monomers. Docking studies of SLKP peptoid and its counter peptide with A42 peptide (PDB ID 1IYT)65 revealed that SLKP peptiod binds to the hydrophobic region (B.E4.1Kcal/mol) through polar and non-polar interaction (Figure. 4A-C). But the SLKP peptide interacts very weakly with A42 (B.E- 2.3 Kcal/mol). Moreover, molecular docking of SLKP peptoid and peptide with A42 fibril structure was carried out to understand the binding partners and parameter. This study revealed that the peptoid strongly binds with the fibril structure than its counter peptide, the binding partners and the binding parameters were shown in the figure (Figure. 4D-F) (Figure. S21, S22, ESI†). 2.11 Understanding the interaction of A42 with the SLKP peptoid by Isothermal titration calorimetry (ITC) experiment To understand the amyloid aggregation and fibrillation inhibiting ability of SLKP peptoid, the binding affinity of the peptoid with the A42 was determined by ITC experiment. The heat data profiles of the ITC experiment66 was used to calculate the binding parameters between SLKP peptoid and A42 peptide. According to the net enthalpy change, the binding was exothermic in nature and binding stoichiometry was 1:1. Binding constant of SLKP peptoid with A42 was estimated to be (2.35  1.03) ×105 M-1 (Figure. S23 ESI†). This analysis indicated that SLKP peptoid has a strong interaction with A42 peptide. This strong affinity between SLKP peptoid and A42 peptide inhibits the progression of one A42 peptide to the other A42 peptide. In this way, SLKP peptoid inhibits the oligomeric and fibril formation of A42. Other thermodynamic parameters like enthalpy change (H= -45.31  4.2 Kcal/mol), entropy change (S= -128 cal/mol/deg) were also estimated upon the interaction between A42 and SLKP peptoid.


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2.12 Stabilisation of microtubule with the treatment of peptoid In AD, neurofibrillary tangles (NFT), is the another major neuropathological hallmarks. NFT formed due to hyper phosphorylation of Tau (Microtubule associated protein-MAP), which then dissociates from microtubule lattice, starts aggregating, leads to the formation of NFT. Detachment of hyperphosphorillated Tau from microtubule leads to the severe destabilization of microtubule networks and thus during AD microtubule networks severly disrupted. Therefore, the treatment of AD through stabilization of microtubule is considered to be one of the important strategy. Previous study shown that microtubule stabilizing agent like paclitaxel is used for the treatment of AD in tauopathy model.67 Considering the previous background, we were curious to study whether the SLKP peptoid has any role in stabilizing microtubule or not. For this purpose, we first checked the turbidity assay of the tubulin. This assay is the preliminary indicator if any small molecules or peptide interact with tubulin and whether it affects in tubulin dynamics.68 The result reveals that in presence of SLKP peptoid, the turbidity of the solution was increased which signified that SLKP peptoid interacted and helped to polymerize the tubulin (Figure. S24, ESI†). Again to validate the turbidity assay, microtubule assembly assay was performed using 4',6-diamidino-2-phenylindole (DAPI) as a dye. It was previously reported that with increasing tubulin polymerization, the fluorescence intensity of DAPI molecule increased and the fluorescence intensity of the DAPI molcule is proportional to tubulin polymerization.69 Results showed that the fluorescence intensity of the DAPI molecule in presence of the SLKP peptoid enhanced as compared to the control experiment (absence of peptoid) and also the intensity increasd with increasing the peptoid concentration (Figure. 5A). These data clearly supports that the SLKP peptoid provides stabilization of microtubule and promotes its polymerization, which is an encouraning reslut to perform further intracellular experiments.



2.13 Tryptophan quenching experiment to calculate the binding constant between tubulin and SLKP peptoid Before performing the intracellular experiment, we first checked how strongly the SLKP peptoid interacts with the tubulin. For that purpose, quenching of tryptophan fluorescence intensity was performed. It was previously reported that after binding small molecule or peptide with the tubulin, the intrinsic tryptophan fluorescence intensity decreases. Measuring the fluorescence intensity of tubulin with the peptoid, we can easily calculate the binding constant of SLKP peptoid with the tubulin. Measuring the fluorescence intensity of tubulin with the peptoid, we can easily calculate the binding constant of SLKP peptoid with the tubulin. The result showed that the peptoid had moderate binding enegy about 2.37X104 M (Figure. 5B). 2.14 Cell viability and cellular uptake study of 5(6)-carboxyfluorescein attached SLKP peptoid To perform various cell based assay, cell viability assay of the SLKP peptoid was performed. The MTT assay revealed that this peptoid was not cytotoxic in the PC12 derived neurons (Figure. S25, ESI†). Moreover, the cellular uptake of the peptoid was studied by microscope as well as FACS study. The microscope images revealed that a significant amount 5(6)carboxyfluorescein attched SLKP peptoid enters into the PC12 derived neurons (Figure. S26, ESI†). The FACS study also suggests that with increasing peptoid concentration, the cellular uptake of the peptoid also increases (Figure. S27, ESI†). Therefore, above data confirms the cellular uptake of SLKP peptoid. 2.15 Neurite outgrowth assay with the treatment of SLKP peptoid by staining the microtubules So far results suggest that SLKP peptoid provides microtubule stabilization as well as its promotion. This result further encourages us to analyse whether our SLKP peptoid has any


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neuro-regeneration capability or not. For this purpose, first we were interested to check whether

SLKP

peptoid

provides

intracellular

microtubule

stability

or

not.

Immunocytochemistry experiments were performed by staining the microtubule using primary and secondary antibody.70 Microscopic images revealed that even after the treatment with the peptoid, the PC12 derived neurons were healthy and showed significant neurite outgrowth as compared to its peptide analogue (Figure. 5C-D). Further, we quantified the neurite length, which showed a significant increase in the neurite length after the treatment of peptoid unlike its peptide analouge (Figure S28, ESI†). This result clearly showed that SLKP peptoid not only shows its potential in in vitro but also it shows its potential in PC12 derived neurons. 2.16 Immunoblotting experiment with the treatment of SLKP peptoid for stabilization of microtubule For further confirmation of microtubule stabilisation in presence of SLKP peptoid, western blot was performed with acetylated tubulin, which is used as a marker for stabilised microtubules. We used SLKP peptide as a control for this experiment. Western blot data showed higher expression of the protein for peptoid than its counter peptide, which suggested that the peptoid was able to provide better stabilisation of microtubule compared to its peptide analogue (Figure. 5E-F, S29, ESI†)). All these results support that the peptoid interacts with the intracellular microtubule networks and helps to stabilize the microtubles. 2.17 Neuroprotective effect of SLKP peptoid using anti-NGF assay Next, we were eager to perform cell based assay for further confirmation of neuro-protection ability of SLKP peptoid through fibril inhibition. Therefore, the nerve growth factor (NGF) deprived PC12 cellular model was used for neuro-protection assay. Previously, it was shown that NGF deprivation leads to the over production of intracellular Aβ, which causes neuronal cell death of PC12 derived neurons.71 This process can be inhibited by inhibiting Aβ



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production using β- and γ-secretase inhibitors or Aβ peptide targeted antibodies.72 This NGF deprived PC12 cell death is now commonly used to understand the mechanism of the cell death in neurodegenerative diseases.72 Therefore, we performed this assay for neuroprotection using our SLKP peptoid. For this assay, PC12 cells were treated with NGF for 7 days to differentiate PC12 cells into neurons. Then, the neuron morphology was checked after the treatment of anti-NGF alone and anti-NGF with the peptoid. The microscopic images clearly revealed that in presence of the peptoid, the NGF deprived neurons were able to maintain the neuronal morphology (Figure. 6A-C). To further illuminate the neuroprotection assay MTT reduction method was performed. Interestingly, the result revealed that the peptoid provides significant neuroprotection to the NGF deprived PC12 neurons, which suggests that the SLKP peptoid at 20 µM was able to rescue about 76% cells from amyloid toxicity (Figure. 6D). 2.18 Neuroprotective effect of SLKP peptoid using MTT assay From the above studies, it is known that SLKP peptoid has the capacity to ability to rescue the PC12 derived neurons from anti-NGF mediated toxicity. Further, we wanted to check whether the SLKP peptoid might be effectively protecting the neurons from A-mediated toxicity

or

not.

For

this

purpose,

MTT

[3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazoliumbromide] assay was performed. The MTT result showed that A42 decreased the cell viability but with the addition of SLKP peptoid, the cell death was suppressed in a dose-dependent manner (Figure S30, ESI†). This result suggests that SLKP has neuro-protection effect against A-mediated cytotoxicity in PC12 derived neurons. 2.19 Evalution of serum stability of Peptoid Above data reveals the remarkable potential of SLKP peptoid as Aβ inhibitor and promotion of neuroprotection as well as neuro-regeneration. Now, the question is whether this peptoid is a potential candidate for performing in vivo (animal model) experiment or not. For that


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purpose as a preliminary initiative, serum stability of the peptoid was checked upto 24 h using reverse phase high performance liquid chromatography (RP-HPLC). The result clearly showed that the peptoid was stable in serum (Figure. S31, ESI†). This is due to the side chain of the peptoid is append to nitrogen rather than carbon. Therefore, serum protease can not identify the peptoid backbone. The serum stability increases the bioavailability of the peptoid. Hence, the peptoid can be considered as a potential candidate for the evaluation in animal model for the development of anti-AD therapeutics. 2.20 Evaluation of the cytotoxicity of peptoid in primary cortical neuron after treatment with peptoid The serum stability of the peptoid motivated us to use this peptoid in in vivo model system. For that purpose, the toxicity of the peptoid was checked in freshly prepared rat primary cortical neurons. The images of primary cortical neurons with the treatment of peptoid showed that the primary neurons maintained healthy morphology as the untreated neurons (Figure. 7A-D). From this study it is clear that SLKP is non cytotoxic in primary cortical neurons (Figure. S32, ESI†). Therefore, this data provides us important benchmark for evaluation of this peptoid in in vivo mice model in our future studies. 2.21 Blood-Brain Barrier (BBB) crossing experiment of SLKP peptoid One major difficulty for the successful treatment with the drugs is to cross the blood-brain barrier (BBB). Therefore, blood-brain barrier (BBB) experiment was performed with SLKP peptoid to check whether it can penetrate the blood-brain barrier (BBB). For this experiment, SLKP peptoid was injected intra-peritoneally. Then, after 6 h brain was extracted and a homogenate was prepared in acetonitrile solution. The presence of SLKP mass in the mass spectra reveals that SLKP peptoid was able to cross Blood-Brain Barrier (Figure. 7E). As negative control, sucrose73 was used which did not show any molecular peak (Figure. S33 ESI†). This method has been previously validated with positive control of an already known



brain penetrating peptide YPFF (from the Brain Peps database) which showed molecular peaks.74 These results clearly indicate the strong potential of SLKP peptoid as anti-AD therapeutic candidate.

3. CONCLUSION In summary, we have demonstrated for the first time that amphibian neuropeptide contains an active neuroprotective peptide, which shows potential in eukaryotic neurons. Further, we implemented here a top down approach to develop a proteolytically stable short peptoid for inhibition of Aβ toxicity and neuro-regeneration. We identified a short tertrapeptide SLKP through truncation of dodecapeptide R-RFa from frog brain. Further, SLKP peptoid was designed and synthesized with envision to develop a proteolytically stable neuroprotective molecule. Results showed that SLKP peptoid significantly inhibits Aβ oligomerisation, fibrillization, moderately binds with tubulin and promotes tubulin polymerization as well as stabilization of microtubule networks. Moreover, we found that SLKP peptoid is stable in serum and showed significant neuroprotection against Aβ mediated toxicity, promotes significant neurite outgrowth and maintain healthy morphology of rat primary cortical neurons. SLKP peptoid is also able to cross the blood brain barrier. To the best of our knowledge, SLKP peptoid is the first shortest peptoid showing significant neuroprotection as well as neuro-regeneration against Aβ toxicity. Therefore, we envision that this peptoid could be a potential lead for the development of anti-AD therapeutic in near future. 4. EXPERIMENTAL SECTION 4.1 Chemicals All the fmoc protected amino acids and Rink Amide AM resin were purchased from Novabiochem (Germany). All the amines, Pipyridine, 1,1,1,3,3,3-Hexafluoroisopropanol


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(HFIP) and Uranyl acetate dehydrate were bought from Spectrochem. Sucrose was taken from SRL. Dichloromethane (DCM), N, N′-Dimethylformamide (DMF), methanol, Thioflavine-T, Trifluoroacetic acid, Hydrogen peroxide (30% solution) and diethyl ether were purchased from Merck (Germany) Ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), N, N′ Diisopropylcarbodiimide (DIC), 4, 6-diamidino-2-phenylindole (DAPI), Guanosine 5′-triphosphate sodium salt hydrate (GTP), 3-(4, 5-dimethylthiazol-2-yl)- 2, 5diphenyltetrazolium bromide (MTT), Dulbecco’s Modified Eagle’s Medium (DMEM), 4Piperazinediethanesulfonic acid (PIPES) and Cell cultured DMSO were purchased from Sigma Aldrich. 2-[4-(2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid (HEPES) was taken from Himedia. Various serum and culture medium were purchased from Invitrogen. Human recombinant NGF was purchased from Sigma (St. Louis, MO, USA) and Anti-NGF was taken from Abcam. Water and acetonitrile (HPLC grade) were purchased from J. T. Baker. β-amyloid (1-42) was purchased from Alxotec (sweeden). Nitrocellulose membrane was taken from Merck Millipore. Primary antibody 6E10, A11 and OC were taken from Bio Legend, Thermo Scientific and Millipore respectively. RP-HPLC (Shimadzu) with Symmetry C-18 (Waters) was used for purification of peptoid and peptide. Without further purification all compounds were used. 4.2 Peptide Synthesis and Purification All the amino acids for peptide synthesis were used in the Fmoc protected form. 20% piperidine in DMF was used to deprotect fmoc group of the resin and amino acid. All the peptides were synthesized following solid phase peptide synthesis (SPPS) method using rink amide AM resin. Cleavage cocktail solution containing TFA (91%) EDT (3%), Milli Q (3%), Phenol (3%) was used to cleave peptides from the resin. Then, the peptides were precipitated in the ice cold diethyl ether. The crude peptides were collected after centrifugation and purified by RP-HPLC. Finally the peptides were characterized by ESI Mass Spectroscopy.



4.3 Peptoid Synthessis56 and Purification For peptoid synthesis, 100 mg Rink amide resin was taken and swelled in dimethylformamide (DMF) for 2h. The fmoc group of the resin was removed by using 20% piperidine in DMF. Then the free amine group of the resin was acylated using 1 mL of bromoacetic acid solution (0.6 M in DMF) and 0.2 mL N,N'-diisopropylcarbodiimide solution (DIC, 50% in DMF v/v) under continuous shaking. After acylation step, the resin was washed with DMF five times. Then the resin was treated with amine of the corresponding amino acids to substitute the bromine group. For this displacement step, 1 mL (2M) amine was used. After this displacement step, the resin was again washed with DMF five times. Repeating the acylation and followed by displacement step, the desired length of the peptoid was obtained. Cleavage cocktail solution containing trifluoroacetic acid (95%) and water (5%) was used to cleave the peptoid from the resin. TFA was removed by gentle nitrogen flow. Then, water and acetonitrile (1:1) was added. This solution was lyophilized to get the dry product. Crude peptoid was purified by RP-HPLC to get the pure product and characterized by ESI mass spectrometry. Finally, the pure peptoid was stored in -20 0C. 4.4 Preparation A42 solution A42 was stored in -20 0C with the suspension of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to keep the monomer state of A42. Just before the experiment A42, A42 solution in HFIP was taken out and the HFIP was removed under gentle nitrogen flow to get the residue which was dissolved using minimum amount of 1% NH4OH solution. The desired concentration of A was made using required amount of phosphate buffer. 4.5 Thioflavin T (Th T) fluorescence Assay Using the PBS solution, the stock solution of the ThT was prepared. This solution was stored in 4 0C and covered with aluminium foil to prevent degradation. For ThT assay, we have used the freshly prepared ThT solution.


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4.6 Inhibition self-induced Aβ42 aggregation by SLKP peptoid by Th T assay For this experiment, Aβ42 (10 μM) peptide was incubated with five different concentrations (2.5, 5, 10 & 20 μM) of SLKP peptoid with constant agitation for 24h. For the control experiment PBS was used in place of peptoid. For the fluorescence study, incubated solution (40 μL) was mixed with ThT solution (200 μL, final concentration 50 μM) and the final volume was made up to 400 μL using PBS solution. For this study, the excitation wavelength was at 435 nm and emission wavelength was from 450 nm to 600 nm using a slit width of 1.25 nm in Quanta Master Spectrofluorometer (QM-40). Then data was plotted by using Origin Pro 8.5 software. 4.7 Inhibition of self-induced Aβ42 fibrillation by Th T assay This experiment was performed after completion of Aβ42 aggregation. For this experiment, the Aβ42 solution was continuously agitated at 37 °C for 24 h. Then, five different concentrations of SLKP peptoid were added and incubated at 37 °C for another 24 h. The final concentration of Aβ42 was 10 M and peptoid concentrations were 4, 8, 12, 16 & 20 μM. Then the measurement of the intensity of ThT was performed following the above method. 4.8 Preparation of Aβ42 Fibrils and Oligomers A42 was taken in PBS buffer at pH-7.4 and incubated at 37 °C for 48 h with constant shaking. Then ThT assay was performed to confirm the formation of fibril structure. DMSO was used to dissolve the Aβ42 peptide and diluted in PBS buffer (pH 7.4) to 100 μM. Then, the solution was incubated for 1 h at 37 °C and incubation was continued for 24 h at 4 °C. After that, supernatant was taken after centrifugation. The supernatant contained the oligomers. Further, dot blot analysis was performed for the confirmation of the formation of oligomers. 4.9 Dot Blot Analysis



Peptide and peptoid were added to a freshly prepared Aβ42 (10 μM) sample individually. This experiment was performed in PBS buffer (10 mM, pH 7.4). Then, these solutions were incubated for 1 h at 37 °C and again further incubation was continued for 24 h at 4 °C. After incubation, samples were centrifuged and the supernatant solutions were separated out. The supernatant was spotted on the nitrocellulose membrane. This experiment was performed in triplicate. Blocking buffer containing 5% BSA (bovine serum albumin) in TBST buffer was used to block the nonspecific sites by soaking for 1 hr at room temperature. Then, the membranes were incubated with primary antibody 6E10 (1:3000) to detect Aβ peptide, A11 (1:3000) to detect Aβ oligomer, OC (1:3000) to detect Aβ42 fibrillar aggregates overnight at 4 °C. After incubation, the membrane was washed with TBST buffer for 5 min three times. Then, the membrane was again incubated with the anti-mouse secondary antibody (1:10,000) conjugated with horseradish peroxidase (HRP) for 1 hr at RT. The membrane was washed with TBST buffer for 5 min three times. Finally, enhanced chemiluminescence (ECL) reagent was added and incubated for 2 min. Then, chemiluminescence was recorded in Biorad chemitouch. The chemiluminescence intensity was compared to that of the control (Aβ42). 4.10 Isothermal Titration Calorimetric analysis study This titration experiment was performed at a fixed temperature (298 K) in PBS (0.1 mM) between A42 (20 M) and SLKP peptoid. Each injection of 10 L of peptoid was mixed at an interval of 5 min by a computer control programming with continuous stirring. For this experiment, we performed 28 injections for each experiment. Each peak in the binding isotherm represents the interaction of peptoid with the A42 receptor protein solution. With successive addition of the peptoid solution, the heat was released. Then, the amount of heat released is plotted against the molar ratio of peptoid with the A42 peptide. 4.11 Transmission Electron Microscopy (TEM)


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Two solutions, one solution containing 10 M of A control experiment and another solution containing 10 M of A with 20 M peptoid were incubated for 7 days at 37 °C. For this experiment, 300 mesh copper grid was used. Over this grid, 10 μL from each of the incubated solutions was placed. After 1 min, the grid was stained with 2% uranyl acetate solution in water. TECNAI G2 SPIRIT BIOTWIN CZECH REPUBLIC 120 KV electron microscope operating at 120 kV was used for capturing the images. 4.12 Fourier Transform Infrared Spectroscopy A (10 M), A (10 M) with SLKP (20 M) and only SLKP (20 M) solutions were incubated for seven days at 37 oC. FT-IR spectra of SLKP peptoid was also recorded after 1h incubation. For all other solutions FT-IR was recorded after 7 days of incubation. PerkinElmer Spectrum 100 FT-IR spectrometer was used to record the all spectrum with speed 0.2 cm/s at a resolution of 1.6 cm-1. The KBr pellets were used for this study recoding 5 times scan. The LiTaO3 detector was used to plot the data. For each data background correction was performed to eliminate air interference. 4.13 Isolation of Tubulin75 Tubulin was isolated from goat brain and purification was performed following the previously described method. The purified tubulin was stored at -80 oC freeze. 4.14 Tubulin Polymerization Assay (Tubulin turbidity assay) Turbidity assay of tubulin was performed in Brinkley Reassembly Buffer 80 (BRB 80) buffer containing 4 mM GTP, 20 μM tubulin and 10% dimethyl sulfoxide (DMSO). DMSO was used to initiate the polymerization of the tubulin. All the components of the experiment were mixed in ice. Then, the absorbance of the solution was recorded at 350 nm for 40 min in the UV-Vis Spectrophotometer (G6860A Cary 60 UV-Vis Spectrophotometer, Agilent Technologies) at 37 °C. In case of control experiment, the peptoid was replaced with BRB 80 buffer.



4.15 Microtubule assembly assay Microtubule assembly assay was also performed in BRB80 buffer containing 35 μM DAPI, 100 μM tubulin, 10 mM GTP and 25, 50 μM SLKP. For this experiment, the excitation wavelength was at 355 nm and the emission spectra were recorded at 400 nm to 600 nm wavelength for 60 min at different time intervals in Quanta Master Spectrofluorometer (QM40). This instrument was equipped with Peltier to control the temperature for the experiment at 37 °C. In case of control experiment, BRB80 buffer was used in place of peptoid. Origin Pro 8.5 software was used to plot the data. 4.16 Determination of binding affinity of SLKP Peptoid with the tubulin by fluorescence intensity quenching study of intrinsic Tryptophan residue of tubulin For this experiment, tubulin was incubated in presence of different concentrations of SLKP peptoid in BRB80 buffer at  C after 30 min. The excitation wavelength was at 295 nm. The emission spectra were recorded at wavelength 310 to 450 nm. Quanta Master Spectrofluorometer (QM-40) equipped with Peltier were used to record all fluorescence data. 4.17 Fluorescence microscopic study to check the effect of SLKP peptoid on microtubule networks For this experiment, after seeding the PC12 cells over the cover glass bottom dish, PC12 cells were treated with NGF upto 7 days for its differentiation into neurons. After that, the differentiated PC12 derived neurons were treated with SLKP peptoid (20 M) for 24h. Then, cells were fixed and permeabilized using 4% formaldehyde and 0.2% Triton-X respectively. Primary antibody (alpha-Tubulin, Novus Biologicals) in 1:300 dilutions was added and incubated overnight at 4 C. Further incubation was performed with mouse IgG fluor (Millipore) at 37 C for another 2h. Then, the cells were washed using PBS. Again, cells were incubated with 1% Hoechst 33258 (Sigma-Aldrich) at 37 C for 1h. Finally, cells were


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washed using PBS and microscopy was performed to capture the images of PC12 derived neurons. 4.18 Immunoblotting Experiment76 PC12 derived neurons were treated with 10 μM of SLKP peptoid and its peptide analogue for 12h at 37 °C. After that, RIPA cell lysis buffer supplemented with 1% protease inhibitor cocktail was used to lyse the cells. This process was performed on ice. Then, the solution was centrifuged at 12000g at 4 °C. Bradford reagent was used to quantify the protein concentration present in the supernatant. 12% SDS-polyacrylamide gel electrophoresis was exposed to the cell lysates. Then the solution again was relocated onto PVDF membranes. Anti-α-tubulin (acetyl K40) primary antibody was used with proper dilution to probe the membranes. The process was performed for overnight at 4 °C. Next, 1×TBS buffer supplemented with tween 20 was used to wash the membrane three times. Again, the membrane was incubated with anti-mouse HRP-conjugated secondary antibody for 2 h. Luminata Forte chemiluminescence reagent was used to detect all the proteins. Densitometry was used to quantify the protein. For this experiment, α-tubulin was used as a loading control. 4.19 PC12 Cell Culture Rat adrenal pheochromocytoma cell line (PC12 cells) was purchased from NCCS Pune, India. DMEM medium supplemented with 10% horse serum (HIMEDIA) and 5% fetal bovine serum (GIBCO) were used to grow the cells under 5% CO2 at 37 oC temperature. For the differentiation of PC12 cells into neurons, 1% Horse Serum and 100 ng/mL NGF were used. For all the biological assays, we have used the differentiated neurons. 4.20 Cell Viability Assay Cell viabilities of four peptides and peptoid were checked in PC12 derived neurons using MTT reduction method. In live cells, cellular reductase enzymes reduce the MTT into formazan which is purple in colour. For dead cells, this reduction step does not occur. From



this assay, we can easily quantify the healthy (viable cells) and dead cells. For this experiment, cells were seeded in 96-well plate. After 24h of plating, the cells were treated with 6.25, 12.5, 25, 50, 100, 200 μM of peptide and peptoid and incubated for 24 h. Then, MTT solution (10 mg/mL) prepared in PBS was added into each well and further incubated for 4 h at 37 °C. Next, 1:1 (v/v) DMSO:MeOH was used to dissolve purple formazan. Then, at 570 nm the absorbance was measured using microplate ELISA reader. Using these absorbance data, the %viability of the cells were calculated following the below method.

%viability= [(A570 treated cells-A570 backgrounds) / (A570untreated cells –A570backgrounds)] X100 4.21 Microscopy and FACS study for cellular uptake of SLKP peptoid Cellular uptake of SLKP peptoid was studied in differentiated PC12 Neurons. For microscopy, cells were plated in confocal dishes and differentiated with 100 ng/mL NGF in serum free media for 5 days. The cells were then treated with 10 µM fluorescein conjugated SLKP peptoid for 2 h. Then the cells were washed, and fixed in 4% formaldehyde for 30 min. The cells were subsequently washed with PBS and then nucleus was stained with Hoechst 33258 for 30 min. The cells were again washed and observed under Olympus (IX83) microscope equipped with Andor iXon3 897 EMCCD camera. For studying cellular uptake through FACS, PC12 cells were plated in 6 well plates and differentiated using NGF for 5 days. The cells were then treated for 2 h with 20 µM, 10 µM and 5 µM fluorescein conjugated SLKP peptoid and then the cells were trypsinized and taken for cellular uptake analysis using green channel of BD LSRFORTESA flow cytometer having emission filter at 530 nm. 4.22 NGF Study


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For NGF assay, the differentiated PC12 neurons were used. For the differentiation of PC12 cell into neurons, PC12 cells were treated with NGF (100 ng/mL) and 1% horse serum for 7 days. After differentiation, neurons were treated with anti-NGF (2 g/mL) alone and antiNGF with three different concentrations of peptoid for another 20 hours. MTT assay was performed to quantify the percentage of viable cells. MTT assay was performed following the above procedure. 4.23 MTT based assay for cell rescue study PC12 cells from the rat pheochromocytoma were differentiated into the neurons using neurite growth factor. The neurons were cultured in Dulbecco’s minimum essential medium (Gibco) containing 10% horse serum and 5% fetal bovine serum. Then, the neurons were treated with A42 (5 M) in the absence or presence of SLKP peptoid and neurons were cultured for another 24 hrs at 37 °C. MTT reduction method was used to evaluate the cytotoxicity. 4.24 Serum stability of Peptoid77 Peptiod (150 M) was incubated at 37 C with 50% human serum. At different time intervals, 100 L of the incubated solutions were taken out. Then, 100 L acetonitrile was added to precipitate serum proteins and these solutions were kept in 4 oC for 30 min to stop the protease activity. Next, these solutions were centrifuged to get the supernatant and RP-HPLC was performed to analyses the serum stability with the supernatant. 4.25 Docking Autodock-Vina software (version 1.1.2)78 was used to perform blind docking of the SLKP peptoid, its counter peptide with the receptor A peptide (PDB ID: 1IYT) and Aβ42 fibril (PDB ID 5KK3)79. The H-bonding and energy were shown in the figure. 4.26 Effect of peptoid on primary cortical neuron Primary cortical neurons were cultured following the previous described method.80,81,82 Briefly, brains were isolated from of E18 embryos of timed-pregnant Sprague Dawley rat.



The isolated brain cortices were micro-dissected, digested, dissolved, filtered and suspended in MEM medium containing 10% horse serum and glucose (0.6% wt/vol). Then, confocal dishes coated with poly-D-lysine were used to culture suspended the cells with 5% CO2 environment at 37 oC for 4h. After 4h incubation, medium was changed with neurobasal media supplemented with B27. The cells were treated with the peptoid (10 µM) for 4 days. 4.27 Blood-Brain Barrier (BBB) crossing experiment of SLKP peptoid For this experiment, six mice were taken and divided in two groups (3 mice/group). 100 µL SLKP peptoid, sucrose (dosage of 10 mL/Kg body weight of mice) solution were injected intraperitoneally and for the control experiment, 100 µL saline solution was injected into the second group of mice. After 6 h, mice were anaesthetized with avertin (i.p) to sacrifice. To drain out blood from the body, transcardial perfusion was performed. After removing blood vessel and meninges the mice brains were collected in PBS. The brain cortical region was dissected out and used for analysis. Then, the cortex was homogenized in liquid nitrogen and extracted in acetonitrile. The acetonitrile solution was centrifuged to get the supernatant and mass spectroscopy was performed with the supernatant. 4.28 Data Analysis For various spectroscopic data and bar diagram calculation, Origin 8.5 pro was used. Image J software was used to analysis the various microscopic images. Tow tailed Student’s t-test and one-way ANOVA were performed to calculate statistical analysis. Statistical values were for *P≤0.05, **P≤0.03 in various experiment. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website http://pubs.acs.org. Synthetic scheme for preparation of all the peptides and peptoid; HPLC, mass chromatogram of peptides and peptoid; FT-IR spectrum of A42, SLKP peptoid and A42 with SLKP


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peptoid; ThT assay with SLKP peptide and peptoid; Docking study of SLKP peptide and peptoid with A peptide; MTT assay of the SLKP peptides and peptoid; Microscopic images of neurite out growth by SLKP peptoid in PC12 derived neurons, rat primary cortical neurons and their bar diagram for quantitative 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 Surajit Ghosh: 0000-0002-8203-8613 Prasenjit Mondal: 0000-0003-0767-449X Gaurav Das: 0000-0002-8432-5384 Abbreviation



A: amyloid beta, AD: Alzheimer’s disease, NGF: nerve growth factor, MTT: 3-(4,5Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide, PDB: Protein data bank. Author contributions K.P. carried out synthesis, purification and characterization of peptides and peptoid,

in vitro assays and computational study such as ThT assay, FT-IR, ITC, microtubule assembly assay and molecular docking. K.P. and G.D. performed various dot blot experiment and BLAST. G.D. along with V.G. executed cell based assays such as MTT assay, neuro-regeneration, intracellular microtubule imaging and western blot. P.M. guided K.P. to perform the molecular docking experiments. J.K., G.D. and VG executed the rat primary cortical neuron culture experiment and blood brain barrier experiment. S.B. supports K.P. and G.D. for performing various experiments and edited the manuscript. S.G. conceived the idea supervised the project and wrote the manuscript. Notes The authors declare no competing financial interest ACKNOWLEDGMENT The authors wish to thank Mr J. Mandal for ITC experiment. KP, SB thank UGC, GD thanks ICMR, VG and JK thank DST-Inspire and PM thanks CSIR for awarding their fellowships.


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SG kindly acknowledges SERB, India (EMR/2015/002230) for financial assistance and CSIR-IICB for infrastructure.

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Figure 1. Cartoon represents design of three tetrapeptides from a dodeca-neuropeptide [SLKPAANLPLRF (R-RFa)] isolated from the frog brain. .


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Figure 2. (A) ThT assay reveals that R-RFa, SLKP peptide has better effect in Aβ42 aggregation than AANL and PRLF peptides. (Error bar corresponds to standard deviation of the value *p

Discovery of Neuro-regenerative Peptoid from Amphibian

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