Competitive Inhibition Mechanism of Acetylcholinesterase without

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Competitive Inhibition Mechanism of Acetylcholinesterase without Catalytic Active Site Interaction: Study on Functionalized C60 Nanoparticles via in vitro and in silico Assays Yanyan Liu, Bing Yan, David A. Winkler, Jianjie Fu, and Aiqian Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 11 May 2017 Downloaded from http://pubs.acs.org on May 12, 2017

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ACS Applied Materials & Interfaces

Competitive Inhibition Mechanism of Acetylcholinesterase without Catalytic Active Site Interaction: Study on Functionalized C60 Nanoparticles via in vitro and in silico Assays Yanyan Liu1,2, Bing Yan3,4, David A. Winkler5-8, Jianjie Fu1,2, Aiqian Zhang1,2,* 1

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China 2 College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100190, China 3 School of Chemistry and Chemical Engineering, Shandong University, Jinan, China 4 School of Environment, Guangzhou Key Laboratory of Environmental Exposure and health and Guangdong Key Laboratory of Environmental Pollution and Health, Jinan University, Guangzhou 510632, China 5 CSIRO Manufacturing, Clayton 3168, Australia 6 Monash Institute of Pharmaceutical Sciences, Parkville 3052, Australia 7 Latrobe Institute for Molecular Science, Bundoora, 3046, Australia 8 School of Chemical and Physical Science, Flinders University, Bedford Park 5042, Australia

*Corresponding author：Tel: 86-10-62849157; Fax: 86-10-62849339; E-mail address: [email protected]

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Abstract Acetylcholinesterase (AChE) activity regulation by chemical agents or, potentially, nanomaterials is important for both toxicology and pharmacology. Competitive inhibition via direct catalytic active sites (CAS) binding or noncompetitive inhibition through interference with substrate/product entering/exiting has been recognized previously as an AChE inhibition mechanism for bespoke nanomaterials. The competitive inhibition by peripheral anionic site (PAS) interaction without CAS binding remains unexplored. Here we proposed and verified the occurrence of a presumed competitive inhibition of AChE without CAS binding for hydrophobically functionalized C60 nanoparticles (NPs) by employing both experimental and computational methods. The kinetic inhibition analysis distinguished six competitive inhibitors, probably targeting the PAS, from the pristine and hydrophilically modified C60 NPs. A simple quantitative nanostructure-activity relationship (QNAR) model relating the pocket accessible length of substituent to inhibition capacity was then established to reveal how the geometry of the surface group decides the NP difference in AChE inhibition. Molecular docking identified the PAS as the potential binding site interacting with the NPs via a T-shape plug-in mode. Specifically, the fullerene core covered the enzyme gorge as a lid through π-π stacking with Tyr72 and Trp286 in the PAS, while the hydrophobic ligands on the fullerene surface inserted into the AChE active site to provide further stability for the complexes. The modelling predicted that inhibition would be severely compromised by Tyr72 and Trp286 deletions, and the subsequent site-directed mutagenesis experiments proved this prediction. Our results demonstrate AChE competitive inhibition of NPs without CAS participation to gain further understanding of both the neurotoxicity and curative effect of NPs. 2


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Keywords: C60 nanoparticle, acetylcholinesterase, competitive inhibition, peripheral anionic site, quantitative nanostructure-activity relationship, molecular docking, site-directed mutation

Introduction Acetylcholinesterase (AChE) is an important enzyme in the central nervous system. It controls neurotransmission by fast hydrolysis of the cationic neurotransmitter acetylcholine (ACh). Neurodegenerative disorders such as Alzheimer’s disease (AD) are characterized by a reduced level of ACh; thus, the inhibition of AChE is one of the key therapeutic solutions for the treatment of AD.1 Various small-molecule drug AChE inhibitors have been designed.2-3 Moreover, AChE has also been regarded as an important biomarker of pollutant neurotoxicity, and chemicals of diverse structure have presented the AChE inhibition potential.4 Various pollutants other than organophosphate and carbamate pesticides exhibited strong AChE inhibition capacity, including heavy metals, polycyclic aromatic hydrocarbons, and detergents. Recently, different classes of nanoparticles (NPs) showed high AChE affinity and could inhibit AChE activity in a dose-dependent manner.5 Fig. 1A shows a sketch of the active pocket of AChE enzyme, also known as the AChE gorge. Two regions are particularly important for the AChE normal catalysis activity: the catalytic activity site (CAS) at the bottom of the gorge approximately 20 Å deep; and the peripheral anionic site (PAS) at the upper part of the gorge. The key residues involved in the substrate reaction, hydrolyzing acetylcholine to acetic acid and choline, is the catalytic triad consisting of Ser203, Glu334 and His447 in CAS, while the dominant feature of PAS is a group of 14 highly conserved aromatic residues lining the gorge upper part leading to CAS. These aromatic residues are 3



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responsible for both substrate and inhibitor binding. Although various kinds of inhibitors have been found or developed for AChE, such as metal ions,6 small organic chemicals like TZ2PA6,7 tacrine8 and stryphnusin analogs,9 bespoke nanomaterials,10 and even peptide11 and aptamer,12 only competitive inhibition via direct CAS binding,13 both CAS and PAS interaction,7, 13-14 or noncompetitive inhibition through interference with the substrate/product entering/exiting has been recognized previously as AChE inhibition mechanisms.13,

15

Fullerene (C60) and its derivatives possess interesting physicochemical properties that have resulted in studies of their potential in biomedical applications, in particular. Numerous studies have revealed the direct interaction of fullerene and its derivatives with biomolecules, including enzymes,16-18 transport proteins,19-22 peptides,23 and DNA.24 Experiments have shown that C60 NPs penetrate cell membranes,25 and computer simulations have illustrated how they permeate lipid bilayers without major membrane disruption.26 Moreover, C60 NPs have been shown to penetrate the blood–brain barrier and enter the brain,27 consistent with the reported neuroprotective effects of C60 NPs.28-29 However, very little is known about the molecular targets for, and mechanisms of, the neuroprotective activity of C60 NPs. As the cholinergic signaling system is clearly involved in important neurological diseases, we presumed AChE as the potential target for C60 NPs and investigated the interaction of surface functionalized fullerenes with AChE.

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A

Front view

10 Å

B

CAS inhibitor

5Å

Plug-in mode

Mid-gorge

5Å

C

PAS inhibitor

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Vertical section

EP

Figure 1. (A) The opaque surface of the active pocket of AChE enzyme from the front view, including the catalytic active sites (CAS), peripheral anionic sites (PAS) and the narrowest mid-gorge. (B) The vertical section of the pocket. The lipophilic potential (LP) of the surface is characterized with color ramps from brown (the highest lipophilic area of the molecule) to blue (the highest hydrophilic area). The electrostatic potential (EP) of the surface is characterized with color ramps from red (most positive) to purple (most negative). (C) The plug-in mode between the surface-modified C60 NPs and the PAS of the pocket.

As mentioned above, the reported competitive inhibition, especially for C60 nanoparticles, up to now, must involve direct CAS binding, either through exclusive CAS interaction30 or by simultaneous interaction of both CAS and PAS via a structural linker,13-14, 30-31 which can pass through the narrowest part, of about 5 Å in length, in the AChE active gorge. The reason is rather clear. The entrance to the gorge has a diameter of approximately 10 Å, much larger than that of a single fullerene molecule (7 Å). An aggregated C60 cluster evidently cannot 5



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enter the gorge. However, a protruding fullerene unit on the edge of the aggregate may interact with the PAS of AChE.32-33 The binding of the fullerene moiety of C60 NPs to the PAS may block the gorge entrance, thus interfering with the access for the substrate and egress of the product. Nevertheless, if strong ligand-protein interaction only occur at the entrance of the funnel-shape PAS regions based on π-π stacking between fullerene molecule and aromatic residues and electrostatic interaction, typically demonstrated by the cationic fullerene

nanoparticles

like

bis-N,N-dimethylfulleropyrrolidinium

functionalized-C60

derivatives,33 enzyme inhibition via this Lid-type mechanism are non-competitive. A study on the bait proteins of C60 also found the cationic fulleropyrrolidinium ions occupying the hydrophobic PAS cavity of AChE and acting as noncompetitive and low-affinity inhibitors by hindering the access of a substrate.34 Both experimental and modeling results showed that, in addition to the cationic ones, anionic ones35 and neutral hydrophilic fullerene28 also cannot get incremental complex stability from its surface group because the upper part of the gorge is of high hydrophobicity. On the contrary, some functionalized fullerenes with long surface chain exhibited competitive inhibition potential for AChE because they can directly interact with the key residues in the CAS and block the substrate access tunnel through PAS binding as well.30 Unfortunately, the competitive inhibition initiated by PAS interaction without CAS binding remains unexplored. Questions remain regarding which kind of surface modification can make C60 NPs become competitive inhibitors without CAS binding and what are the key molecular interactions that facilitate bindings between such C60-like NPs and AChE?

Considering the two sites, CAS and PAS, are nearly 15 Å apart, a potential AChE competitive inhibition mechanism was proposed here for functionalized C60 nanoparticles, symbolized by a special T-shape plug-in interaction mode with its one fullerene core covering the funnel-shape gorge entrance as a lid and its functional group inserting into the upper gorge 6


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to provide further stability for the complexes (Fig. 1C). According to the structural feature of the PAS, such plug-in mode requires the hydrophobical surface modification on the fullerene cage because the upper part of the gorge is of high hydrophobicity. The high hydrophobicity of the upper channel makes the hydrophilic surface group energetically less favorable for such mechanism. Moreover, the size of the surface group should be optimized to fit that of the upper channel with the length of about 10 Å. Different from efficient competitive inhibitors directly bound to CAS, such as edrophonium,36 our attention has been paid on the hydrophobically functionalized C60 targeting PAS in the proposed T-shape plug-in mode to present its competition. Therefore, 6 hydrophobically functionalized fullerenes with certain size of surface groups were used to verify the presumed mechanism by both in vitro and in silico methods, and unmodified fullerene and two hydrophilic fullerenes were used as control and contrast, respectively (Fig. 2). The similar methodology has been successfully used to reveal the toxicity mechanism of fullerene and its derivatives,17, 37-38 in which computational methods have already been proved to be reliable tools to reveal the structural basis for their interaction with potential target proteins.30,

39-44

The enzyme inhibition kinetics result

identified the competitive NP inhibitors from other NPs, and a simple quantitative nanostructure-activity relationship (QNAR) model45 was constructed to elucidate key structural features deciding the difference of the competitive C60 inhibitors in the AChE inhibition capacity. The subsequent molecular docking not only indicated PAS as a binding site for the plug-in competitive inhibitors, but also revealed key residues predominating the putative π-π stacking interactions between C60 core and aromatic residues at the PAS. This finding was finally confirmed by site-directed mutagenesis experiments. Although we previously described how surface functionalized gold NPs interacted with the active site of AChE and generated a quantitative model linking surface chemistry to AChE inhibition,38 this

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is the first study investigating the AChE competitive inhibition by C60 NPs through PAS interactions without the synergy of CAS to our knowledge.

C60-1

C60-2

C60-3

C60-4

C60-5

C60-6

C60-7

C60-8

C60-9

Figure 2. Structures and labels of tested surface-modified C60 NPs.

EXPERIMENTAL SECTION Chemicals and Materials Nine fullerene derivative NPs were all purchased from Sigma-Aldrich(St. Louis, MO, USA): [6,6]-phenyl C61 butyric acid methyl ester (>99.5%), N-methylfulleropyrrolidine (99.0%), [6,6]-thienyl C61 butyric acid methyl ester (≥99%), fullerene-methyl nipecotate (≥95.0%), 1’,4’-dihydro-naphtho[2’,3’:1,2][5,6]

fullerene

C60

(97.0%),

8


(1,2-methanofullerene

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C60)-61-carboxylic

acid(97.0%),

C60

pyrrolidine

Tris-acid

(97.0%),

tert-butyl

(1,2-methanofullerene C60)-61-carboxylate purum (≥95.0%), and fullerene C60 (98.0%). AChE, substrate acetylthiocholine iodide (ATC) (≥98.0%) ， chromogenic reagent 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) (≥98.0%), the positive control neostigmine bromide (NB) (≥98%) and propidium iodide (PI) (≥94%) were all purchased from Sigma-Aldrich(St. Louis, MO). The phosphate-buffer saline solution (PBS) of 0.1 M was purchased from HyClone. Ultrapure water with a resistivity of 18.2 MΩ·cm was produced using an ultrapure water system (Barnstead International, Dubuque, IA, USA). Toluene was purchased from Thermo Fisher Science and its purity was above 99.0%. The Eppendorf tubes and the 96-well microtiter plates were purchased from Corning Costar Corp (Cambridge, MA, Britain). Luria–Bertani broth (LB) was used to culture the Escherichia coli strain BL21 (DE3) as the host strain. LB broth agar was provided by Sangon Biotech Corporation. The E. coli expression vector pET28a was obtained from Novagen and was used to produce the fusion protein. Isopropyl-β-D-1-thiogalactopyranoside (IPTG) and all the antibiotics were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Inhibition of AChE Activity by C60 NPs AChE activity was assessed using the microtiter plate adaptation method of the Ellman assay46 by an enzyme-linked immunosorbent assay (ELISA) plate reader.47 The AChE from Electrophorus electricus (eeAChE) has been widely used as an enzyme model in biochemical studies.48-49 The AChE family of enzymes from various species have a high sequence 9



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homology. For example, the sequence homologies between eeAChE and human or murine AChE proteins are 88.5% or 100%, respectively. Moreover, the amino acids lining the surface of the active pocket are highly conserved, in which 70% in AChE from different species are identical,50 while those of the catalytic site are even more conserved.51 The root-mean-square-deviation (RMSD) values of Cα atoms of the gorge structure from eeAChE (residue D5~T543) and human AChE (hAChE) (residues D5~P258, N265~P492, P495~T543) was 0.6 Å, much smaller than the relevant crystal resolution (2.76 Å for hAChE). Therefore, eeAChE was used as a model for AChE enzymes in this study. More detailed information for the inhibition assay is given in the Supporting Information. The interference of surface groups, possibly releasing from the NPs, were also evaluated by using surface-group-like substances for the AChE activity inhibition assay (Supporting Information). Moreover, the kinetic study was carried out to obtain information on the AChE inhibition mechanism of the NPs using two types of Lineweaver-Burk plots, one with varying NP concentrations to explore their enzyme inhibition mode and the other demonstrating ATC hydrolysis by eeAChE in the presence and absence of PI, a reversible inhibitor targeting PAS,52 to identify the potential binding site in comparison with two theoretically distinguishable lines suggested by Krupka.52 More detailed information for the kinetic assays is given in the Supporting Information.

QNAR Model Development and Validation The structures of the functional groups attached to fullerene were constructed and optimized by the Gaussian 09 package53 using the popular B3LYP hybrid functional with the 10


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standard 6-311G(d,p) basis set. The molecular length of the functional group was then calculated by measuring the distance between the carbon atoms linked to the fullerene core and the terminal atoms using Gaussian 09 (lg), and the group’s lipophilicity expressed as the logarithm of the octanol-water partition coefficient (logP) was obtained from ChemBioDraw Ultra 12.0 for both neutral and ionized speciations (Table 1). The negative logarithm of the concentration of the inhibitors causing 50% inhibition of the enzymatic reaction (pIC50) was defined as a biological activity index (dependent variable) for QNAR modelling as is traditionally the case (Table 1). The structural effect on AChE inhibition was explored using the linear regression. All data were used in the training set for the regression analysis with no compulsory estimation of the degree of freedom because the data set was too small to be divided into a test set and training set for robustness testing of the QNAR model. Therefore, the statistical significance of the QNAR model was verified by Monte Carlo simulation, and the reliability of the model was judged by comparing the correlation coefficient of the model (R) with that of the pseudo regression equation (R*). 54-55 (Supporting Information)

Molecular Docking Molecular docking has been applied here to elucidate the binding mode of the nanoparticles with AChE since the active gorge of the enzyme is well defined,30, 34, 39, 41, 43, 56 and necessary molecular dynamics simulation has also performed for the C60-AChE complexes to assist in finding adequate pose of the fullerene and its derivatives by taking the structural plasticity of the protein into considerations.40-44 In fact, even the large scale adaptive allosteric effect can be precisely revealed by well-designed molecular dynamics 11



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study.42 All molecular simulation studies on the competitive inhibitors were performed on DELL Precision 370 work station with SYBYL-X 1.2 software package from Tripos, Inc. Co. (SYBYL-X, version 1.2; Tripos International: St. Louis, MO, 2010) to obtain insight into the enzyme-inhibitor interaction. We selected the protruding single fullerene core at the surface of the clusters as the simplified NP model. The X-ray crystal structure of eeAChE PDB coded 1C2B was obtained from the Protein Data Bank (PDB, http://www.rcsb.org/pdb). The Tripos Surflex-Dock program was then used to explore the interaction mode of fullerene derivatives because it incorporated a base fragment matching algorithm into the automated docking procedure, allowing the fragment shift from its original position to a certain degree during pose optimization.57 Surflex-Dock’s scoring function, which contained hydrophobic, polar, repulsive, entropic, and salvation terms, was trained as the total score (TS) to estimate the dissociation constant (Kd) expressed in –log (Kd) unit,58 and the free energy of binding (kcal/mol) could be calculated from the Surflex pKd results, as previously described.59 Therefore, both the TS values and the binding modes were used to evaluate the binding affinity of the compounds in the PAS of eeAChE. Additionally, computational site-directed mutagenesis was carried out to verify the role of key residues identified by the docking analysis. The alanine mutant structure was generated from the wild-type (WT) enzyme structure by replacing the sidechain of the targeted residues at the Cγ position with a hydrogen atom simultaneously. Based on the assumption that the local perturbation of alanine mutant induces no significant AChE structural changes, the mutagenesis effect on the ligand binding could be calculated according to the TS values obtained before and after mutation. More detailed information for the docking operation is given in the Supporting Information. 12


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Expression and Purification of Recombinant AChE One microliter of the pET28a expression vector was transformed into BL21 (DE3)-competent cells via heat-shock transformation. The competent cells with pET28a were put back on ice for 2 min after 90 s of heat-shocking at 42°C. The obtained culture was plated on LB broth agar plates containing 30 µg /mL of kanamycin and incubated at 37°C overnight. The positive transformants harboring pET28a were grown overnight at 37°C in LB supplemented with the antibiotics ingredients mentioned above. The culture was inoculated in fresh LB medium (1:100 dilution) of 4 L containing 30 µg/mL of kanamycin and allowed to grow to the mid-log phase (OD600 =0.6) at 37 °C for test expression. Expression of the eeAChE mutant was induced with 0.5 mM IPTG at 20 °C, and expression was continued overnight. For the eeAChE mutant purification, the cells were harvested by centrifugation, and the pellet was resuspended in a buffer (20mM PBS, 300mM NaCl, 0.1%TritonX-100, 1mM DL-Dithiothreitol(DTT), pH 8.0) and sonicated to break the cell-wall using a probe sonicator. The soluble and insoluble fractions of the cell lysate were separated by centrifugation at 12000 rpm at 4 °C for 20 min. The supernatant was used for further purification. The protein purification of target eeAChE was conducted using an Ni-IDA column, and the column was first washed at a rate of 5 mL/min with 10 column volumes (CV) of binding buffer (20 mM PBS, 300 mM NaCl, 0.1% TritonX-100, 1 mM DTT, pH 8.0) and then was eluted at a rate of 2 mL/min with wash buffer (10 mM imidazole, 20 mM PBS, 300 mM NaCl, 0.1% TritonX-100, 1mM DTT, pH 8.0). The fusion protein was eluted with a pH 8.0 buffer solution 13



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containing 500 mM imidazole，20 mM PBS, 300 mM NaCl, 0.1% TritonX-100 and 1 mM DTT. Further purification was carried out using gel-filtration chromatography. The purity of the obtained preparation was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

RESULTS AND DISCUSSION

Inhibition of AChE activity by surface-modified C60 NPs Fullerenes have low cytotoxicity (early reports of toxicity were mostly artifacts of residual solvent), and aqueous C60 solutions were prepared at a nontoxic concentration of 200 µM.60 The brownish suspensions were subjected to transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential measurements for characterization. As mentioned above, TEM images showed that all 9 C60 NPs formed spherical clusters with a diameter of approximately 40-60 nm, and the individual C60 molecule cannot exist in water stably because of an extremely high surface area (Fig. S1). Besides, the hydrodynamic diameters of 9 C60 NPs were 85-160 nm as determined by DLS, indicating that the spherical clusters of 9 kinds of C60 NPs were well-dispersed in water and their aqueous solution is rather stable (Fig. S2). The zeta potential values of the NPs further verified their stability (Table S1). Such ensures the successful application of the prepared stock solution to the following in vitro bioassay. In addition, the major difference of NPs in the AChE inhibition potential can be ascribed to their surface structures since they share a similar particle size and comparable stable dispersion capacity in aqueous solution. 14


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Figure 3. (A-B) Concentration–response curves of the tested chemicals on WT eeAChE inhibition for (A) the CAS positive inhibitor NB, the PAS positive inhibitor PI, and 9 C60 NPs; (B) pristine and 8 surface modified C60 NPs. The experimental concentration of AChE was 0.023 µM. The initial concentration for the 9 NPs was set as 150 µM and then 750 times dilution for another twelve concentrations. (C-D) Concentration–response curves of the tested chemicals on double mutant eeAChE inhibition for (C) CAS-positive inhibitor NB, PAS-positive inhibitor PI, and 6 hydrophobically modified C60 NPs; (D) 6 hydrophobically modified C60 NPs. The final concentration of AChE was 0.04 µM. The initial concentration for the 6 NPs was set as 150 µM and then 150 times dilution for another 11

eleven concentrations. The initial concentration for 2 positive inhibitors was set as 10 mM and then 10

times dilution for another eleven concentrations. The error bar represents the standard deviation of three independent measurements.

The enzyme activity inhibition assay was used to determine the eeAChE inhibition by C60 NPs. Fig. 3A and Table 1 showed that all nine C60 NPs can significantly inhibit the activity of eeAChE in a dose-dependent manner at micromolar levels, a similar concentration at which the neuroprotective effect was observed for carboxyfullerenes.29 Just as expected, carboxyfullerenes with negative logP values and fullerene itself presented lower eeAChE 15



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activity inhibition efficacy than the other 6 nano-C60 materials with positive logP values (Table 1 and Fig. 3). The IC50 data of two carboxyfullerenes were 90.7±4.6 µM for C60-2 and 76.1±4.2 µM for C60-3, comparable to that of fullerene (115.9±14.8 µM). In contrast, as Fig.1(C) demonstrated, the derivatives with long side chains of positive logP values are required for the binding via proposed T-shape plug-in mode. Therefore, the nano-C60 with hydrophobic surface groups of similar length may present comparable enzyme inhibition capacity. Obviously, C60-8 and C60-9, the hydrophobically functionalized C60 NPs with the same longest surface group of about 7Å, the methyl valerate, showed lowest concentrations (~10µM) needed to inhibit 50% of eeAChE activity, followed by C60-5 and C60-6 (~24µM) with the surface groups of the similar logP value and length (~6Å). C60-4 and C60-7 with the shortest surface groups showing positive logP exhibited lowest inhibition ability(~50µM). Moreover, other chemical features of the surface groups also play a role in enzyme inhibition in addition to their hydrophobicity and length. For example, methyl valerate and other surface groups on C60-8 and C60-9 can insert into the upper gorge of about 10Å deeply to stabilize the ligand-protein complexes. Moreover, due to the conserved aromatic residues in the PAS, the derivatives with aromatic tails showed enhanced inhibition potential. It is no surprise that C60-9, the C60 derivative with an aromatic tail show higher inhibition rate than C60-8 with a thienyl. Besides, the narrow configuration of the upper gorge made the eeAChE inhibition potential of C60-7 lower than that of C60-4 although the indane functional group on C60-7 is longer and less hydrophilic than the trimethylamine on C60-4.In addition, the enzyme activity interference of unbonded surface substances (Fig. S4) possibly released from the C60 NPs was excluded since the inhibition rate of the surface substance-like 16


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chemicals was lower than 10% even at the exposure level of 10 µM with one exception, nitrilotriacetic acid which is the surface group modified on C60-3(15% at 10 µM) (Fig. S4). To further exclude the possible synergistic effect between the C60 NPs and the corresponding surface chemicals, the combined effect of the selected binary mixtures of fullerene nanoparticles and the surface chemicals have been investigated (Supporting Information). Fig. S5A and Fig. S5B showed no statistically significant difference between the inhibition rates of single exposure of pristine fullerene, the weakest non-competitive inhibitors, and those of its coexposure with the addition of 10% or even 100% methyl 5-phenylvalerate. For example, 80 µM C60-1 presented 32.43±2.78% inhibition rate, while the addition of 8 µM or 80 µM methyl 5-phenylvalerate obtained 32.58±1.62% or 31.17±2.51% inhibition efficiency. The same combination ratios for methyl 5-phenylvalerate, and C60-9, the strongest competitive inhibitors in the present study, were used to check the potential synergistic effect of the corresponding mixtures. Likewise, 9 µM C60-9 presented 47.85±3.29% inhibition rate, while the addition of 0.9 µM or 9 µM methyl 5-phenylvalerate obtained 47.91±2.66% or 49.64.17±2.59% inhibition. No enhancement of methyl 5-phenylvalerate to the inhibition potential of C60-9 was observed. Fig. S5C and Fig. S5D revealed no synergistic effect for the selected binary mixtures of methyl 5-phenylvalerate with C60-9. Moreover, the 1:10 binary mixtures of the other 7 surface chemicals with the corresponding C60 NPs at the final concentrations around IC50 values also presented no synergistic effect (Fig. S5E).

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Table 1 Structures and properties of surface groups and IC50 values and TS of C60 NPs. NP Label 1

2

3

4 5 6 7 8 9

Surface Group --

AlogPa neutral /ionized

ClogP neutral /ionized

0.00/0.00

0.00/0.00

-0.07/-0.19

lg-(Å)

ld (Å)

IC50b (µM)

pIC50

TS

--

--

115.9±14.8

3.94

--

-4.19/-0.07

3.82

--

90.7±4.6

4.04

--

-2.71/-0.94

-9.33/-2.71

5.58/ 3.21/ 2.41

--

76.1±4.2

4.12

--

0.22/0.02

0.42/0.22

4.09

4.50

46±4.0

4.34

2.49

0.16/0.53

0.53/0.16

5.83

6.40

26.4±2.9

4.59

3.21

0.83/1.42

1.42/0.83

5.73

6.00

21.2±2.9

4.68

4.42

2.93/3.15

3.15/2.93

4.79

4.10

51.5±5.0

4.28

2.15

1.61/2.83

2.83/1.61

7.90

11.0±0.4

4.96

4.54

3.05/3.19

3.19/3.05

7.90

9.0±0.3

5.05

5.15

6.85/ 4.99 6.98/ 5.40

a

The logP values correspond to the surface molecule attached via a methylene linker and were calculated using ChemBioDraw. b The IC50 values represent the Mean±SEM from at least three independent experiments. The asterisk (*) highlights the carbon atoms linked to the surface.

Furthermore, the kinetic studies were performed to obtain more information on the AChE inhibition mechanism of nine C60 NPs. The steady-state inhibition data were graphically analyzed by Lineweaver–Burk plots (Fig. 4), which were reciprocal rates versus reciprocal ATC concentrations. Double-reciprocal plots yielded a group of lines, in which the y-intercept corresponds to the reciprocal of the maximal velocity (vm) and the slope corresponds to the reciprocal of vm/Km. Fig. S6 presented constant vm and variant Km at 18


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different inhibitor concentrations for 6 hydrophobically modified C60 NPs (C60-4~C60-9), suggesting a reversible competitive AChE inhibition (Table S2), as the proposed plug-in mechanism assumed. This type of inhibition can be very attractive in terms of the ability of the NPs to bind to the AChE PAS. In contrast, 3 other C60 NPs(C60-1~C60-3), the pristine and hydrophilically modified ones, demonstrated a different AChE binding mode, characterized by increased Km and decreased vm with an increase in the inhibitor concentration (Table S2). Moreover, Fig. 4 provided the binding site information of 6 competitive NP inhibitors of AChE. As discussed above, two possible inhibition mechanisms have been proposed so far to explain the inhibition of AChE except for the backdoor mechanism in which inhibitors may induce a conformational change in the Trp86 residue next to the catalytic triad at the catalytic site and interfere with the opening and closing of an additional product-releasing channel.61 Firstly, a competitive inhibitor may insert into the gorge for about 20Å depth, thus interacting with the catalytic triad.7,

13-14, 31, 36

Secondly, an inhibitor may bind to the PAS through

cation-π or π-π interactions, thus preventing the entry of a substrate into the gorge or restricting the exit of a product.62-63 PI, a specific PAS inhibitor of AChE, was used here as a marker inhibitor in the kinetic studies based on its well-known ability to bind to the PAS sites of AChE.63 The Lineweaver–Burk plots displayed in Fig. 4 identified the PAS as the potential binding site of 6 hydrophobically modified C60 NPs, as described by Krupka.52 All of these NPs could compete with PI, presumably at the PAS of eeAChE, when the NPs were co-incubated with PI in the reaction solutions. As shown in Fig. 1, the access to the CAS is only possible for small molecules with a diameter lower than 5 Å. Clearly, it is not possible for a C60 cluster with a diameter over 400 Å to enter the gorge with the entrance diameter of 19



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approximately 10 Å, go through the narrow middle part of the gorge (~5 Å), and finally reach the gorge bottom and interact with the Ser203-Glu334-His447 catalytic triad or induce conformational changes in Trp86 (Fig. 1). The active site gorge is approximately 20 Å long and the catalytic site is located 4 Å from the bottom of the enzyme. Therefore, surface modified C60 NPs may act as competitive inhibitors either via stable PAS binding like C60-4~C60-9 or through direct CAS interaction if individually functionalized fullerenes can detach from the cluster. There is another possibility for competitive inhibition, bridging the PAS and CAS by the C60 NPs if the surface group covalently bound to the fullerene is sufficiently long to make the end of the surface group interfere with the catalytic triad function when the surface fullerene cores bind to the PAS simultaneously. Clearly, the kinetic data denied the other possibilities but supported the T-shape plug-in mode for the 6 C60 NPs, C60-4~C60-9.

20


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C60-4

B

D

F

C60-8

C60-9

1/V (L*s/mol)

E

C60-7

1/V (L*s/mol)

C60-6

1/V (L*s/mol)

C

C60-5

1/V (L*s/mol)

1/V (L*s/mol)

A

1/V (L*s/mol)

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1/[S] (mM-1)

1/[S] (mM-1)

Figure 4. (A-F) Lineweaver–Burk plots for the hydrolysis of ATC by eeAChE in the presence and absence of reversible inhibitors C60-4~C60-9, respectively. Dixon plots demonstrated the interference of PI with binding of surface-modified C60 NPs. The dotted lines represent the theoretical plots for no interference of PI with the binding of C60 NPs and for interference, namely, complete competition for the same binding site. The final concentration of AChE was 0.023 µM, and the ATC solution concentration were ranging from 0.56 mM to 7.5 mM. The concentrations of the inhibitors were 20 µM for C60 NPs and 1 µM for PI. The results represent the Mean±SEM from at least three independent experiments.

QNAR study on eeAChE inhibition by C60 NPs

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It is clear that there is a strong dependence of the inhibition on the physicochemical properties of the functionalized fullerenes. Because both the AChE inhibition kinetic assay and PI competition experiment indicated PAS and T-shape plug-in mode as the possible binding site and binding mode for six of the modified C60 NPs, respectively, we next used simple statistical models to further verify this hypothesis. Fourteen highly conserved aromatic residues, including Tyr72, Tyr124, Tyr341, Trp286, Phe295, and Phe297 line the binding pocket and make up 40% of the PAS surface, while Trp236, Phe337 and Phe338 locates at the narrow channel linking the PAS to the CAS region.64 The presumed common feature of the PAS binding with six NPs is that the aromatic residues in the PAS may establish strong π-π interactions with the protruding fullerene unit, thus helping to position the hydrophobic surface group within the gorge. The only distinct structure trait of the NPs comes from their surface groups. According to the descriptor lg (Table 1), the surface groups of six competitive C60 NP inhibitors can reach about half of the upper part of the enzyme gorge when they adopt extended conformations along the vertical axis of the gorge. To further evaluate the relationship between C60 NP structures and enzyme inhibition, we carried out QNAR studies for the six NPs of the same mode of action. It is not surprising that the logP values were not statistically significantly correlated with the lg values for this data set (p>0.05) due to the low structural similarity of the 6 surface groups (Table 1). The small size of the data set precluded more sophisticated QNAR analysis, and the differences in the eeAChE inhibition capacity of the NPs may be described by simple descriptors such as lg and the logP (Table 1). We tried to construct simple QNAR models describing the relationships between the pIC50 values and the substituent length or logP of neutral and ionized substituent speciations. 22


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The single-descriptor QNAR models predicting pIC50 (-logIC50) for six competitive NPs inhibitors were rather unsatisfactory when logP was used as the independent variable no matter which speciation was applied for modeling (R20.05). However, we could obtain a linear model with P=0.01 for pIC50 (Eqn.1, R2=0.63) even for all nine NPs using the calculated lg. When the model was restricted to the six competitive inhibitors interacting with eeAChE through an assumed T-shape plug-in mode at the PAS, we obtained a statistically significant QNAR model with P

Competitive Inhibition Mechanism of Acetylcholinesterase without

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