Subscriber access provided by YORK UNIV
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
Power of Tyrosine Assembly in Microtubule Stabilization and Neuroprotection Fuelled by Phenol Appendages Surajit Barman,1 Gaurav Das,1, 2 Prasenjit Mondal, 1, 2 Krishnangsu Pradhan,1 Debmalya Bhunia,1 Juhee Khan,1 Chirantan Kar,1 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: Microtubules play crucial role in maintenance of structure, function, axonal extensions, cargo transport and polarity of neurons. During neurodegenerative diseases, microtubule structure and function gets severely damaged due to destabilization of its major structural proteins. Therefore, design and development of molecules that stabilize these microtubule networks have always been an important strategy for development of potential neurotherapeutic candidates. Towards this venture, we designed and developed a tyrosine rich tri-substituted triazine molecule (TY3) that stabilizes microtubules through close interaction with the taxol binding site. Detailed structural investigations revealed that the phenolic protons are the key interacting partners of tubulin. Interestingly, we found that this molecule is non-cytotoxic in
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
PC12 derived neurons, stabilized microtubules against nocodazole induced depolymerization and increases expression of acetylated tubulin (K-40), an important marker of tubulin stability. Further, results show that TY3 significantly induces neurite sprouting as compared to the untreated control as well as the two other analogues (TS3 and TF3). It also possesses anti-Aβ fibrillation property as confirmed by ThT assay, which leads to its neuro-protective effect against amyloidogenic induced toxicity caused through NGF deprivation in PC12 derived neurons. Remarkably, our result reveals that it reduces the expression of pY490 TrkA associated with NGF deprived amyloidogenesis, which further proves that it is a potent amyloid beta inhibitor. Moreover, it promoted the health of the rat primary cortical neurons through higher expression of key neuronal markers such as MAP2 and Tuj1. Finally, we observed that it has good serum stability and has the ability to cross the blood brain barrier (BBB). Overall, our work indicates the importance of Phenolic –OH in promoting neuroprotection and its importance could be implemented in the development of future neuro-therapeutics. KEYWORDS: Triazine, Microtubule, FRET, STD NMR, TR-NOESY, Neuroprotection, Blood Brain-Barrier 1. INTRODUCTION The embellished shape of neuron and its polarity has been a centre point of interest for wide range of researchers in the field of neurobiology for the last several decades.1-3 To maintain its incredible structure and polarity several factors play crucial role and microtubules are one of the most important players.4-7 Microtubules (MT) are dynamic polymers of α, β-tubulin heterodimers that along with actin and intermediate filaments forms the key cytoskeletal components of the eukaryotic cells including neurons.1-7 Along with other eukaryotic cells, microtubules have
ACS Paragon Plus Environment
Page 2 of 37
Page 3 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
special importance in neurons where apart from providing them the much-needed shape and structure, they also have important functional aspects.8 In neurons, microtubules are involved in a variety of tasks like cargo transport through gigantic axonal projections with the aid of the associated molecular motors.9 This role of axonal transport in neurons is so significant that even slightest perturbations in the microtubule structure or functions create severe damage to the normal neuronal physiology as observed in several neurodegenerative diseases.10 In the axons, the linear arrays of microtubules comprising of two ends- plus ends towards synapse and minus ends towards cell body provides directionality and structural support to the neurons.11, 12 Many studies have indicated that neurodegenerative diseases like Alzheimer’s Disease (AD) have been caused due to defects in axonal microtubules and this has given rise to the importance of MTstabilizing molecules.13-19 During AD progression, microtubule networks in neurons is severely damaged due to disintegration of microtubule associated protein Tau, which further aggregates and generates neurofibrillary tangles (NFT). Moreover, it has been also reported that Aβ aggregation damages the microtubule network. Many microtubule stabilizing molecules have been so far reported to have shown promising effects in restoration of microtubules in axonal transport as well as improving cognition in animal models. As a result, such compounds may be considered as potential candidates for the treatment of AD and related taupathies. Microtubule stabilising agents (MSA) accelerates the equilibrium from tubulin to the polymeric microtubule and promotes tubulin polymerization. MSA’s are already well established chemotherapeutic agents like taxol but the action of these neuro-protective microtubule stabilizing agents are somewhat different as their order of stabilization is not so pronounced as to arrest the cell division and cause cell death.20-22 Though a number of neuroprotective MT-stabilizing agents have been reported, most of them so far have produced clinically insignificant results. Moreover,
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
many naturally occurring and synthesized molecules with phenolic -OH moiety like in neurotransmitter molecules such as dopamine and Ferulic acid have been reported to possess neuroprotective effects23-27 but none of the studies directly or indirectly have explored the importance of phenolic -OH in neuroprotection and their possible cross talk with tubulin or microtubule. Talking about phenolic –OH, several groups have already reported tyrosine analogues with effective biological activity and is one of the hot arenas in current research among the chemical biologists. 28-30 To address this fundamental aspects and to provide more detailed understanding in this manuscript, we have designed novel tripodal molecules based on 1, 3, 5-triazine core with tri-substitution by tyrosine (Y), phenylalanine (F) and serine (S) named as TY3, TF3 and TS3 respectively. Among the three molecules, TY3 and TF3 binds close to the taxol binding pocket of the tubulin. Next, the identification of pharmacophore of TY3 for the interaction with tubulin was investigated through STD NMR and TR-NOESY experiment. Further, it was observed that TY3 confers significant microtubule stabilization in PC12 derived neurons against nocodazole induced depolymerization, increases the expression of acetylated tubulin (K-40) and facilitates neurite outgrowth, while TF3 and TS3 does not show any activity in neurons. Interestingly, we found that TY3 also inhibits Aβ fibrillation and confers significant neuroprotection against Aβ mediated toxicity in PC12 derived neurons. It has been reported earlier that NGF deprivation is associated with an increase in the expression of a particular phosphorylation of TrkA (pY490).31
We found that TY3 reduces this TrkA associated
phosphorylation, thus reinstating the fact that it has anti-amyloidogenic activity. Moreover, we observed that TY3 crosses the blood-brain barrier, shows significant serum stability and promotes healthy morphology of the cultured primary rat cortical neurons by upregulation of neuronal markers (Tuj1 and MAP2). Overall, results reveal that our newly designed molecules
ACS Paragon Plus Environment
Page 4 of 37
Page 5 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
confers significant microtubule stabilization and neuroprotection, where spatially positioned phenolic-OH group interacts close to the taxol binding pocket of tubulin and plays an important role. We strongly believe that this novel tripodal molecule with tyrosine substitution has significant potential to serve as a unique neurotherapeutics template.
2. RESULTS AND DISCUSSION 2.1. Synthesis of triazine based small molecules. We have synthesized three triazine based compounds (TS3, TF3 and TY3) and characterized them by ESI-mass spectroscopy, 1H-NMR, 13C-NMR, DEPT-135 and HPLC (Figure 1A and S110). The fluorescein attached TF3 and TY3 were synthesized by solid phase peptide synthesis (SPPS) method using CEM, purified them by HPLC and characterized by MALDI-TOF mass spectroscopy (Figure S11, 12). 2.2 Microtubule assembly assay. To check whether our compounds have any interaction with tubulin, we have performed microtubule assembly assay using the change in fluorescence intensity of 4', 6-diamidino-2phenylindole (DAPI) as an indicator during tubulin polymerization.32, 33 Remarkably, we found that fluorescence intensity of DAPI increased in case of TF3, TY3 as compared to untreated control (tubulin alone). But, there was no significant change in fluorescence intensity in case of TS3. Next, we performed microtubule assembly assay of the TF3 and TY3 in dose dependent manner (Figure 1B, D and S13). These results clearly shows that TF3 and TY3 are promoting the tubulin polymerization.
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
2.3 Determination of binding constant by Surface Plasmon Resonance (SPR) study. Above results encouraged us to further explore the affinity of TF3 and TY3 with tubulin and their association and dissociation kinetics. For this, we performed Surface Plasmon Resonance (SPR)34 for TF3 and TY3. The SPR studies suggest that the association constant (Ka) for TF3 and TY3 are 1.42 × 103 M-1, 1.05 ×103 M-1 respectively and the dissociation constant of TF3 and TY3 are 158 μM and 16.4 μM respectively (Figures 1 C, E). 2.4 Determination of binding affinity using tryptophan fluorescence quenching study. Next, we have performed the tryptophan fluorescence quenching study to determine the binding affinity with tubulin. Binding affinity of TF3 and TY3 were determined using modified SternVolmer equation. The binding constants of TF3 and TY3 are 2.68 × 103 M-1 and 7.75 × 103 M-1 respectively (Figure S14). These results are in agreement with the association constant measured through previous SPR experiments. 2.5 Förster Resonance Energy Transfer (FRET) study to determine the binding location in tubulin. To know more in details about the binding location of tubulin, we performed Förster Resonance Energy Transfer (FRET) experiment using these two compounds (TF3 and TY3). Here, we used three florescent probes (colchicine, 8-anilinonaphthalene-1-sulfonic acid (ANS) and TAMRAE3NAP) as FRET partner with fluorescein tagged TF3 and TY3. The binding sites of colchicine, ANS and TAMRA-E3NAP in tubulin are well understood and the Förster distance (R0) between tubulin bound colchicine and fluorescein dye attached peptide is 29.5 ± 1 Å, tubulin bound ANS and fluorescein-peptide is 50.3 ± 2 Å and tubulin bound TAMRA-peptide with fluoresceinpeptide is 62.9 ± 2 Å.35, 36 We performed the FRET experiment with the above FRET partners
ACS Paragon Plus Environment
Page 6 of 37
Page 7 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
with TY3 compound resulting in a distance of 34.64 ± 1 Å, 53.4 ± 1 Å and no FRET with TAMRA-E3NAP complex (Figure 2A-C). Similarly, when we performed FRET experiments with TF3 and the above FRET partners, we found similar result like TY3 (34 ± 1 Å from colchicine site, 54 ± 1 Å from ANS site and no FRET between F-TF3 with TAMRA-E3NAP complex) (Figure 2D-F). Then we calculated the distance obtained from the FRET results that revealed TY3 and TF3 binds close to the taxane binding site of tubulin (Figure 2G). 2.6 Conformational analysis and subsequent docking study. We performed molecular docking to know the binding affinity and interacting partners of TY3 and TF3 at taxol pocket using DFT minimized conformers of TY3 and TF3 molecules (Figure S15-25). The docking results reveal that the interacing partners of TY3 are ARG278, ARG284, PRO274, ARG320 and GLY370 with binding affinity of -7.7 kcal/mol and for TF3 are GLY370, PRO274, THR276 and LEU371 with binding affinity of -7.9 kcal/mol (Figure 2H, I and S26). TY3 and TF3 are shown in docking image bound to their respective binding sites in tubulin which are shown in Figure 2J. 2.7 STD NMR and TR-NOESY experiments. In order to obtain more minute details of their molecular recognition, STD and TR-NOESY were performed to derive a tubulin-bound conformer of TY3. First, we have assigned different protons of TY3 using 1D NMR (1H-NMR, 13C-NMR and DEPT study) and 2D NMR experiments (COSY, HSQC, HMBC and NOESY) (Figure S27-30). To study more in details, we have performed saturation transfer difference (STD) and transfer nuclear overhauser spectroscopy (TR-NOESY) experiment. These studies are useful for the epitope mapping of a molecule with a protein. In STD-NMR, the bound ligand only shows the STD signals and higher STD signals
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
indicate the close proximity of the epitope to the protein. The STD results revealed that aromatic protons of ligands TY3 are showing the highest STD effect while less signal for the aliphatic group was observed (Figure 3A-C). From this result, we can conclude that aryl moiety is essential for its interaction with tubulin. In TR-NOESY experiment, the negative cross peaks were detected in the presence of tubulin. We have deduced the tubulin bound conformation of TY3 where two aromatic protons of TY3 (ArH6.67 and ArH6.97) are in close proximity to the –OMe,-CH2 and -CH protons (Figure 4A). Further, we deduced the plausible conformation of tubulin bound TY3. For this, we performed the molecular docking study with TY3 using DFT energy minimized conformer of TY3. The results of molecular docking of TY3 are in agreement with the TR-NOESY data (Figure 4B, C and S31). The STD NMR and TR-NOESY experiments clearly shows that the phenyl moiety is crucial for its interaction with tubulin. 2.8 Cell viability assay So far we have performed several in vitro assays that indicate the microtubule stabilizing nature of TY3 and TF3. In order to perform further cell based screening, it is pertinent to confirm their cyto-toxic nature. Hence, we performed the cell viabilty assay using differentiated PC12 cell lines upto 200uM concentrations of both TY3 and TF3. The results revealed that all the compounds are non-cyto-toxic to the differentiated neurons (Figure 5A, B). 2.9 Nocodazole induced depolymerization and expression of Acetylated Tubulin (AcK-40) Previous in vitro studies like SPR and microtubule assembly assay using DAPI have proven TY3 and TF3 both to be microtubule polymerizing compound. Now, it was to be tested, whether it shows similar results in neurons. After reversibly depolymerizing microtubules of the PC12
ACS Paragon Plus Environment
Page 8 of 37
Page 9 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
derived neurons by 4 h treatment with 10 µM nocodazole, the cells were washed free of the nocodazole and divided into three groups one being control replaced with only fresh medium while the other two with TY3 and TF3. After 2 h of treatment, the cells were fixed and immunostained with primary antibodies against alpha-tubulin. The cells treated with TY3 showed healthy microtubule network completely recovered from the nocodazole treatment while the control cells and TF3 treated cells still suffered from the depolymerizing effect of nocodazole (Figure 5C and S32). This proves that our cellular data is in line with our in vitro observation and TY3 is indeed a microtubule stabilizer. The treated cells also showed a higher expression of acetylated tubulin (AcK 40), a known marker of tubulin stabilization compared to the untreated control (Figure 5D, E and S33). 2.10 TY3 promoted significant neurite outgrowth among the other screened molecules. Many microtubule stabilizing drugs like Methyl 3, 4-dihydroxybenzoate and GIT 1 have also shown to promote neurite outgrowth.37,
38
All these factors prompted us to check the neurite
outgrowth potential of our microtubule stabilizing compounds TY3 and TF3. When these compounds were incubated with undifferentiated PC12 cells, TY3 showed neurite outgrowth after 24 h of incubation while the others (TF3 and TS3) failed to do so (Figure 6A, B). This above results motivated us to conduct more studies for TY3 in neurons. It was also seen that TY3 promoted the healthy morphology and architecture of microtubule network (Figure 6 C, D). It is important to note that although TY3 binds close to the taxol binding site, yet it does not cause toxicity due to the fact that it binds at the taxol binding site 3428 fold times weaker and also dissociates in a faster rate (Kd=16.4 μM) compared to taxol.39 Due to this reasons, TY3 can provide microtubule stabilization through weak binding as reported before.34
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
2.11 ThT assay indicates that TY3 inhibits Aβ (1-42) aggregation We initially performed ThT assay with TY3 and incubated it with Aβ (1-42). To our astonishment, TY3 inhibited Aβ (1-42) aggregation (Figure 7A) and this proves that TY3 would be able to show significant neuroprotective effect when tested in some cellular model of neurotoxicity. For this, it was very important to first ascertain the fact that our compound TY3 is non-toxic to the neurons. We have already screened TY3 on PC12 derived neurons with a wide range of concentrations and it was observed that TY3 is completely non-toxic to the neurons at even very high concentrations which paved way for the future studies in cell. 2.12 TY3 confers neuroprotection in NGF deprived PC12 derived neurons by reducing the phosphorylation of TrkA. It is now well documented that NGF deprivation in PC12 derived neurons leads to amyloidogenesis40 and that may cause an unexpected phosphorylation of TrkA at Y490, thus activating phospholipase C (PLC) associated cell death pathway causing severe neurotoxicity. This phosphorylation is usually connected with two kinases namely Src and CDK5 which are known to be involved in amyloidogenesis and and mediated p75 processing. Our in vitro ThT assay data encouraged us to pursue the neuroprotective potential of TY3 in this NGF deprived model of PC12 derived neurons. We observed that TY3 confers neuroprotection upto 1.5 µM concentrations (Figure 7B-E) and causes inhibition of the neuronal death inducing TrkA phosphorylation (pY490). But no change in the expression of TrkA was observed (Figure 7F, G and S34), which denotes that TY3 specifically inhibits amyloidogenesis. Here, we would like to point out that TF3 and TS3 does not show any neuroprotection ability although TF3
ACS Paragon Plus Environment
Page 10 of 37
Page 11 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
contains phenyl group and TS3 contains -OH group. This fact clearly indicates that combination of both phenyl and -OH group that is phenol group is essential to confer neuroprotection. 2.13 TY3 promotes the healthy morphology of cultured primary cortical neurons and upregulates several neuronal markers. All the cellular studies concerning TY3 have been so far studied in PC12 derived neurons but what about its effect on primary neurons. On treating the rat primary cortical neurons with TY3 for 2 days, we observed that TY3 was well tolerated by them and also promoted the healthy morphology of the neurons (Figure 8A-C) when compared with the untreated control. Moreover, we observed significant increase in the expression of important neuronal markers MAP2 and Tuj1 in TY3 treated neurons as revealed from the immunoblots (Figure 8D, E and S35). 2.14 Serum stability of TY3 performed by high-performance liquid chromatography (HPLC). For any candidate to qaulify as a neuroprotective drug, it must cross the blood brain barrier (BBB). But before performing the blood brain barrier crossing, we checked the stability of TY3 in serum. TY3 was incubated with serum up to 24 h and monitored using HPLC. It was observed that more than 40% TY3 remained stable after 24 h of incubation with serum. This result suggests significant serum stability of TY3 (Figure S36). 2.15 Blood-Brain Barrier (BBB) crossing experiment. Since TY3 showed serum stability, we finally needed to check whether it can cross the blood brain barrier (BBB). To address this, we have performed the MALDI-Mass spectroscopy of brain extract obtained from mice treated with TY3. The MALDI-Mass spectrum showed molecular peaks at (M=661 Da) and (M+K+ =699 Da). This data indicated the presence of TY3 in the brain
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 12 of 37
extract, which confirmed its potential to cross the blood-brain barrier (BBB) (Figure 8F and S37, 38). Hence we can now consider this as a promising neurotherapeutic candidate. 3. Conclusion In summary, we have shown in this paper the power of phenolic –OH appendages in neuroprotection and neurite outgrowth through its interaction with microtubule. Our results reveal that tyrosine substitution on a tripodal platform plays an important role where this phenol group acts like a key player. Moreover, we found that phenolic –OH group interacts with tubulin close to the taxol binding pocket by FRET and NMR where the aromatic substituent binds in the hydrophobic pocket. Interestingly, we found that TF3 and TS3 did not have any significant effect on microtubules and that further indicated that the phenolic –OH moieties are the key partners for interaction with tubulin. The cellular assays in PC12 derived neurons and primary rat cortical neurons also reciprocated similar results. Moreover, it also showed serum stability and crossed blood brain barrier. Finally, the tripodal core substituted with the phenolic –OH group in TY3 could serve as a potential candidate for the development of future neuro-therapeutics. 4. Experimental Section 4.1 Chemicals Cyanuric chloride, Tyrosine methyl ester, Phenyl alanine methyl ester and Serine methyl ester, Thiazolyl Blue Tetrazolium Bromide (MTT), Kanamycin sulfate, Dulbecco’s Modified Eagle’s Medium (DMEM), Guanosine 5′-triphosphate sodium salt hydrate (GTP), 4, 6-diamidino-2phenylindole (DAPI), Trypsin-EDTA solution, Colchicine, 5(6)-carboxyfluorescein, PIPES, Ethylene
glyolbi(2-aminoethyl
ether)-N,N,N′,N′-tetra
acetic
acid
(EGTA),
N,N′-
Diisopropylcarbodiimide (DIC), 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS) and
ACS Paragon Plus Environment
Page 13 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
Cell culture grade DMSO were purchased from Sigma-Aldrich. Sodium chloride, Sodium hydrogen carbonate, Di-sodium hydrogen phosphate dihydrate, Potassium hydroxide, Magnesium chloride hexahydrate, Trifluoroacetic acid (TFA) and Potassium dihydrogen phosphate were purchased from Merck. Potassium chloride and Agarose were purchased from Fisher Scientific. Dimethyl sulfoxide, N, N’-Diisopropylethylamine (DIPEA), Ethyl acetate, Chloroform and Hexane were purchased from Spectrochem. Triton-X-100 was purchased from SRL. Amyloid-beta 1-42 (Aβ42) was purchased from AnaSpec. NGF-β (7S) was purchased from Sigma (St. Louis, MO, USA). Neurobasal media, B27, glutaMAX and Pen/strep were bought from Gibco, Life technologies. 2-[4-(2-Hydroxyethyl) piperazin-1-yl] ethanesulfonic acid (HEPES) and Horse Serum were purchased from Himedia. Penicillin-Streptomycin and Fetal bovine serum were purchased from Invitrogen. Anti-alpha Tubulin clone EP 1332Y, AntiTubulin Anti-body, Beta III isoform (Tuj 1) were purchased from Merck Millipore. Anti-TrkA (pY490), Anti-TrkA and Anti-alpha Tubulin (acetyl K-40) were purchased from Abcam. Bisbenzimide H 33258 (hoechst) was purchased from Calbiochem. MAP2 Monoclonal Antibody (M13) was purchased from Thermofisher scientific. HPLC-grade H2O and ACN were purchased from J.T. Baker. All the NMR solvents were purchased from Cambridge isotope. All compounds were used without further purification. 4.2 Cell culture Pheochromocytoma of the rat adrenal medulla (PC12) were purchased from NCCS, Pune (India). The PC12 cells were cultured in complete DMEM with 10% horse serum and 5% FBS. The differentiation of PC12 cells into neurons was achieved by culturing the cells in serum free medium with 1% horse serum and 100 ng/mL NGF (Nerve Growth Factor) for 5 days.
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
4.3 Protein purification After the isolation of tubulin from goat brain, we purified it by two cycle’s polymerization and depolymerization procedure followed by storing it in liquid nitrogen cryochamber using 10% glycerol.41 4.4 Synthesis, characterization of TY3, TS3 and TF3 and its fluorescein derivatives. In 100mL round bottom flask, methyl ester of amino acids (4 equivalents) and cyanuric chloride (1 equivalent) in dry acetonitrile was stirred at room temperature. Then N, N′Diisopropylethylamine (DIPEA) was added to the reaction mixture. The reaction mixtures were refluxed overnight under inert atmosphere. Finally, the compounds were purified by column chromatography and characterized by ESI-mass spectroscopy, 1H-NMR, 13C-NMR, and DEPT135. Fluorescein tagged triazine derivatives (F-TY3 and F-TF3) were synthesized using solid phase peptide synthesis (SPPS) method in a microwave-based (35 watt) automated peptide synthesizer (CEM, Liberty 1). All the synthesized fluorescein tagged small molecules were purified through C-18 reverse phase HPLC column at 210 nm and characterized by MALDITOF in acetonitrile and water (1:1) mixture. 4.5 Conformational Analysis. We deduced the energy minimized conformers of the TY3 and TF3 molecules using Spartan’1642-45 and Gaussian ’09 program46. Geometry optimizations of each molecules were performed in multistep. In first step 1000 most probable conformers were chosen and a conformational search was performed to predict their corresponding energies using molecular mechanics force field
ACS Paragon Plus Environment
Page 14 of 37
Page 15 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
(MMFF). In the next step 100 lowest energy conformers chosen from the MMFF calculation were further refined by semi-empirical calculation at PM6 level. Next, the 20 lowest energy conformers taken after the semi-empirical calculation were optimized applying Hartree-Fock method at 3-21G level. 4.6 Docking study We performed blind docking study using the software Autodock-Vina version 1.1.2.47 98×60×64 affinity grids were centered on the tubulin [PDB ID: 1Z2B]48 for docking TY3 and TF3. All the images were visualized and developed in PyMOL (The PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC.) 4.7 Microtubule assembly assay A stock solution of 10 μM DAPI in BRB80 buffer containing 100 μM tubulin, 10 mM GTP and different concentration of TF3 and TY3 were mixed. Emission spectra of solutions were recorded in region ranging from 400 nm to 600 nm wavelength at 37 0C after excitation at 355 nm. 4.8 Surface Plasmon Resonance (SPR) experiment The SPR experiment was performed using NTA biosensor chip, Ni2+ and streptavidin-His6 were immobilized on to the NTA biosensor chip. Next, we washed the surface with BRB80 buffer and flowed biotinylated tubulin (20 μg/mL) followed by incubation for 15 min. The surface was washed and desired compounds were added at various concentrations as analytes. The recorded data were analyzed by plotting the curve with a local fitting. 4.9 Tryptophan fluorescence quenching experiment
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Tubulin was mixed with different concentrations of TF3 and TY3 separately in BRB80 and these mixtures were incubated for 40 min at 25 °C. Then, the intrinsic tryptophan fluorescence data was recorded and calculated by modified Stern-Volmer equation. 4.10 Förster resonance energy transfer (FRET) experiment We performed the FRET study to know the binding pocket of TF3 and TY3 in the tubulin. Tubulin-colchicine, tubulin-ANS and tubulin-TAMRA-E3NAP complexes were used to find out the distance of fluorescein attached small molecules from their specific binding pocket in tubulin following previously described method. The excitation and emission ranges were set at 355 nm and 450 to 650 nm respectively. 4.11 STD NMR sample preparation and experiments49, 50 The samples of the ligands were prepared with a 300 μM concentration of the TY3 and 10 μM of tubulin in D2O, 10 mM NaPi, 0.1 mM GTP. Small amount of D6-DMSO were used to solubilize the TY3 ligand . We recorded the STD-NMR by Bruker AVANCE 600 MHz spectrometer with saturation time 2s. Percentage of relative STD effects was calculated by the following equation ASTD = (I0 - Isat)/I0 = ISTD/I0
where intensity of the signals in the STD NMR spectrum (ISTD)
with signal intensities of a reference spectrum (I0). The maximum STD signal was considered as 100% STD effect and others were calculated relatively. 4.12 TR-NOESY experiments Strong negative NOE cross peaks were detected by TR-NOESY experiments (mixing times of 200 ms) in presence of tubulin receptor compared to the free state of the TY3 ligand. All the datas were calculated using Mnova software.
ACS Paragon Plus Environment
Page 16 of 37
Page 17 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
4.13 Neurite outgrowth experiment For neurite outgrowth experiment, all the three triazine derivatives were treated with the undifferentiated PC12 cells and incubated for 24h at 370C.Then the treated cells were imaged under a microscope at DIC mode. Evaluation of neurite length was carried out using the CellSens software. 4.14 Effect of TF3 and TY3 on neuronal microtubule PC12 cells (density~3000-5000) were grown in a confocal dish on the day before the treatment. We treated TY3 for 24 h. Next day, cells were fixed using 4% paraformaldehyde and incubated with 5% BSA and 0.2% triton-X in PBS for 1 h. Then we washed the fixed cells with PBS and treated with primary antibody polyclonal anti-α-tubulin IgG antibody. After 2 h, we treated secondary antibody (Cy3.5 pre-absorbed goat anti-rabbit IgG) after washing the cells with PBS and kept for 2 h. On the day of the imaging of the cells, cells were washed with PBS and treated with Hoechst 33258 (1 μg/mL) for half an hour. Images of various zone of culture dish were captured using microscope. 4.15 Thioflavin T (ThT) assay Thioflavin-T (ThT) is used to identify the amyloid fibrils both in vitro and in vivo. There is an enhancement of its fluorescence intensity upon binding with the fibrils. To monitor the Aβ peptide aggregation, we performed the following experiment.51 Different concentrations of the TY3 were mixed with 10 μM Aβ42 peptide and incubated for 48h. Then, we added ThT solution to the incubated solution and recorded the fluorescence in PTI QM-40 spectrofluorimeter (excitation: 435 nm and emission: 460-650 nm).
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
4.16 Neuroprotection study using differentiated PC12 cells For the event of differentiation, PC12 cells were cultured for 24 h and treated with serum free DMEM containing 1% horse serum and 100 ng/mL NGF and incubated for 5 days. Differentiated PC12 cells were seeded in 96-well plates (1 × 104 cells/mL) and then cells were treated with anti-NGF alone and along with different concentrations of TY3 for 24 h. MTT solution were added to each well and incubated at 37oC for 4 h. Then the MTT solutions were replaced by 1:1 DMSO-MeOH. Then the percentage of cell viability was analyzed using microplate ELISA reader (Thermo; Multiskan GO Microplate Spectrophotometer) at 550 nm wavelength. 4.17 Effect of TY3 on primary cortical neuron culture Primary cortical neurons were cultured by previously described protocol.52 In brief, we have received timed-pregnant Sprague Dawley rat. Then we isolated the brain from the dissected E18 embryos. After the micro-dissection of the brain, cortices were dissected out in the MEM medium containing 10% horse serum and glucose (0.6% wt/vol). ~3-5 × 105/mL cells were plated on confocal dishes coated with poly-D lysine and was incubated in CO2 incubator for about 4 h. Then this culture medium was changed with neurobasal media supplemented with B27, Pen/Strep and GlutaMAX and cultured for another 14 days untill the neurons matured. We treated these neurons with TY3 and assessed its effect on primary cortical neurons. 4.18 Serum stability Stability of any small/large molecules is important for their biological aspects.53 For this purpose, we checked the stability of TY3 in horse serum. Quantitative stability of the compounds
ACS Paragon Plus Environment
Page 18 of 37
Page 19 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
was monitored by C18 reverse phase HPLC system upto 24 h. It was found that almost 40% of the TY3 remained intact after 24 h. 4.19 Blood-Brain Barrier (BBB) crossing experiment. 54, 55 C57BL/6J female mice of around 20 g have been used for our study. Animals were divided into two groups (3 mice /group) and the mice were injected intraperitoneally with TY3 dissolved in saline solution at the concentration of 50 µg/Kg body weight of mice. For control, mice were treated with only saline solution (2nd group). After 6 h of treatment, animals were deeply anaesthetized with ketamine (i.p) and were sacrificed by transcardial perfusion. The mice brains were isolated and directly transferred into liquid nitrogen, crushed using a mortar and pestle. Acetonitrile and water mixture (1:1) was added into the crushed brain. Mixture was centrifuged to separate out the soluble part. HPLC and mass analysis were performed to analyze the data and for the identification of TY3 mass. ASSOCIATED CONTENT Supporting Information This materials are available free of charge via the internet at http://pubs.acs.org. Synthesis and characterization of TS3, TF3, TY3, F-TF3 and F-TY3 using 1H-NMR, 13C-NMR, DEPT-135 and mass spectroscopy. HPLC chromatograms, Microtubule assembly assay, Quenching of Tryptophan fluorescence experiment, DFT study of tri-substituted triazine derivatives. 2D NMR (COSY, NOESY, HSQC and HMBC) of TY3 and TF3. Control experiments for TR-NOESY and BBB experiment. Serum stability of TY3. Immunoblots of AcK-40, phospho-TrkA (pY490), TrkA, MAP2 and Tuj1. (PDF) AUTHOR INFORMATION: Corresponding Author Dr. Surajit Ghosh Principal Scientist
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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 Barman: 0000-0003-3584-4716 Gaurav Das: 0000-0002-8432-5384 Prasenjit Mondal: 0000-0003-0767-449X Surajit Ghosh: 0000-0002-8203-8613
Author contributions SB performed synthesis, characterization, major experiments and analyzed the data. GD performed various cell based assays like neurite outgrowth, microtubule associated depolymerization and the immunoblots. PM and KP performed FRET, computational experiment and in vitro inhibition of Aβ aggregation. DB helped GD and JK in performing the BBB experiment. JK and GD performed the primary neuron culture. CK performed the DFT calculations .SG conceived the idea, designed and monitored all the experiments, analyzed the data, prepared figures and supervised GD and SB in writing the manuscript. Notes The authors declare no competing financial interest ACKNOWLEDGMENT We thank Ms. Varsha Gupta for critically reading the manuscript. We also want to thank Dr. E. Padmanban for helping in STD and TR-NOESY experiments and Mr. K Suresh Kumar for SPR
ACS Paragon Plus Environment
Page 20 of 37
Page 21 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
experiments. SB, KP thanks UGC, PM thanks CSIR, DB, JK thanks DST and GD thanks ICMR, India for their fellowships. CK thanks SERB for his postdoctoral fellowship. SG kindly acknowledges DST, India for providing support (EMR/2015/002230) and CSIR-IICB for infrastructure. Animal experiments were performed following IICB ethical guidelines.
Reference: 1. Craig, A. M., and Banker, G. (1994) Neuronal polarity. Annu. Rev. Neurosci., 17, 267-310. 2. Arimura, N., and Kaibuchi, K. (2007) Neuronal polarity: from extracellular signals to intracellular mechanisms. Nature Rev. Neurosci., 8, 194-205.
3. Conde, C., and Cáceres A. (2009) Microtubule assembly, organization and dynamics in axons and dendrites. Nat Rev Neurosci., 5, 319-32.
4. Baas, P. W. (2002) Neuronal polarity: microtubules strike back. Nature Cell Biol., 4, 194-195. 5. Witte, H., and Bradke, F. (2008) The role of the cytoskeleton during neuronal polarization. Curr. Opin. Neurobiol., 18, 1-9.
6. Desai, A., and Mitchison, T. (1997) Microtubule polymerization dynamics. Annu. Rev. Cell Dev. Biol., 13, 83-117.
7. Nogales, E. (2001) Structural insights into microtubule function. Annu. Rev. Biophys. Biomol. Struct., 30, 397-420.
8. Akhmanova, A., and Hoogenraad, C.C. (2015) Microtubule minus-end-targeting proteins. Curr. Biol., 25, 162-171.
9. Baas, P. W., and Ahmad, F. J. (2001) Force generation by cytoskeletal motor proteins as a regulator of axonal elongation and retraction. Trends Cell Biol., 11, 244-249.
10. Baas, P. W., and Mozgova, O. I. (2012) A novel role for retrograde transport of microtubules in the axon. Cytoskeleton, 69, 416-425. 11. Baas, P. W., and Lin, S. (2011) Hooks and comets: The story of microtubule polarity orientation in the neuron. Dev Neurobiol., 71, 403-418.
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
12. Lasser, M., Tiber, J., and Lowery, L. A. (2018) The Role of the Microtubule Cytoskeleton in Neurodevelopmental Disorders. Front Cell Neurosci., 12, 165.
13. Ballatore, C., Smith, A. B., Lee, V. MY., Trojanowski, J. Q., and Brunden, K. R. (2016) Microtubule-Stabilizing Agents for Alzheimer’s and Other Tauopathies. In: Wolfe M. (eds). Alzheimer’s Disease II. Topics in Medicinal Chemistry, 24. Springer, Cham
14. Ballatore, J. C., Brunden, K. R., Huryn, D. M., Trojanowski, J. Q., Lee, V. M.-Y. and Smith, A. B. (2012) Microtubule Stabilizing Agents as Potential Treatment for Alzheimer’s Disease and Related Neurodegenerative Tauopathies. J. Med. Chem., 55, 8979-8996.
15. Varidaki, A., Hong, Y., and Coffey, E. T. (2018) Repositioning Microtubule Stabilizing Drugs for Brain Disorders. Front Cell Neurosci., 12, 226.
16. Michaelis, M. L., Chen, Y., Hill, S., Reiff, E., Georg, G., Rice, A., and Audus, K. (2002) Amyloid peptide toxicity and microtubule-stabilizing drugs. J. Mol. Neurosci., 19, 101-105.
17. Roy, S., Zhang, B., Lee, V. M., and Trojanowski, J. Q. (2005) Axonal transport defects: a common theme in neurodegenerative diseases. Acta Neuropathol., 109, 5-13
18. Liu, X., Rizzo, V., and Puthanveettil, S. V. (2012) Pathologies of axonal transPort in neurodegenerative diseases. Transl Neurosci., 3, 355-372.
19. Fanara, P., Banerjee, J., Hueck, R. V., Harper, M. R., Awada, M., Turner, H., Husted, K. H., Brandt, R., and Hellerstein, M. K. (2007) Stabilization of hyperdynamic microtubules is neuroprotective in amyotrophic lateral sclerosis. J Biol Chem., 282, 23465-23472.
20. Mukhtar, E., Adhami, V. M., and Mukhtar, H. (2014) Targeting Microtubules by Natural Agents for Cancer Therapy. Mol Cancer Ther., 13, 275-284.
21. Weaver, B. A. (2014) How Taxol/paclitaxel kills cancer cells. Mol Biol Cell., 25, 2677-2681. 22. Dumontet, C., and Jordan, M. A. (2010) Microtubule-binding agents: a dynamic field of cancer therapeutics. Nat Rev Drug Discov., 9, 790-803.
ACS Paragon Plus Environment
Page 22 of 37
Page 23 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
23. Sarkar, S., Raymick, J., and Imam, S. (2016) Neuroprotective and Therapeutic Strategies against Parkinson’s disease: Recent Perspectives. Int. J. Mol. Sci., 17, 904
24. Spencer, G. E., Klumperman, J., and Syed, N. I. (1998) Neurotransmitters and neurodevelopment. Role of dopamine in neurite outgrowth, target selection and specific synapse formation. Perspect Dev Neurobiol., 5, 451-67.
25. Spencer, G. E., Lukowiak, K., and Syed, N. I. (1996) Dopamine regulation of neurite outgrowth from identified Lymnaea neurons in culture. Cell Mol Neurobiol., 16, 577-589.
26. Ojha, S., Javed, H., Azimullah, S., Abul Khair, S. B., and Haque, M. E. (2015) Neuroprotective potential of ferulic acid in the rotenone model of Parkinson's disease. Drug Des Devel Ther., 9, 5499-510.
27. Ren, Z., Zhang, R., Li, Y., Li, Y., Yang, Z., and Yang, H. (2017) Ferulic acid exerts neuroprotective effects against cerebral ischemia/reperfusion-induced injury via antioxidant and anti-apoptotic mechanisms in vitro and in vivo. Int J Mol Med., 40, 1444-1456.
28. Neidhardt, M. M., Schmitt, K., Baro, A., Schneider, C., Bilitewski, U. and Laschat, S. (2018) Self-assembly and biological activities of ionic liquid crystals derived from aromatic amino acids. Phys Chem Chem Phys., 20, 20371-20381.
29. Augustyn, E., Finke, K., Zur, A. A., Hansen, L., Heeren, N., Chien, H. C., Lin, L., Giacomini, K. M., Colas, C., Schlessinger, A. and Thomas, A. A. (2016) LAT-1 activity of meta-substituted phenylalanine and tyrosine analogs. Bioorg Med Chem Lett., 26, 2616-2621.
30. Bader, K., Neidhardt, M. M., Wöhrle, T., Forschner, R., Baro, A., Giesselmann, F. and Laschat, S. (2017) Amino acid/crown ether hybrid materials: how charge affects liquid crystalline selfassembly. Soft Matter, 13, 8379-8391.
31. Matrone, C., Maroldaa, R., Ciafre`a, S., Ciottia, M. T. Mercantia, D., and Calissanoa, P. (2009) Tyrosine kinase nerve growth factor receptor switches from prosurvival to proapoptotic activity via Abeta-mediated phosphorylation, Proc Natl Acad Sci U S A., 27, 11358-11363.
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
32. Bonne, D., Heusele, C., Simon, C., and Pantaloni, D. (1985) 4', 6-Diamidino-2-phenylindole, a Fluorescent Probe for Tubulin and Microtubules. J. Biol. Chem., 260, 2819-2825.
33. Ghosh, J. G., Houck, S. A., and Clark, J. I. (2007) Interactive Domains in the Molecular Chaperone Human αB Crystallin Modulate Microtubule Assembly and Disassembly. PLoS ONE, 2, e498.
34. Mondal, P., Das, G., Khan, J., Pradhan, K., and Ghosh, S. (2018) Crafting of Neuroprotective Octapeptide from Taxol-Binding Pocket of β-Tubulin. ACS Chem. Neurosci., 9, 615-625.
35. Jana, B., Mohapatra, S., Mondal, P., Barman, S., Pradhan, K., Saha, A., and Ghosh, S. (2016) αCyclodextrin Interacts Close to Vinblas-tine Site of Tubulin and Delivers Curcumin Preferentially to the Tubulin Surface of Cancer Cell. ACS Appl. Mater. Interfaces, 8, 1379313803.
36. Adak, A., Mohapatra, S., Mondal, P., Jana, B., and Ghosh, S. (2016) Design of a novel microtubule targeted peptide vesicle for delivering different anticancer drugs. Chem. Commun., 52, 7549-7552.
37. Zhang, Z., Zhou, X., Zhou, X., Zhou, X., X, Xiao., Liao, M., Yan, L., Lv, R., and Luo, H. (2012) Methyl 3,4-dihydroxybenzoate promotes neurite outgrowth of cortical neurons cultured in vitro. Neural Regen Res., 7, 971-977.
38. Li, Y., Qin, L., Liu, J., Xia, W., Li, J., Shen, H., and Gao, W. (2016) GIT1 enhances neurite outgrowth by stimulating microtubule assembly. Neural Regen Res., 11, 427-434.
39. Ross, J. L., Santangelo, C. D., Makrides, V., and Fygenson, D. K. (2004) Tau induces cooperative Taxol binding to microtubules. Proc Natl Acad Sci U S A., 101, 12910-12915.
40. Carmelaa, M., Annaa, D. L., Giovannib, M., Simonac, D., Cinziaa, S., Teresaa, C. M., Antoninob, C., and Pietroa, C. (2008) Activation of the Amyloidogenic Route by NGF Deprivation Induces Apoptotic Death in PC12 Cells. J Alzheimers Dis., 13, 81-96.
ACS Paragon Plus Environment
Page 24 of 37
Page 25 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
41. Hyman, A., Drechsel, D., Kellogg, D., Salser, S., Sawin, K., Steffen, P., Wordeman, L., and Mitchison, T. (1991) Preparation of Modified Tubulins. Methods Enzymol 196, 478-485.
42. Becke, A. D. (1993) Density‐functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648-5652.
43. Lee, C., Yang, W., and Parr, R. G. (1988) Development of the Colle Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B, 37, 785-789.
44. Andrae, D., Haeussermann, U., Dolg, M., Stoll, H., and Preuss, H. (1990) Energy-adjustedab initio pseudopotentials for the second and third row transition elements. Theor. Chim. Acta, 77, 123-141.
45. Gaussian 09, Revision D.01, M. J. Frisch et al Gaussian, Inc., Wallingford CT, 2013. 46. Kong, J., White, C. A., Krylov, A. I., Sherrill, D., Adamson, R. D., Furlani, T. R., Lee, M. S., Lee, A. M., Gwaltney, S. R., Adams, T. R., Ochsenfeld, C., Gilbert, A. T. B., Kedziora, G. S., Rassolov, V. A., Maurice, D. R., Nair, N., Shao, Y., Besley, N. A., Maslen, P. E., Dombroski, J. P., Daschel, H., Zhang, W., Korambath, P. P., Baker, J., Byrd, E. F. C., Van Voorhis, T., Oumi, M., Hirata, S., Hsu, C.P., Ishikawa, N., Florian, J., Warshel, A., Johnson, B. G., Gill, P. M. W., Head-Gordon, M., and Pople, J. A. (2000) Q‐Chem 2.0: a high‐performance ab initio electronic structure program package. J. Comput. Chem., 21, 1532-1548.
47. Trott, O., and Olson, A. J. (2010) AutoDock Vina: Improving the Speed and Accuracy of Docking with a New Scoring Function, Efficient Optimization, and Multithreading. J. Comput. Chem., 31, 455-461.
48. Gigant, B., Wang, C., Ravelli, R.B., Roussi, F., Steinmetz, M.O., Curmi, P.A., Sobel, A., and Knossow, M. (2005) Structural basis for the regulation of tubulin by vinblastine. Nature, 435, 519-522.
49. Kotar, A., Tomašič, T., Živković, M. L., Jug, G., Plavec, J., and Anderluh, M. (2016) STD NMR and molecular modelling insights into interaction of novel mannose-based ligands with DC-
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
SIGN. Org. Biomol. Chem., 14, 862-875.
50. Canales, A., Salarichs, J. R., Trigili, C., Nieto, L., Coderch, C., Andreu, J. M., Paterson, I., Barbero, J. J., and Díaz, J. F. (2011) Insights into the Interaction of Discodermolide and Docetaxel with Tubulin. Mapping the Binding Sites of Microtubule-Stabilizing Agents by Using an Integrated NMR and Computational Approach. ACS Chem. Biol., 6, 789-799.
51. Biswas, A., Kurkute, P., Saleem, S., Jana, B., Mohapatra, S., Mondal, P., Adak, A., Ghosh, S., Saha, A., Bhunia, D., Biswas, S. C. and Ghosh, S. (2015) Novel hexapeptide interacts with tubulin andmicrotubules, inhibits Aβ fibrillation, and shows significant neuroprotection. ACS Chem. Neurosci., 6, 1309-1316.
52. Xu, S. Y., Wu, Y. M., Ji, Z., Gao, X. Y., and Pan. S. Y. (2012) A Modified Technique for Culturing Primary Fetal Rat Cortical Neurons. J. Biomed. Biotechnol., 2012, 803930.
53. Bhunia, D., Mondal, P., Das, G., Saha, A., Sengupta, P., Jana, J., Mohapatra, S., Chatterjee, S., and Ghosh, S. (2018) Spatial Position Regulates Power of Tryptophan: Discovery of a MajorGroove-Specific Nuclear-Localizing, Cell Penetrating Tetrapeptide. J. Am. Chem. Soc., 140, 1697-1714.
54. Gynther, M., Petsalo, A., Hansen, S. H., Bunch, L., and Pickering DS (2015) Blood-brain barrier permeability and brain uptake mechanism of kainic acid and dihydrokainic acid. Neurochem Res., 40, 542-549.
55. Alonso, E., Vieira, A. C., Rodriguez, I., Alvariño, R., Gegunde, S., Fuwa, H., Suga, Y., Sasaki, M., Alfonso, A., Cifuentes, J. M., and Botana, L. M. (2017) Tetracyclic Truncated Analogue of the Marine Toxin Gambierol Modifies NMDA, Tau, and Amyloid β Expression in Mice Brains: Implications in AD Pathology. ACS Chem. Neurosci., 8, 1358-1367.
ACS Paragon Plus Environment
Page 26 of 37
Page 27 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
Figure 1: (A) Synthesis scheme and structure of three triazine based compound. Reagents and conditions: (a) Methyl ester of amino acids, DIPEA, Dry THF, Reflux for overnight. Microtubule polymerization assay using DAPI of TY3 (B) and TF3 (D). SPR sensogram for TY3 (C) and TF3 (E) in presence of tubulin.
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 2: Energy transfer graph between (A) Tub-Colchicine complex and F-TY3; (B) Tub-ANS complex and F-TY3; (C) Tub-TAMRA E3NAP and F-TY3 complex; (D) Tub-Colchicine complex and FTF3; (E) Tub-ANS complex and F-TF3; (F) Tub-TAMRA E3NAP and F-TF3 complex. (G) Diagram for the distance calculation from FRET experiments. Molecular docking image showing interaction of (H) TY3 with PRO 274, ARG 278, ARG 284, ARG 320 and GLY 370 and (I) TF3 with PRO 274, THR 276, GLY 370 and LEU 371. (N) Binding position of TF3 (shown in grey colored sphere model) and TY3 (shown in cyan colored sphere model) in dimeric tubulin.
ACS Paragon Plus Environment
Page 28 of 37
Page 29 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 3: (A) Off-resonance NMR spectrum (600MHz) of TY3 (upper line) STD experiment (lower line) with TY3 in presence of tubulin. Zoom spectrum for aromatic region (Inset). Aromatic region shown in zoom spectrum (Inset). (B) Epitope mapping of TY3 (C). (D) % STD of different proton of TY3.
Figure 4: (A) TR-NOESY spectrum (mixing time: 300ms) of presence of TY3. (B)Tubulin bound conformation of TY3 and (C) its interacting partners.
ACS Paragon Plus Environment
Page 30 of 37
Page 31 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Paragon Plus Environment
Page 32 of 37
Page 33 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
Figure 5: Cell viability assay of (A) TY3 and (B) TF3 in PC12 cell lines. (C) Nocodazole induced depolymerization Experiment. (D) Immunoblot depicts the increase of acetylated tubulin (AcK-40) as compared to control with TY3 treatments. (E) Bar diagram reveals the relative expressions of acetylated tubulin normalized with loading control. All experiments have been performed in triplicate. The error bar corresponds to the standard deviation of the value. (*p < 0.05, performing two tailed student’s t-test).
Figure 6: (A) Neurite outgrowth of PC12 derived neurons after treatment with triazine derivatives (TF3, TY3 and TS3). (B) Quantative analysis for neurite outgrowth of PC12 derived neurons. Microscopic images of microtubule networks of PC12 cells for control (C) and after treatment with TY3 (D) at merged images. All experiments have been performed in triplicate. The error bar corresponds to the standard deviation of the value. (*p < 0.05, performing two tailed student’s t-test).
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 7: (A) ThT assay of TY3. (B) Bar diagram analysis represents cell viability of differentiated PC12 derived neurons by NGF assay in absence or presence of various concentrations of TY3 and Anti-NGF. Microscopic images of the (C) control PC12 cells, (D) Anti-NGF treated cells and (E) TY3 treated cells. (F) Western blot of phospho-TrkA (pY490) (pTrkA) and full-length TrkA (TrkA) protein levels in PC12 derived neurons exposed to NGF (+NGF) or deprived of NGF for 24 h (-NGF). The expression of pTrkA increases due to 24 h of NGF deprivation, while decreases on treatment with TY3. On the other hand the expression of full length TrkA remains the same. (G) Bar diagram represents the quantitative analysis of the immunoblots. All experiments have been performed in triplicate. The error bar corresponds to the standard deviation of the value. (*p < 0.05, performing two tailed student’s t-test).
ACS Paragon Plus Environment
Page 34 of 37
Page 35 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
Figure 8: Microscopic images of primary cortical neurons after treatment with TY3 at (A) bright field, (B) 561 nm channel and (C) merged channel. (D) Immunoblotting experiment showing higher activation of MAP2 and β-III tubulin in primary rat cortical neurons after TY3 treatment as compared to Control and (E) their quantitative analysis. (F) Blood Brain Barrier crossing experiment. All experiments have been performed in triplicate. The error bar corresponds to the standard deviation of the value. (*p < 0.05 and **p < 0.03, performing two tailed student’s t-test).
ACS Paragon Plus Environment
ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Table of content
ACS Paragon Plus Environment
Page 36 of 37
Page 37 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
A tyrosine rich tripodal neuroprotective molecule has been designed, where phenol group plays crucial role in interacting with microtubule and confers neuroprotection. Results reveal that molecule has significant serum stability and capability to cross blood-brain barrier. 417x394mm (300 x 300 DPI)
ACS Paragon Plus Environment