Cholinergic inhibitors replace thioflavin-T from acetylcholinesterase binding pocket: A potential fluorescence based molecular switch

Accepted Manuscript Research paper Cholinergic inhibitors replace thioflavin-T from acetylcholinesterase binding pocket: A potential fluorescence based molecular switch Mullah Muhaiminul Islam, Sivaprasad Mitra PII: DOI: Reference:

S0009-2614(16)30763-1 http://dx.doi.org/10.1016/j.cplett.2016.09.079 CPLETT 34220

To appear in:

Chemical Physics Letters

Received Date: Revised Date: Accepted Date:

12 July 2016 26 September 2016 30 September 2016

Please cite this article as: M. Muhaiminul Islam, S. Mitra, Cholinergic inhibitors replace thioflavin-T from acetylcholinesterase binding pocket: A potential fluorescence based molecular switch, Chemical Physics Letters (2016), doi: http://dx.doi.org/10.1016/j.cplett.2016.09.079

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Cholinergic

inhibitors

replace

thioflavin-T

from

acetylcholinesterase binding pocket: A potential fluorescence based molecular switch

Mullah Muhaiminul Islam, and Sivaprasad Mitra*

Centre for Advanced Studies in Chemistry, North-Eastern Hill University, Shillong – 793022, India

*Author to whom all correspondence should be addressed. Phone: (91)-364-2722634. Fax: (91)-364-2550076. E-mail: [email protected], [email protected]. 1

Abstract

The fluorescence intensity of acetylcholinesterase (AChE) bound thioflavin-T (ThT) is found to decrease regularly in presence of cholinergic inhibitors. The quenching phenomenon is not consistent with typical Stern-Volmer mechanism and can be explained on the basis of dynamic replacement of the probe from AChE gorge into the bulk aqueous medium. The resulting modulation in steady state intensity and/or time-dependent fluorescence depolarization forms the basis of a molecular switch between ThT and inhibitors, which can be correlated with AChE inhibition activity of the drugs.

Key-words: Acetylcholinesterase (AChE) inhibitors; thioflavin-T (ThT); fluorescence anisotropy; quenching; molecular switch.

2

1. Introduction The cationic organic dye, 3,6-dimethyl-2-(4-dimethylaminophenyl)-benzothiazolium ion (most commonly known as thioflavin-T, ThT), is a fluorescent molecule and found wide application in the detection and quantification of amyloid fibrils [1, 2]. The various fluorescence parameters of the dye are strongly dependent on the concentration, solvent polarity and/or viscosity and also on the excitation wavelength. The three different types of species, viz. protonated, the locally excited (LE) and the dimerized state of the molecule, are the main origin to give fluorescence in this molecule. ThT displays extremely low fluorescence yield in aqueous buffer medium or in low viscous solvents. This is primarily due to the fact that in low viscous medium, the ThT molecule undergoes twisted internal charge transfer (TICT) process to generate a nonfluorescent state from where the molecule relaxes through a nonradiative pathway. The torsional motion of the benzthiazole and aminobenzene rings relative to each other is responsible for the origin of TICT state of ThT in low viscous solvents [3, 4]. ThT can be used as a molecular rotor as its fluorescence intensity is strongly increased due to the restricted rotation of the benzthiazole ring in a solution with high viscosity [3]. The photophysical property of ThT is found to be strongly altered when the dye binds with deoxyribonucleic acid (DNA), as compared to that in the bulk aqueous phase [5, 6]. The dye is generally used to detect the amyloid beta (A) fibrils, an insoluble filamentous aggregate, of proteins mostly due to the intercalation of ThT molecules within grooves between solvent-exposed side chains of the amyloid fibril that run parallel to the fibril axis [7]. Binding within the channels is believed to provide rigidity in the molecule and to prevent the formation of TICT state. While the formation and deposition of amyloid-beta (A) fibrils in brain tissues [8] is considered to be one of the key pathways towards the development of a chronic and progressive neurodegenerative disorder called Alzheimer’s disease (AD), the over activity of

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acetylcholinesterase (AChE) resulting insufficient release of neurotransmitter acetylcholine (ACh) in the neuromuscular junction has also been reported in the literature to be an important factor (cholinergic pathway) [9]. The A formation pathway has been extensively monitored by using ThT as a fluorescent marker in a variety of experimental conditions including in-situ real-time or single-time point dilution ThT assay methods [10]. On the other hand, the fluorescence of ThT is also known to be markedly enhanced in presence of AChE [11]. However, surprisingly, the number of literatures dealing with detailed understanding of AChE bound ThT fluorescence and its modulation in presence of cholinergic drugs is relatively scarce [11, 12]. Fluorescence based molecular switches have gained significant interest in recent times. Besides their application in sensing ions [13-15], solution pH [16], small molecules like nitric oxide [17] and hydrogen peroxide [18]; recent progress in the detection of large biological samples like membranes [19], proteins [20] and enzymes [21] has opened new possibilities in the fields of analytical, pharmaceutical and medical sciences. Some of the recent reports also demonstrate the use of sequestering agents like simple surfactant [22, 23], rotaxanes [24] as well as curcurbituril [25] etc. in controlling the response of suitably designed fluorophores towards desired application. In this communication, we report the detailed mechanistic understanding on the “on-off” fluorescence behavior of ThT under saturated bound condition with AChE in presence of few standard cholinergic inhibitors, some of which are already prescribed as FDI approved AD drugs. Interestingly, modulation of the characteristic ThT fluorescence parameters in presence of the inhibitors correlate directly with IC50 values of the drugs. 2. Materials and Method The type V-S, lyophilized powder form of acetylcholinesterase, activity ≥1,000 units /mg protein, from electrophorus electricus (electric eel) was purchased from Sigma—Aldrich

4

Chemical Company (product no C2888). The chemicals like thioflavin T (ThT, cat. No. T3516), tacrine hydrochloride (TAC, purity ≥99%, cat. No. A79922), galanthamine hydrobromide from lycoris sp. (GAL, purity ≥94% TLC, cat No. G1660), donepezil hydrochloride monohydrate (DON, purity ≥ 98% HPLC, cat No. D6821), (-)-Huperzine A (HuPA, product no H5902), eserin (ESE, purity ≥99% HPLC, cat No. E8375) were all received from Sigma—Aldrich Chemical Company and used directly as received without any further purification. The gelatin were received from Qualigens fine chemicals (a division of GlaxoSmithkline Pharmaceuticals Ltd.), India. The anhydrous GR form of di-potassium hydrogen phosphate, sodium di-hydrogen phosphate monohydrate and extra-pure analytical grade of sodium hydroxide pellets were received from Merck and Sisco Research Laboratories (SRL), India respectively. For making the buffer solution, the analytical grade type

ater (resistivity

10 MΩ.cm at room temperature)

as obtained from Elix 10

ater

purification system (Millipore India Pvt. Ltd.). The solution pH was checked with Systronics μ-pH system 361. The spectroscopic studies were done in 0.1 M phosphate buffer of pH 8.0. All the solutions were prepared afresh and kept for at least half-an-hour for settling before the spectroscopic measurements were performed. Steady state absorption measurements were performed in a Perkin-Elmer model Lambda25 absorption spectrophotometer. Fluorescence spectral measurements were done in Quanta Master (QM-40) steady state apparatus obtained from Photon Technology International (PTI) and all the spectra were corrected for the instrument response function. For monitoring the ThT fluorescence emission the samples were excited at 412 nm. The corresponding steady-state data, thus obtained from three separate experiments, were averaged and further analysed using Origin 6.0 (Microcal Software, Inc., USA). Although all the compounds except ThT used in this study have no significant absorption at the excitation

5

wavelength, each fluorescence spectrum in presence of various species was corrected for any possible inner filter effect using the following equation [26]: (1) Where, A represents the absorbance of the free ThT, and Atot is the total absorbance of the solution at the excitation wavelength (

). The corrected spectrum can be taken as the

contribution from ThT only. The fluorescence decay curves were obtained by using 410 nm LED excitation in a Pico Master time correlated single photon counting (TCSPC) lifetime apparatus (PM-3) supplied by Photon Technology International (PTI), USA. The instrument response function (IRF, ~1.8 ns FWHM) was obtained by using a dilute colloidal suspension of dried non-dairy coffee whitener. The experimentally obtained fluorescence decay traces I(t), collected at the magic angle (54.7°) to eliminate any contribution from the anisotropy decay, were expressed as a sum of exponentials (Eq. 2) and analysed by non-linear least-square iterative convolution method based on Levenberg–Marquardt algorithm [27] as implemented in the data analysis software (Felix GX version 4.0.3) from PTI. (2) Where, ai is the amplitude of the ith component associated with fluorescence lifetime τi such that Σai = 1. The reliability of fitting was checked by numerical value of reduced chi-square (χ2), Durbin– Watson (DW) parameter and also by visual inspection of residual distribution in the whole fitting range [28]. The fluorescence anisotropy decay experiment was done by using 408 nm picosecond laser diode (PiL040X obtained from A.L.S. GmbH) excitation in a Pico Master time correlated single photon counting (TCSPC)–polarization lifetime apparatus (PM3) supplied by PTI. The time dependent anisotropy, r(t) was calculated for each of the experimental sample by using the following equation: 6

(3) Where, G (= IHV/IHH) is the instrument geometry factor.

IVV and IVH represent the

fluorescence intensities for the vertically and horizontally polarized emission respectively, with the vertically polarized excitation. On the other hand, IHV and IHH represent the fluorescence intensities for the vertically and horizontally polarized emission respectively, with the horizontally polarized excitation. The anisotropy data, r(t) so obtained, were expressed as a sum of exponentials (Eq. 4) and analysed in an iterative nonlinear regression (NLR) program [27, 28]. (4) Where, αi is the amplitude of the ith component associated with the rotational correlation time Θi such that

=1.

3. Results and Discussion 3.1. ThT fluorescence in aqueous buffer medium. The absorption and fluorescence emission spectra of 2.0 M ThT in aqueous buffer of pH = 8.0 show maximum at around 412 nm and 478 nm, respectively. This result is consistent with the previously reported data for the dye; where the absorption spectral peak position was reported at ca. 412 nm. However, the fluorescence emission peak position appeared at ca. 489 nm when the sample was excited at 418 nm in pure water [4]. Interestingly, in one of the earlier reports, the excitation and emission peak positions for free ThT in aqueous environment was reported to be at 350 and 440 nm, respectively [29]. Although this difference in the reported spectral peak positions for ThT, particularly for fluorescence emission studies, is not clear; it may be possibly due to different experimental condition (buffer solution in this case, in contrast to the aqueous medium in reported literature) as well as excitation wavelengths (410 nm in the present study). Nevertheless, the 7

fluorescence yield of ThT in homogeneous buffer medium was found to be very low (f = 0.06) and consistent with the literature reports [30]. Time-resolved fluorescence analysis of 2 M ThT excited with 410 nm LED in homogeneous buffer solution yields non-exponential decay profile. Critical inspection of statistical parameters reveals that at least a sum of three exponential decay function is needed to fit the data adequately (supplementary Figure S1). However, the combined contribution of two longer components (1.4 and 6.9 ns, respectively) is always found to be < 2%; the major component (>98%) is within the IRF limit (~1.8 ns FWHM) and cannot be measured with confidence by the present experimental set-up. These observations are consistent with the very fast fluorescence decay of ThT reported earlier [4] or even in some of the very recent communications [31]. 3.2. Binding of ThT with acetylcholinesterase (AChE). The absorption spectrum of ThT does not change significantly (supplementary Figure S2) in presence of AChE; however, the fluorescence emission is strongly affected in the enzyme environment. The intensity of fluorescence increases significantly in presence of AChE. Also, the fluorescence emission peak of ThT is found to be shifted ca. 15 nm towards the red side of the spectrum along with sharpening of the spectral profile (Figure 1a). The increase in fluorescence intensity as well as shift in spectral peak position towards the longer wavelength region is consistent with ThT fluorescence modulation when bound with amyloid fibrils [32, 33]. While the increase in intensity is due to restricted non-radiative motion of benzothiazole and aminobenzene rings in the bound condition, the fluorescence shift is rationalized on the basis of the formation of a more stable fluorescing state upon binding. It is to be noted that in some of the earlier papers, the intensity of ThT was claimed to have increased over 800 times while bound with AChE [12]. However, no spectral data was

8

provided in those reports to support this claim. In the present study, the fluorescence intensity of ~ 3.75 M ThT undergoes about seven fold increase with increase in AChE concentration from 0 to 0.58 M (Figure 1b). A similar range of intensity increase has also been reported for ThT bound

ith amyloid fibrils formed by Aβ(1-40) and Aβ(1-42) [31] and a maximum

of about three- to six- hundred fold increase is reported for specific RNA G-quadruplex sequences [34] due to restricted rotational motion and planarization of the dye. The enhancement of ThT fluorescence intensity in presence of AChE was used to calculate the binding constant (Kb) of the dye with the enzyme using Benesi-Hilderband (BH) relation (Eq. 5a) assuming 1:1 binding ratio (supplementary Figure S3). The binding constant value obtained in this way results Kb = 1.30(±0.03)  106 M-1. (5a) (5b) On the other hand, a reverse titration keeping [AChE] fixed at 0.35 M and varying the [ThT] was also carried out to cross check the AChE-ThT binding (figure 1b). The ThT fluorescence intensity at 490 nm was used to calculate Kb using BH relation with varying [ThT] (Eq. 5b). The Kb value, so obtained, was used as an initial guess in an iterative nonlinear regression (NLR) program based on the Levenberg-Marquardt algorithm for the nonlinear curve fitting of the fluorescence data using Eq. 6 [35] and shown in figure 1b (inset). (6) Where, F1 = aF0 for the complex and “a” is the ratio of the fluorescence intensities of the complexed and uncomplexed chromophore; F0 and F are the fluorescence intensities in the

9

absence and presence of ThT. The refined value of binding constant, so obtained, yields the value of Kb = 3.7(±0.6) 105 M-1. Time-resolved fluorescence decay of ThT remains practically unaffected in bound AChE medium while comparing with pure aqueous buffer (supplementary Table ST1). However, it is well known that ThT fluorescence lifetime increases substantially to several nanoseconds in presence of beta amyloid and/or nucleic acid binding [31, 34] in comparison with < 1 ps decay time in aqueous buffer medium. This dramatic change in lifetime is again inferred as due to the restricted rotation of ThT under bound condition. This difference in modulation of ThT fluorescence (relatively weak fluorescence intensity enhancement as well as almost no effect on fluorescence lifetime) in presence of AChE can be rationalized on the basis of the binding behaviour of the dye. While the intensity enhancement and detection of A fibrils is due to the formation of a tight complex of ThT with cross- structure formed ith β-strands oriented perpendicular to the fibril long axis [36], the binding with nucleic acids proceeds either through end-stacking or groove binding mode [31, 34]. On the other hand, it is already well-known in the literature that the ThT binding site of AChE can be considered as the upper side of a narrow but 20 Å deep gorge with the benzothiazole ring pointing inside [37]. Although the binding efficiency is quite strong (as revealed from the fluorescence titration results) due to involvement of a series of amino acid residues in the acylation site, complete blocking of the C-C rotational non-radiative pathway is not achieved due to volume mismatch in AChE binding site. Therefore, no significant change is noticed in fluorescence lifetime of ThT; however, only moderate increase in ThT fluorescence efficiency is observed. The situation is quite similar to the fact that although ThT exhibits a reasonable binding affinity (ca. 1.5 × 105 M-1) with dsAf16 nucleotide of duplex DNA/RNA, the dye exhibits relatively weak fluorescence enhancement due to the flexible orientations at the binding site [38].

10

It is important to note that although some steady state anisotropy measurement of ThT is reported to explore its binding in nucleic acids/A fibrils [33, 39], the time dependent anisotropy decay behaviour of ThT is relatively less explored, particularly with AChE bound condition. The analysis of the anisotropy decay data (Figure 2) indicates that although the ThT anisotropy decay is single exponential both in aqueous buffer medium as well as in the enzyme environment with saturated bound condition, the rotational correlation time (Θ) of ThT is found to be increased from 2.30.13 ns in aqueous buffer medium to 3.80.25 ns in the saturated enzyme bound condition. The increased value of Θ is consistent

ith the

restricted motion of ThT inside the enzyme binding pocket. 3.3. Quenching of the bound ThT fluorescence in the presence of AChE inhibitors. On addition of varying concentration of all the inhibitors to the ThT-AChE system, the absorption spectra do not undergo any significant change. However, the emission spectral peak position of the AChE-ThT-drug ternary system is observed to be shifted by ca. 5 nm towards free ThT peak position side with broadening in the spectral profile as observed in homogeneous buffer (figure 3a). In addition, the fluorescence intensity of ThT bound with AChE is shown to decrease continuously with addition of different concentrations of the drug (figure 3b). The fluorescence excitation spectra of ThT-AChE binary system also remain unaffected with the addition of drug. The efficiency of quenching is highest for DON and TAC, lowest in the cases of GAL and ESE; whereas, HuPA displays moderate quenching behaviour. Normally, a variety of reasons may be responsible for the observation of quenching in fluorescence which include ground state complex formation (static), collisional (dynamic) quenching, excited state reactions including conformation rearrangement of the fluorophore and also energy transfer between the fluorophore in the excited state (donor) and suitable acceptor (quencher) in the ground state. Identification of the quenching mechanism normally involves analysis of the steady state and/or time-resolved fluorescence data using 11

Stern-Volmer

(SV)

analysis

in

combination

with

careful

inspection

of

the

absorption/excitation and fluorescence emission spectra of both the fluorophore as well as quencher independently and also in presence of each other [28]. The SV plot for the quenching of AChE bound ThT fluorescence in presence of cholinergic drugs does not show any linear correlation; instead a downward curvature is observed in all the cases. Some of the representative plots are shown in figure 4. This type of quenching data is usually associated with fractional accessibility of the fluorophores to the quenchers in a bio-macromolecular environment. Modified SV analysis incorporating only the accessible fluorophores results a straight line with characteristic quenching parameters. However, even the modified SV plots do not show acceptable linear correlation in the present case (supplement figure S4). Also, the fluorescence lifetime data remains practically constant over the whole quencher (inhibitor) concentration used in the present study, ruling out the involvement of both static and/or dynamic quenching processes. Finally, the absorption spectrum of all these drugs normally shows a peak at ca. 310 nm within the 250 ~ 350 nm wavelength region and does not possess any spectral overlap with the ThT fluorescence. Therefore, energy transfer from the excited fluorophore is not considered as a possible mechanism of fluorescence quenching. To get a clear insight into the fluorescence quenching mechanism, a controlled fluorescence titration experiment was performed to check the effect of TAC, which shows highest quenching ability on the ThT fluorescence, in presence of glycerol. The fluorescence intensity of ThT is known to increase in presence of glycerol as compared with pure buffer [40] and can be rationalized on the basis of increased viscosity of the medium. In this set of experiment, the ThT fluorescence was monitored in 25 % (v/v) glycerol-buffer mixture on addition of increased TAC concentration. Interestingly, the data indicates that the fluorescence emission intensity of ThT in glycerol remains practically unaffected in the 12

presence of entire concentration range of TAC used in this study (supplement figure S5), which confirms the involvement of AChE in this quenching process. In fact, the quenching of AChE bound ThT fluorescence in presence of inhibitor drugs can be considered to be a direct consequence of the partial replacement of the dye from the AChE gorge to the aqueous buffer phase with extremely low fluorescence yield (section 3.1). The cholinergic drugs are known to have the tendency to bind either at the peripheral anionic site (PAS) or acylation site (Asite) of the AChE binding region and inhibits the approach of ACh towards its hydrolysis by the enzyme.

Therefore, addition of these drugs in AChE – ThT system causes the

replacement of a fraction of bound ThT from the AChE gorge resulting significant decrease in emission intensity as well shift in fluorescence emission peak. The competing affinity of the inhibitors (see molecular docking calculation result described below) with ThT, towards the AChE binding pocket, results a molecular s itch system

ith “on-off” fluorescence

response and can form the basis of assaying the inhibition efficiency of the drugs. Such kind of mechanisms related to a molecular switch, which is able to induce quenching phenomena, have already been reported in the literature [41, 42]. To get further justification on the replacement of ThT from AChE binding site in presence of inhibitor drugs, molecular docking calculation was performed on each of the systems against AChE using AutoDock version 4.0. The characteristic docking parameter like binding energy (or, inhibition constant, Ki) of ThT was estimated to be -8.14 kcal mol-1 (Ki = 0.108 m). On the other hand, the corresponding parameters for the inhibitors like DON, GAL, ESE, HuPA and TAC were found to be -11.56 (0.003), -8.94 (0.28), -8.76 (0.38), -8.51 (0.58) and -7.36 (4.0), respectively. The binding affinities of all the ligands are either higher or very close to that in the case of ThT, which gives thermodynamic justification of the replacement of the later in presence of inhibitors. However, from the maximum decrease in fluorescence intensity data, it can be inferred that complete removal of 13

ThT cannot be achieved even in presence of excess quencher (drug) concentration; rather, equilibrium between the bound and free drug is established in AChE – ThT – drug ternary system. 3.4. Fluorescence anisotropy decay in presence of AChE inhibitors. The ThT replacement can also be confirmed by the fluorescence anisotropy decay data of AChE-ThT system titrated against the inhibitor drugs. In sharp contrast to the single exponential anisotropy decay of ThT either in aqueous buffer or in saturated bound condition in AChE (figure 2), the anisotropy decay of ThT in presence of quencher drugs needs at least two exponential decay function corresponding to both the free and bound fluorophores with Θ = 2.30.13 and 3.80.25 ns, respectively. Further, with increasing the drug concentration, the contribution of longer rotational correlation time (2) from the bound component decreases gradually with the concomitant increase in contribution from the bulk component (1) with lower rotational correlation time. This observation is consistent with the fact that in presence of inhibitor drugs, the fluorescence reporter ThT is knocked out of the AChE binding pocket and moves into the aqueous buffer medium. Figure 5 reflects the representative variation in amplitudes of fast and slow anisotropy decay components (1 and 2, respectively) with varying drug concentration in different cases. Interestingly, the cross over point for 1 and 2 appears at the individual quencher concentration of 0.46, 0.24, 0.30, 7.90 and 11.52 M, respectively for DON, TAC, HuPA, ESE and GAL. The required quencher concentration leading to this cross over in the pre-exponential factors of fast and slow anisotropy decay component is in qualitative agreement with the trend of their respective AChE inhibition efficiency characterized by IC50 values of the individual drugs [43, 44].

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Conclusion The decrease in AChE bound ThT fluorescence intensity in presence of several standard AD drugs is due to the partial replacement of the fluorophore from the enzyme binding site. The drug concentration representing the cross-over point for amplitudes associated with short and long anisotropy decay components (corresponding to free and bound ThT, respectively) is consistent with reported IC50 values of the individual drugs. These modulated fluorescence parameters representing the ThT knocking off from the AChE gorge in presence of inhibitors can form the basis for the development of highly sensitive fluorescence based molecular switch to assay the potency of already existing and/or newly developed AD drugs working in cholinergic pathway.

Acknowledgements. Thanks are due to Dept. of Science & Technology (DST), Govt. of India for supporting the Chemistry Department through FIST program. MMI is the recipient of a research fellowship from UGC.

Supporting Information. Supplementary material associated with this article can be found online.

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Figure captions Figure 1:

(a) Normalized fluorescence emission spectrum of 3.75 M ThT in aqueous buffer solution of pH = 8.0 in absence (soild circle) and presence of 0.35 M AChE (open circles). (b) Variation in fluorescence intensity with increasing concentration of ThT from 0 to 3.75 M (along the arrow direction) in presence of fixed concentration of AChE (0.35 M). Inset: Non-linear regression analysis (solid line) of AChE – ThT binding data (open circles) obtained by subtracting the blank (open triangle) intensity from the raw data with varied ThT concentration.

Figure 2:

Fluorescence anisotropy decay of 3.75 M ThT in buffer (squares) and in presence of 0.35 M AChE (circles).

Figure 3:

(a) Normalized fluorescence emission spectra of AChE bound ThT under saturated condition (solid line). Same data in presence of 2 M GAL (dashed line) and TAC (dotted line) are also shown to compare the spectral blue shift and broadening in presence of inhibitors. (b) Fluorescence quenching of AChE bound ThT in presence of HuPA.

Figure 4:

Stern-Volmer (SV) plot for the fluorescence quenching data of AChE bound ThT in presence of different cholinergic drugs. The solid line guides the eye along the data points.

Figure 5:

Change in amplitudes of long (triangle) and short (circle) anisotropy decay components with increasing concentration of different inhibitor drugs like DON (a), GAL (b), HuPA (c) and ESE (d). The solid line guides the dye along the data points. The concentration of drug at which these two parameters make cross over is also indicated in each case. 19

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Graphical_Abstract

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Cholinergic

inhibitors

replace

thioflavin-T

from

acetylcholinesterase binding pocket: A potential fluorescence based molecular switch

Mullah Muhaiminul Islam, and Sivaprasad Mitra*

Centre for Advanced Studies in Chemistry, North-Eastern Hill University, Shillong – 793022, India

Highlights

 Addition of cholinergic inhibitors quenches AChE bound ThT fluorescence ;  Fluorescence quenches due to knocking off the bound fluorophore by the drug;  No quenching is observed in glycerol medium;  Fluorescence anisotropy decay modulation corroborates with IC50 values of drugs;

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