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Article
Power of Tyrosine Assembly in Microtubule Stabilization
and Neuroprotection Fuelled by Phenol Appendages
Surajit Barman, Gaurav Das, Prasenjit Mondal, Krishnangsu Pradhan,
Debmalya Bhunia, Juhee Khan, Chirantan Kar, and Surajit Ghosh
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00497 • Publication Date (Web): 19 Dec 2018
Downloaded from http://pubs.acs.org on December 20, 2018

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Page 1 of 37 ACS Chemical Neuroscience

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Power of Tyrosine Assembly in Microtubule
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Stabilization and Neuroprotection Fuelled by Phenol
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Appendages
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17 Surajit Barman,1 Gaurav Das,1, 2 Prasenjit Mondal, 1, 2 Krishnangsu Pradhan,1 Debmalya Bhunia,1
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19 Juhee Khan,1 Chirantan Kar,1 Surajit Ghosh1, 2 *
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22 1. Organic & Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, 4, Raja
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25 S. C. Mullick Road, Jadavpur, Kolkata-700032, West Bengal, India. Fax: +91-33-2473-
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27 5197/0284; Tel: +91-33-2499-5872
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29 2. Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical
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Biology Campus, 4 Raja S. C. Mullick Road, Kolkata 700032, India.
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36 ABSTRACT: Microtubules play crucial role in maintenance of structure, function, axonal
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38 extensions, cargo transport and polarity of neurons. During neurodegenerative diseases,
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microtubule structure and function gets severely damaged due to destabilization of its major
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43 structural proteins. Therefore, design and development of molecules that stabilize these
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45 microtubule networks have always been an important strategy for development of potential
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neurotherapeutic candidates. Towards this venture, we designed and developed a tyrosine rich
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50 tri-substituted triazine molecule (TY3) that stabilizes microtubules through close interaction with
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52 the taxol binding site. Detailed structural investigations revealed that the phenolic protons are the
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54 key interacting partners of tubulin. Interestingly, we found that this molecule is non-cytotoxic in
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3 PC12 derived neurons, stabilized microtubules against nocodazole induced depolymerization and
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6 increases expression of acetylated tubulin (K-40), an important marker of tubulin stability.
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8 Further, results show that TY3 significantly induces neurite sprouting as compared to the
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10 untreated control as well as the two other analogues (TS3 and TF3). It also possesses anti-Aβ
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fibrillation property as confirmed by ThT assay, which leads to its neuro-protective effect against
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15 amyloidogenic induced toxicity caused through NGF deprivation in PC12 derived neurons.
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17 Remarkably, our result reveals that it reduces the expression of pY490 TrkA associated with
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NGF deprived amyloidogenesis, which further proves that it is a potent amyloid beta inhibitor.
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22 Moreover, it promoted the health of the rat primary cortical neurons through higher expression of
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24 key neuronal markers such as MAP2 and Tuj1. Finally, we observed that it has good serum
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26 stability and has the ability to cross the blood brain barrier (BBB). Overall, our work indicates
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29 the importance of Phenolic –OH in promoting neuroprotection and its importance could be
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31 implemented in the development of future neuro-therapeutics.
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34 KEYWORDS:
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36 Triazine, Microtubule, FRET, STD NMR, TR-NOESY, Neuroprotection, Blood Brain-Barrier
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1. INTRODUCTION
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43 The embellished shape of neuron and its polarity has been a centre point of interest for wide
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45 range of researchers in the field of neurobiology for the last several decades.1-3 To maintain its
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47 incredible structure and polarity several factors play crucial role and microtubules are one of the
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most important players.4-7 Microtubules (MT) are dynamic polymers of α, β-tubulin heterodimers
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52 that along with actin and intermediate filaments forms the key cytoskeletal components of the
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54 eukaryotic cells including neurons.1-7 Along with other eukaryotic cells, microtubules have
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3 special importance in neurons where apart from providing them the much-needed shape and
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6 structure, they also have important functional aspects.8 In neurons, microtubules are involved in a
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8 variety of tasks like cargo transport through gigantic axonal projections with the aid of the
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10 associated molecular motors.9 This role of axonal transport in neurons is so significant that even
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slightest perturbations in the microtubule structure or functions create severe damage to the
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15 normal neuronal physiology as observed in several neurodegenerative diseases.10 In the axons,
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17 the linear arrays of microtubules comprising of two ends- plus ends towards synapse and minus
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ends towards cell body provides directionality and structural support to the neurons.11, 12 Many
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22 studies have indicated that neurodegenerative diseases like Alzheimer’s Disease (AD) have been
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24 caused due to defects in axonal microtubules and this has given rise to the importance of MT-
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26 stabilizing molecules.13-19 During AD progression, microtubule networks in neurons is severely
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29 damaged due to disintegration of microtubule associated protein Tau, which further aggregates
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31 and generates neurofibrillary tangles (NFT). Moreover, it has been also reported that Aβ
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33 aggregation damages the microtubule network. Many microtubule stabilizing molecules have
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been so far reported to have shown promising effects in restoration of microtubules in axonal
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38 transport as well as improving cognition in animal models. As a result, such compounds may be
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40 considered as potential candidates for the treatment of AD and related taupathies. Microtubule
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stabilising agents (MSA) accelerates the equilibrium from tubulin to the polymeric microtubule
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45 and promotes tubulin polymerization. MSA’s are already well established chemotherapeutic
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47 agents like taxol but the action of these neuro-protective microtubule stabilizing agents are
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49 somewhat different as their order of stabilization is not so pronounced as to arrest the cell
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52 division and cause cell death.20-22 Though a number of neuroprotective MT-stabilizing agents
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54 have been reported, most of them so far have produced clinically insignificant results. Moreover,
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3 many naturally occurring and synthesized molecules with phenolic -OH moiety like in
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6 neurotransmitter molecules such as dopamine and Ferulic acid have been reported to possess
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8 neuroprotective effects23-27 but none of the studies directly or indirectly have explored the
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10 importance of phenolic -OH in neuroprotection and their possible cross talk with tubulin or
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microtubule. Talking about phenolic –OH, several groups have already reported tyrosine
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15 analogues with effective biological activity and is one of the hot arenas in current research
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17 among the chemical biologists. 28-30 To address this fundamental aspects and to provide more
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detailed understanding in this manuscript, we have designed novel tripodal molecules based on
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22 1, 3, 5-triazine core with tri-substitution by tyrosine (Y), phenylalanine (F) and serine (S) named
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24 as TY3, TF3 and TS3 respectively. Among the three molecules, TY3 and TF3 binds close to the
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26 taxol binding pocket of the tubulin. Next, the identification of pharmacophore of TY3 for the
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29 interaction with tubulin was investigated through STD NMR and TR-NOESY experiment.
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31 Further, it was observed that TY3 confers significant microtubule stabilization in PC12 derived
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33 neurons against nocodazole induced depolymerization, increases the expression of acetylated
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tubulin (K-40) and facilitates neurite outgrowth, while TF3 and TS3 does not show any activity
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38 in neurons. Interestingly, we found that TY3 also inhibits Aβ fibrillation and confers significant
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40 neuroprotection against Aβ mediated toxicity in PC12 derived neurons. It has been reported
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earlier that NGF deprivation is associated with an increase in the expression of a particular
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45 phosphorylation of TrkA (pY490).31 We found that TY3 reduces this TrkA associated
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47 phosphorylation, thus reinstating the fact that it has anti-amyloidogenic activity. Moreover, we
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49 observed that TY3 crosses the blood-brain barrier, shows significant serum stability and
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52 promotes healthy morphology of the cultured primary rat cortical neurons by upregulation of
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54 neuronal markers (Tuj1 and MAP2). Overall, results reveal that our newly designed molecules
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3 confers significant microtubule stabilization and neuroprotection, where spatially positioned
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6 phenolic-OH group interacts close to the taxol binding pocket of tubulin and plays an important
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8 role. We strongly believe that this novel tripodal molecule with tyrosine substitution has
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10 significant potential to serve as a unique neurotherapeutics template.
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16 2. RESULTS AND DISCUSSION
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19 2.1. Synthesis of triazine based small molecules.
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22 We have synthesized three triazine based compounds (TS3, TF3 and TY3) and characterized
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24 them by ESI-mass spectroscopy, 1H-NMR, 13C-NMR, DEPT-135 and HPLC (Figure 1A and S1-
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10). The fluorescein attached TF3 and TY3 were synthesized by solid phase peptide synthesis
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29 (SPPS) method using CEM, purified them by HPLC and characterized by MALDI-TOF mass
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31 spectroscopy (Figure S11, 12).
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34 2.2 Microtubule assembly assay.
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To check whether our compounds have any interaction with tubulin, we have performed
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40 microtubule assembly assay using the change in fluorescence intensity of 4', 6-diamidino-2-
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42 phenylindole (DAPI) as an indicator during tubulin polymerization.32, 33 Remarkably, we found
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44 that fluorescence intensity of DAPI increased in case of TF3, TY3 as compared to untreated
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47 control (tubulin alone). But, there was no significant change in fluorescence intensity in case of
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49 TS3. Next, we performed microtubule assembly assay of the TF3 and TY3 in dose dependent
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51 manner (Figure 1B, D and S13). These results clearly shows that TF3 and TY3 are promoting the
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tubulin polymerization.
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3 2.3 Determination of binding constant by Surface Plasmon Resonance (SPR) study.
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Above results encouraged us to further explore the affinity of TF3 and TY3 with tubulin and
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9 their association and dissociation kinetics. For this, we performed Surface Plasmon Resonance
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11 (SPR)34 for TF3 and TY3. The SPR studies suggest that the association constant (Ka) for TF3
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13 and TY3 are 1.42 × 103 M-1, 1.05 ×103 M-1 respectively and the dissociation constant of TF3 and
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16 TY3 are 158 μM and 16.4 μM respectively (Figures 1 C, E).
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19 2.4 Determination of binding affinity using tryptophan fluorescence quenching study.
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22 Next, we have performed the tryptophan fluorescence quenching study to determine the binding
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24 affinity with tubulin. Binding affinity of TF3 and TY3 were determined using modified Stern-
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Volmer equation. The binding constants of TF3 and TY3 are 2.68 × 103 M-1 and 7.75 × 103 M-1
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29 respectively (Figure S14). These results are in agreement with the association constant measured
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31 through previous SPR experiments.
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34 2.5 Förster Resonance Energy Transfer (FRET) study to determine the binding location in
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37 tubulin.
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40 To know more in details about the binding location of tubulin, we performed Förster Resonance
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42 Energy Transfer (FRET) experiment using these two compounds (TF3 and TY3). Here, we used
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44 three florescent probes (colchicine, 8-anilinonaphthalene-1-sulfonic acid (ANS) and TAMRA-
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47 E3NAP) as FRET partner with fluorescein tagged TF3 and TY3. The binding sites of colchicine,
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49 ANS and TAMRA-E3NAP in tubulin are well understood and the Förster distance (R0) between
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51 tubulin bound colchicine and fluorescein dye attached peptide is 29.5 ± 1 Å, tubulin bound ANS
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and fluorescein-peptide is 50.3 ± 2 Å and tubulin bound TAMRA-peptide with fluorescein-
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56 peptide is 62.9 ± 2 Å.35, 36 We performed the FRET experiment with the above FRET partners
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3 with TY3 compound resulting in a distance of 34.64 ± 1 Å, 53.4 ± 1 Å and no FRET with
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6 TAMRA-E3NAP complex (Figure 2A-C). Similarly, when we performed FRET experiments
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8 with TF3 and the above FRET partners, we found similar result like TY3 (34 ± 1 Å from
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10 colchicine site, 54 ± 1 Å from ANS site and no FRET between F-TF3 with TAMRA-E3NAP
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complex) (Figure 2D-F). Then we calculated the distance obtained from the FRET results that
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15 revealed TY3 and TF3 binds close to the taxane binding site of tubulin (Figure 2G).
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18 2.6 Conformational analysis and subsequent docking study.
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21 We performed molecular docking to know the binding affinity and interacting partners of TY3
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and TF3 at taxol pocket using DFT minimized conformers of TY3 and TF3 molecules (Figure
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26 S15-25). The docking results reveal that the interacing partners of TY3 are ARG278, ARG284,
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28 PRO274, ARG320 and GLY370 with binding affinity of -7.7 kcal/mol and for TF3 are GLY370,
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30 PRO274, THR276 and LEU371 with binding affinity of -7.9 kcal/mol (Figure 2H, I and S26).
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33 TY3 and TF3 are shown in docking image bound to their respective binding sites in tubulin
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35 which are shown in Figure 2J.
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38 2.7 STD NMR and TR-NOESY experiments.
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41 In order to obtain more minute details of their molecular recognition, STD and TR-NOESY were
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performed to derive a tubulin-bound conformer of TY3. First, we have assigned different protons
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46 of TY3 using 1D NMR (1H-NMR, 13C-NMR and DEPT study) and 2D NMR experiments
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48 (COSY, HSQC, HMBC and NOESY) (Figure S27-30). To study more in details, we have
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50 performed saturation transfer difference (STD) and transfer nuclear overhauser spectroscopy
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53 (TR-NOESY) experiment. These studies are useful for the epitope mapping of a molecule with a
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3 indicate the close proximity of the epitope to the protein. The STD results revealed that aromatic
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6 protons of ligands TY3 are showing the highest STD effect while less signal for the aliphatic
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8 group was observed (Figure 3A-C). From this result, we can conclude that aryl moiety is
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10 essential for its interaction with tubulin.
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13 In TR-NOESY experiment, the negative cross peaks were detected in the presence of tubulin.
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16 We have deduced the tubulin bound conformation of TY3 where two aromatic protons of TY3
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18 (ArH6.67 and ArH6.97) are in close proximity to the –OMe,-CH2 and -CH protons (Figure 4A).
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20 Further, we deduced the plausible conformation of tubulin bound TY3. For this, we performed
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23 the molecular docking study with TY3 using DFT energy minimized conformer of TY3. The
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25 results of molecular docking of TY3 are in agreement with the TR-NOESY data (Figure 4B, C
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27 and S31). The STD NMR and TR-NOESY experiments clearly shows that the phenyl moiety is
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crucial for its interaction with tubulin.
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33 2.8 Cell viability assay
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36 So far we have performed several in vitro assays that indicate the microtubule stabilizing nature
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38 of TY3 and TF3. In order to perform further cell based screening, it is pertinent to confirm their
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40 cyto-toxic nature. Hence, we performed the cell viabilty assay using differentiated PC12 cell
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43 lines upto 200uM concentrations of both TY3 and TF3. The results revealed that all the
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45 compounds are non-cyto-toxic to the differentiated neurons (Figure 5A, B).
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48 2.9 Nocodazole induced depolymerization and expression of Acetylated Tubulin (AcK-40)
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51 Previous in vitro studies like SPR and microtubule assembly assay using DAPI have proven TY3
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and TF3 both to be microtubule polymerizing compound. Now, it was to be tested, whether it
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3 derived neurons by 4 h treatment with 10 µM nocodazole, the cells were washed free of the
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6 nocodazole and divided into three groups one being control replaced with only fresh medium
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8 while the other two with TY3 and TF3. After 2 h of treatment, the cells were fixed and
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10 immunostained with primary antibodies against alpha-tubulin. The cells treated with TY3
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showed healthy microtubule network completely recovered from the nocodazole treatment while
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15 the control cells and TF3 treated cells still suffered from the depolymerizing effect of nocodazole
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17 (Figure 5C and S32). This proves that our cellular data is in line with our in vitro observation and
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TY3 is indeed a microtubule stabilizer. The treated cells also showed a higher expression of
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22 acetylated tubulin (AcK 40), a known marker of tubulin stabilization compared to the untreated
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24 control (Figure 5D, E and S33).
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27 2.10 TY3 promoted significant neurite outgrowth among the other screened molecules.
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30 Many microtubule stabilizing drugs like Methyl 3, 4-dihydroxybenzoate and GIT 1 have also
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33 shown to promote neurite outgrowth.37, 38 All these factors prompted us to check the neurite
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35 outgrowth potential of our microtubule stabilizing compounds TY3 and TF3. When these
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37 compounds were incubated with undifferentiated PC12 cells, TY3 showed neurite outgrowth
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after 24 h of incubation while the others (TF3 and TS3) failed to do so (Figure 6A, B). This
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42 above results motivated us to conduct more studies for TY3 in neurons. It was also seen that TY3
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44 promoted the healthy morphology and architecture of microtubule network (Figure 6 C, D). It is
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important to note that although TY3 binds close to the taxol binding site, yet it does not cause
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49 toxicity due to the fact that it binds at the taxol binding site 3428 fold times weaker and also
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51 dissociates in a faster rate (Kd=16.4 μM) compared to taxol.39 Due to this reasons, TY3 can
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53 provide microtubule stabilization through weak binding as reported before.34
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3 2.11 ThT assay indicates that TY3 inhibits Aβ (1-42) aggregation
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We initially performed ThT assay with TY3 and incubated it with Aβ (1-42). To our
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9 astonishment, TY3 inhibited Aβ (1-42) aggregation (Figure 7A) and this proves that TY3 would
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11 be able to show significant neuroprotective effect when tested in some cellular model of
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13 neurotoxicity. For this, it was very important to first ascertain the fact that our compound TY3 is
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16 non-toxic to the neurons. We have already screened TY3 on PC12 derived neurons with a wide
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18 range of concentrations and it was observed that TY3 is completely non-toxic to the neurons at
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20 even very high concentrations which paved way for the future studies in cell.
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2.12 TY3 confers neuroprotection in NGF deprived PC12 derived neurons by reducing the
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26 phosphorylation of TrkA.
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29 It is now well documented that NGF deprivation in PC12 derived neurons leads to
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31 amyloidogenesis40 and that may cause an unexpected phosphorylation of TrkA at Y490, thus
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activating phospholipase C  (PLC) associated cell death pathway causing severe neuro-
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36 toxicity. This phosphorylation is usually connected with two kinases namely Src and CDK5
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38 which are known to be involved in amyloidogenesis and  and  mediated p75 processing. Our
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41 in vitro ThT assay data encouraged us to pursue the neuroprotective potential of TY3 in this
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43 NGF deprived model of PC12 derived neurons. We observed that TY3 confers neuroprotection
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45 upto 1.5 µM concentrations (Figure 7B-E) and causes inhibition of the neuronal death inducing
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TrkA phosphorylation (pY490). But no change in the expression of TrkA was observed (Figure
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50 7F, G and S34), which denotes that TY3 specifically inhibits amyloidogenesis. Here, we would
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52 like to point out that TF3 and TS3 does not show any neuroprotection ability although TF3
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3 contains phenyl group and TS3 contains -OH group. This fact clearly indicates that combination
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6 of both phenyl and -OH group that is phenol group is essential to confer neuroprotection.
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9 2.13 TY3 promotes the healthy morphology of cultured primary cortical neurons and upregulates
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11 several neuronal markers.
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14 All the cellular studies concerning TY3 have been so far studied in PC12 derived neurons but
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what about its effect on primary neurons. On treating the rat primary cortical neurons with TY3
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19 for 2 days, we observed that TY3 was well tolerated by them and also promoted the healthy
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21 morphology of the neurons (Figure 8A-C) when compared with the untreated control. Moreover,
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we observed significant increase in the expression of important neuronal markers MAP2 and
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26 Tuj1 in TY3 treated neurons as revealed from the immunoblots (Figure 8D, E and S35).
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29 2.14 Serum stability of TY3 performed by high-performance liquid chromatography (HPLC).
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32 For any candidate to qaulify as a neuroprotective drug, it must cross the blood brain barrier
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34 (BBB). But before performing the blood brain barrier crossing, we checked the stability of TY3
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37 in serum. TY3 was incubated with serum up to 24 h and monitored using HPLC. It was observed
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39 that more than 40% TY3 remained stable after 24 h of incubation with serum. This result
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41 suggests significant serum stability of TY3 (Figure S36).
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44 2.15 Blood-Brain Barrier (BBB) crossing experiment.
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Since TY3 showed serum stability, we finally needed to check whether it can cross the blood
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50 brain barrier (BBB). To address this, we have performed the MALDI-Mass spectroscopy of brain
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52 extract obtained from mice treated with TY3. The MALDI-Mass spectrum showed molecular
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54 peaks at (M=661 Da) and (M+K+ =699 Da). This data indicated the presence of TY3 in the brain
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3 extract, which confirmed its potential to cross the blood-brain barrier (BBB) (Figure 8F and S37,
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6 38). Hence we can now consider this as a promising neurotherapeutic candidate.
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9 3. Conclusion
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12 In summary, we have shown in this paper the power of phenolic –OH appendages in
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14 neuroprotection and neurite outgrowth through its interaction with microtubule. Our results
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reveal that tyrosine substitution on a tripodal platform plays an important role where this phenol
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19 group acts like a key player. Moreover, we found that phenolic –OH group interacts with tubulin
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21 close to the taxol binding pocket by FRET and NMR where the aromatic substituent binds in the
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hydrophobic pocket. Interestingly, we found that TF3 and TS3 did not have any significant effect
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26 on microtubules and that further indicated that the phenolic –OH moieties are the key partners
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28 for interaction with tubulin. The cellular assays in PC12 derived neurons and primary rat cortical
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30 neurons also reciprocated similar results. Moreover, it also showed serum stability and crossed
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33 blood brain barrier. Finally, the tripodal core substituted with the phenolic –OH group in TY3
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35 could serve as a potential candidate for the development of future neuro-therapeutics.
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38 4. Experimental Section
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41 4.1 Chemicals
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44 Cyanuric chloride, Tyrosine methyl ester, Phenyl alanine methyl ester and Serine methyl ester,
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47 Thiazolyl Blue Tetrazolium Bromide (MTT), Kanamycin sulfate, Dulbecco’s Modified Eagle’s
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49 Medium (DMEM), Guanosine 5′-triphosphate sodium salt hydrate (GTP), 4, 6-diamidino-2-
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51 phenylindole (DAPI), Trypsin-EDTA solution, Colchicine, 5(6)-carboxyfluorescein, PIPES, Eth-
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ylene glyolbi(2-aminoethyl ether)-N,N,N′,N′-tetra acetic acid (EGTA), N,N′-
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56 Diisopropylcarbodiimide (DIC), 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS) and
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3 Cell culture grade DMSO were purchased from Sigma-Aldrich. Sodium chloride, Sodium
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6 hydrogen carbonate, Di-sodium hydrogen phosphate dihydrate, Potassium hydroxide,
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8 Magnesium chloride hexahydrate, Trifluoroacetic acid (TFA) and Potassium dihydrogen
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10 phosphate were purchased from Merck. Potassium chloride and Agarose were purchased from
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Fisher Scientific. Dimethyl sulfoxide, N, N’-Diisopropylethylamine (DIPEA), Ethyl acetate,
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15 Chloroform and Hexane were purchased from Spectrochem. Triton-X-100 was purchased from
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17 SRL. Amyloid-beta 1-42 (Aβ42) was purchased from AnaSpec. NGF-β (7S) was purchased from
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Sigma (St. Louis, MO, USA). Neurobasal media, B27, glutaMAX and Pen/strep were bought
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22 from Gibco, Life technologies. 2-[4-(2-Hydroxyethyl) piperazin-1-yl] ethanesulfonic acid
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24 (HEPES) and Horse Serum were purchased from Himedia. Penicillin-Streptomycin and Fetal
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26 bovine serum were purchased from Invitrogen. Anti-alpha Tubulin clone EP 1332Y, Anti-
27
28
29 Tubulin Anti-body, Beta III isoform (Tuj 1) were purchased from Merck Millipore. Anti-TrkA
30
31 (pY490), Anti-TrkA and Anti-alpha Tubulin (acetyl K-40) were purchased from Abcam.
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33 Bisbenzimide H 33258 (hoechst) was purchased from Calbiochem. MAP2 Monoclonal Antibody
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35
36
(M13) was purchased from Thermofisher scientific. HPLC-grade H2O and ACN were purchased
37
38 from J.T. Baker. All the NMR solvents were purchased from Cambridge isotope. All compounds
39
40 were used without further purification.
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42
43 4.2 Cell culture
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45
46
Pheochromocytoma of the rat adrenal medulla (PC12) were purchased from NCCS, Pune (India).
47
48
49 The PC12 cells were cultured in complete DMEM with 10% horse serum and 5% FBS. The
50
51 differentiation of PC12 cells into neurons was achieved by culturing the cells in serum free
52
53 medium with 1% horse serum and 100 ng/mL NGF (Nerve Growth Factor) for 5 days.
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3 4.3 Protein purification
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After the isolation of tubulin from goat brain, we purified it by two cycle’s polymerization and
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9 depolymerization procedure followed by storing it in liquid nitrogen cryochamber using 10%
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11 glycerol.41
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14 4.4 Synthesis, characterization of TY3, TS3 and TF3 and its fluorescein derivatives.
15
16
17 In 100mL round bottom flask, methyl ester of amino acids (4 equivalents) and cyanuric chloride
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19
20 (1 equivalent) in dry acetonitrile was stirred at room temperature. Then N, N′-
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22 Diisopropylethylamine (DIPEA) was added to the reaction mixture. The reaction mixtures were
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24 refluxed overnight under inert atmosphere. Finally, the compounds were purified by column
25
26
27
chromatography and characterized by ESI-mass spectroscopy, 1H-NMR, 13C-NMR, and DEPT-
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29 135. Fluorescein tagged triazine derivatives (F-TY3 and F-TF3) were synthesized using solid
30
31 phase peptide synthesis (SPPS) method in a microwave-based (35 watt) automated peptide
32
33
synthesizer (CEM, Liberty 1). All the synthesized fluorescein tagged small molecules were
34
35
36 purified through C-18 reverse phase HPLC column at 210 nm and characterized by MALDI-
37
38 TOF in acetonitrile and water (1:1) mixture.
39
40
41 4.5 Conformational Analysis.
42
43
44 We deduced the energy minimized conformers of the TY3 and TF3 molecules using
45
46
47 Spartan’1642-45 and Gaussian ’09 program46. Geometry optimizations of each molecules were
48
49 performed in multistep.
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51
52 In first step 1000 most probable conformers were chosen and a conformational search was
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54 performed to predict their corresponding energies using molecular mechanics force field
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3 (MMFF). In the next step 100 lowest energy conformers chosen from the MMFF calculation
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6 were further refined by semi-empirical calculation at PM6 level. Next, the 20 lowest energy
7
8 conformers taken after the semi-empirical calculation were optimized applying Hartree-Fock
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10 method at 3-21G level.
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12
13 4.6 Docking study
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15
16
17
We performed blind docking study using the software Autodock-Vina version 1.1.2.47 98×60×64
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19 affinity grids were centered on the tubulin [PDB ID: 1Z2B]48 for docking TY3 and TF3. All the
20
21 images were visualized and developed in PyMOL (The PyMOL Molecular Graphics System,
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Version 1.7.4 Schrödinger, LLC.)
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25
26
27
4.7 Microtubule assembly assay
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29
30 A stock solution of 10 μM DAPI in BRB80 buffer containing 100 μM tubulin, 10 mM GTP and
31
32 different concentration of TF3 and TY3 were mixed. Emission spectra of solutions were
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34 recorded in region ranging from 400 nm to 600 nm wavelength at 37 0C after excitation at 355
35
36
37 nm.
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39
40 4.8 Surface Plasmon Resonance (SPR) experiment
41
42
43 The SPR experiment was performed using NTA biosensor chip, Ni2+ and streptavidin-His6 were
44
45 immobilized on to the NTA biosensor chip. Next, we washed the surface with BRB80 buffer and
46
47
flowed biotinylated tubulin (20 μg/mL) followed by incubation for 15 min. The surface was
48
49
50 washed and desired compounds were added at various concentrations as analytes. The recorded
51
52 data were analyzed by plotting the curve with a local fitting.
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54
55 4.9 Tryptophan fluorescence quenching experiment
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3 Tubulin was mixed with different concentrations of TF3 and TY3 separately in BRB80 and these
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6 mixtures were incubated for 40 min at 25 °C. Then, the intrinsic tryptophan fluorescence data
7
8 was recorded and calculated by modified Stern-Volmer equation.
9
10
11 4.10 Förster resonance energy transfer (FRET) experiment
12
13
14 We performed the FRET study to know the binding pocket of TF3 and TY3 in the tubulin.
15
16
17
Tubulin-colchicine, tubulin-ANS and tubulin-TAMRA-E3NAP complexes were used to find out
18
19 the distance of fluorescein attached small molecules from their specific binding pocket in tubulin
20
21 following previously described method. The excitation and emission ranges were set at 355 nm
22
23
and 450 to 650 nm respectively.
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25
26
27
4.11 STD NMR sample preparation and experiments49, 50
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29
30 The samples of the ligands were prepared with a 300 μM concentration of the TY3 and 10 μM of
31
32 tubulin in D2O, 10 mM NaPi, 0.1 mM GTP. Small amount of D6-DMSO were used to solubilize
33
34 the TY3 ligand . We recorded the STD-NMR by Bruker AVANCE 600 MHz spectrometer with
35
36
37 saturation time 2s. Percentage of relative STD effects was calculated by the following equation
38
39
40 ASTD = (I0 - Isat)/I0 = ISTD/I0 where intensity of the signals in the STD NMR spectrum (ISTD)
41
42 with signal intensities of a reference spectrum (I0). The maximum STD signal was considered as
43
44 100% STD effect and others were calculated relatively.
45
46
47
4.12 TR-NOESY experiments
48
49
50
51 Strong negative NOE cross peaks were detected by TR-NOESY experiments (mixing times of
52
53 200 ms) in presence of tubulin receptor compared to the free state of the TY3 ligand. All the
54
55 datas were calculated using Mnova software.
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3 4.13 Neurite outgrowth experiment
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For neurite outgrowth experiment, all the three triazine derivatives were treated with the
8
9 undifferentiated PC12 cells and incubated for 24h at 370C.Then the treated cells were imaged
10
11 under a microscope at DIC mode. Evaluation of neurite length was carried out using the CellSens
12
13 software.
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15
16
17
4.14 Effect of TF3 and TY3 on neuronal microtubule
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19
20 PC12 cells (density~3000-5000) were grown in a confocal dish on the day before the treatment.
21
22 We treated TY3 for 24 h. Next day, cells were fixed using 4% paraformaldehyde and incubated
23
24 with 5% BSA and 0.2% triton-X in PBS for 1 h. Then we washed the fixed cells with PBS and
25
26
27
treated with primary antibody polyclonal anti-α-tubulin IgG antibody. After 2 h, we treated
28
29 secondary antibody (Cy3.5 pre-absorbed goat anti-rabbit IgG) after washing the cells with PBS
30
31 and kept for 2 h. On the day of the imaging of the cells, cells were washed with PBS and treated
32
33
with Hoechst 33258 (1 μg/mL) for half an hour. Images of various zone of culture dish were
34
35
36 captured using microscope.
37
38
39 4.15 Thioflavin T (ThT) assay
40
41
42 Thioflavin-T (ThT) is used to identify the amyloid fibrils both in vitro and in vivo. There is an
43
44 enhancement of its fluorescence intensity upon binding with the fibrils. To monitor the Aβ
45
46
47 peptide aggregation, we performed the following experiment.51 Different concentrations of the
48
49 TY3 were mixed with 10 μM Aβ42 peptide and incubated for 48h. Then, we added ThT solution
50
51 to the incubated solution and recorded the fluorescence in PTI QM-40 spectrofluorimeter
52
53
54
(excitation: 435 nm and emission: 460-650 nm).
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3 4.16 Neuroprotection study using differentiated PC12 cells
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For the event of differentiation, PC12 cells were cultured for 24 h and treated with serum free
8
9 DMEM containing 1% horse serum and 100 ng/mL NGF and incubated for 5 days.
10
11 Differentiated PC12 cells were seeded in 96-well plates (1 × 104 cells/mL) and then cells were
12
13 treated with anti-NGF alone and along with different concentrations of TY3 for 24 h. MTT
14
15
16 solution were added to each well and incubated at 37oC for 4 h. Then the MTT solutions were
17
18 replaced by 1:1 DMSO-MeOH. Then the percentage of cell viability was analyzed using
19
20 microplate ELISA reader (Thermo; Multiskan GO Microplate Spectrophotometer) at 550 nm
21
22
23 wavelength.
24
25
26 4.17 Effect of TY3 on primary cortical neuron culture
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28
29 Primary cortical neurons were cultured by previously described protocol.52 In brief, we have
30
31 received timed-pregnant Sprague Dawley rat. Then we isolated the brain from the dissected E18
32
33
embryos. After the micro-dissection of the brain, cortices were dissected out in the MEM
34
35
36 medium containing 10% horse serum and glucose (0.6% wt/vol). ~3-5 × 105/mL cells were
37
38 plated on confocal dishes coated with poly-D lysine and was incubated in CO2 incubator for
39
40 about 4 h. Then this culture medium was changed with neurobasal media supplemented with
41
42
43 B27, Pen/Strep and GlutaMAX and cultured for another 14 days untill the neurons matured. We
44
45 treated these neurons with TY3 and assessed its effect on primary cortical neurons.
46
47
48 4.18 Serum stability
49
50
51 Stability of any small/large molecules is important for their biological aspects.53 For this
52
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54
purpose, we checked the stability of TY3 in horse serum. Quantitative stability of the compounds
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3 was monitored by C18 reverse phase HPLC system upto 24 h. It was found that almost 40% of
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6 the TY3 remained intact after 24 h.
7
8
9 4.19 Blood-Brain Barrier (BBB) crossing experiment. 54, 55
10
11
12 C57BL/6J female mice of around 20 g have been used for our study. Animals were divided into
13
14 two groups (3 mice /group) and the mice were injected intraperitoneally with TY3 dissolved in
15
16
17
saline solution at the concentration of 50 µg/Kg body weight of mice. For control, mice were
18
19 treated with only saline solution (2nd group). After 6 h of treatment, animals were deeply
20
21 anaesthetized with ketamine (i.p) and were sacrificed by transcardial perfusion. The mice brains
22
23
were isolated and directly transferred into liquid nitrogen, crushed using a mortar and pestle.
24
25
26 Acetonitrile and water mixture (1:1) was added into the crushed brain. Mixture was centrifuged
27
28 to separate out the soluble part. HPLC and mass analysis were performed to analyze the data and
29
30 for the identification of TY3 mass.
31
32
33
ASSOCIATED CONTENT
34
35
36 Supporting Information
37
38 This materials are available free of charge via the internet at http://pubs.acs.org.
39
40 Synthesis and characterization of TS3, TF3, TY3, F-TF3 and F-TY3 using 1H-NMR, 13C-NMR,
41 DEPT-135 and mass spectroscopy. HPLC chromatograms, Microtubule assembly assay,
42
43
Quenching of Tryptophan fluorescence experiment, DFT study of tri-substituted triazine
44 derivatives. 2D NMR (COSY, NOESY, HSQC and HMBC) of TY3 and TF3. Control
45 experiments for TR-NOESY and BBB experiment. Serum stability of TY3. Immunoblots of
46
47
AcK-40, phospho-TrkA (pY490), TrkA, MAP2 and Tuj1. (PDF)
48
49 AUTHOR INFORMATION:
50
51 Corresponding Author
52
53 Dr. Surajit Ghosh
54
55
56 Principal Scientist
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3 Organic and Medicinal Chemistry Division
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6 CSIR-IICB, Jadavpur, Kolkata-700 032, India
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8 Tel: +91-33-2499-5872
9
10 Fax: +91-33-2473-5197/0284
11
12
13
E-mail: sghosh@iicb.res.in
14
15 ORCID ID
16
17 Surajit Barman: 0000-0003-3584-4716
18
19
Gaurav Das: 0000-0002-8432-5384
20
21
22 Prasenjit Mondal: 0000-0003-0767-449X
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24 Surajit Ghosh: 0000-0002-8203-8613
25
26
27
28
29 Author contributions
30
31 SB performed synthesis, characterization, major experiments and analyzed the data. GD
32
33 performed various cell based assays like neurite outgrowth, microtubule associated
34
35
36
depolymerization and the immunoblots. PM and KP performed FRET, computational experiment
37
38 and in vitro inhibition of Aβ aggregation. DB helped GD and JK in performing the BBB
39
40 experiment. JK and GD performed the primary neuron culture. CK performed the DFT
41
42
calculations .SG conceived the idea, designed and monitored all the experiments, analyzed the
43
44
45 data, prepared figures and supervised GD and SB in writing the manuscript.
46
47 Notes
48
49 The authors declare no competing financial interest
50
51
52 ACKNOWLEDGMENT
53
54 We thank Ms. Varsha Gupta for critically reading the manuscript. We also want to thank Dr. E.
55
56 Padmanban for helping in STD and TR-NOESY experiments and Mr. K Suresh Kumar for SPR
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3 experiments. SB, KP thanks UGC, PM thanks CSIR, DB, JK thanks DST and GD thanks ICMR,
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5
6 India for their fellowships. CK thanks SERB for his postdoctoral fellowship. SG kindly
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8 acknowledges DST, India for providing support (EMR/2015/002230) and CSIR-IICB for
9
10 infrastructure. Animal experiments were performed following IICB ethical guidelines.
11
12
13 Reference:
14
15 1. Craig, A. M., and Banker, G. (1994) Neuronal polarity. Annu. Rev. Neurosci., 17, 267-310.
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33 M., Hirata, S., Hsu, C.P., Ishikawa, N., Florian, J., Warshel, A., Johnson, B. G., Gill, P. M. W.,
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46 48. Gigant, B., Wang, C., Ravelli, R.B., Roussi, F., Steinmetz, M.O., Curmi, P.A., Sobel, A., and
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48 Knossow, M. (2005) Structural basis for the regulation of tubulin by vinblastine. Nature, 435,
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55 and molecular modelling insights into interaction of novel mannose-based ligands with DC-
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14 51. Biswas, A., Kurkute, P., Saleem, S., Jana, B., Mohapatra, S., Mondal, P., Adak, A., Ghosh, S.,
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16 Saha, A., Bhunia, D., Biswas, S. C. and Ghosh, S. (2015) Novel hexapeptide interacts with
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18 tubulin andmicrotubules, inhibits Aβ fibrillation, and shows significant neuroprotection. ACS
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27 53. Bhunia, D., Mondal, P., Das, G., Saha, A., Sengupta, P., Jana, J., Mohapatra, S., Chatterjee, S.,
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29 and Ghosh, S. (2018) Spatial Position Regulates Power of Tryptophan: Discovery of a Major-
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31 Groove-Specific Nuclear-Localizing, Cell Penetrating Tetrapeptide. J. Am. Chem. Soc., 140,
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35 54. Gynther, M., Petsalo, A., Hansen, S. H., Bunch, L., and Pickering DS (2015) Blood-brain barrier
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37 permeability and brain uptake mechanism of kainic acid and dihydrokainic acid. Neurochem
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42 55. Alonso, E., Vieira, A. C., Rodriguez, I., Alvariño, R., Gegunde, S., Fuwa, H., Suga, Y., Sasaki,
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44 M., Alfonso, A., Cifuentes, J. M., and Botana, L. M. (2017) Tetracyclic Truncated Analogue of
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46 the Marine Toxin Gambierol Modifies NMDA, Tau, and Amyloid β Expression in Mice Brains:
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48 Implications in AD Pathology. ACS Chem. Neurosci., 8, 1358-1367.
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28 Figure 1: (A) Synthesis scheme and structure of three triazine based compound. Reagents and
29 conditions: (a) Methyl ester of amino acids, DIPEA, Dry THF, Reflux for overnight. Microtubule
30 polymerization assay using DAPI of TY3 (B) and TF3 (D). SPR sensogram for TY3 (C) and TF3 (E) in
31 presence of tubulin.
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30 Figure 2: Energy transfer graph between (A) Tub-Colchicine complex and F-TY3; (B) Tub-ANS
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complex and F-TY3; (C) Tub-TAMRA E3NAP and F-TY3 complex; (D) Tub-Colchicine complex and F-
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33 TF3; (E) Tub-ANS complex and F-TF3; (F) Tub-TAMRA E3NAP and F-TF3 complex. (G) Diagram for
34 the distance calculation from FRET experiments. Molecular docking image showing interaction of (H)
35 TY3 with PRO 274, ARG 278, ARG 284, ARG 320 and GLY 370 and (I) TF3 with PRO 274, THR 276,
36 GLY 370 and LEU 371. (N) Binding position of TF3 (shown in grey colored sphere model) and TY3
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38 (shown in cyan colored sphere model) in dimeric tubulin.
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6 Figure 3: (A) Off-resonance NMR spectrum (600MHz) of TY3 (upper line) STD experiment (lower line)
7 with TY3 in presence of tubulin. Zoom spectrum for aromatic region (Inset). Aromatic region shown in
8 zoom spectrum (Inset). (B) Epitope mapping of TY3 (C). (D) % STD of different proton of TY3.
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44 Figure 4: (A) TR-NOESY spectrum (mixing time: 300ms) of presence of TY3. (B)Tubulin bound
45 conformation of TY3 and (C) its interacting partners.
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7 Figure 5: Cell viability assay of (A) TY3 and (B) TF3 in PC12 cell lines. (C) Nocodazole induced
8 depolymerization Experiment. (D) Immunoblot depicts the increase of acetylated tubulin (AcK-40) as
9 compared to control with TY3 treatments. (E) Bar diagram reveals the relative expressions of acetylated
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tubulin normalized with loading control. All experiments have been performed in triplicate. The error bar
12 corresponds to the standard deviation of the value. (*p < 0.05, performing two tailed student’s t-test).
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TY3 and TS3). (B) Quantative analysis for neurite outgrowth of PC12 derived neurons. Microscopic
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40 images of microtubule networks of PC12 cells for control (C) and after treatment with TY3 (D) at merged
41 images. All experiments have been performed in triplicate. The error bar corresponds to the standard
42 deviation of the value. (*p < 0.05, performing two tailed student’s t-test).
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Figure 7: (A) ThT assay of TY3. (B) Bar diagram analysis represents cell viability of differentiated PC12
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47 derived neurons by NGF assay in absence or presence of various concentrations of TY3 and Anti-NGF.
48 Microscopic images of the (C) control PC12 cells, (D) Anti-NGF treated cells and (E) TY3 treated cells.
49 (F) Western blot of phospho-TrkA (pY490) (pTrkA) and full-length TrkA (TrkA) protein levels in PC12
50 derived neurons exposed to NGF (+NGF) or deprived of NGF for 24 h (-NGF). The expression of pTrkA
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increases due to 24 h of NGF deprivation, while decreases on treatment with TY3. On the other hand the
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53 expression of full length TrkA remains the same. (G) Bar diagram represents the quantitative analysis of
54 the immunoblots. All experiments have been performed in triplicate. The error bar corresponds to the
55 standard deviation of the value. (*p < 0.05, performing two tailed student’s t-test).
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47 Figure 8: Microscopic images of primary cortical neurons after treatment with TY3 at (A) bright field,
48 (B) 561 nm channel and (C) merged channel. (D) Immunoblotting experiment showing higher activation
49 of MAP2 and β-III tubulin in primary rat cortical neurons after TY3 treatment as compared to Control and
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(E) their quantitative analysis. (F) Blood Brain Barrier crossing experiment. All experiments have been
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52 performed in triplicate. The error bar corresponds to the standard deviation of the value. (*p < 0.05 and
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37 A tyrosine rich tripodal neuroprotective molecule has been designed, where phenol group plays crucial role
38 in interacting with microtubule and confers neuroprotection. Results reveal that molecule has significant
39 serum stability and capability to cross blood-brain barrier.
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