Supramolecular Chemistry-Assisted Electrochemical Method for the

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Supramolecular chemistry-assisted electrochemical method for the assay of endogenous peptidylarginine deiminases activities Jing Zhao, Lili Yang, Yingying Tang, Yucai Yang, and Yongmei Yin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13091 • Publication Date (Web): 13 Dec 2016 Downloaded from http://pubs.acs.org on December 15, 2016

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ACS Applied Materials & Interfaces

Supramolecular Chemistry-assisted Electrochemical Method for the Assay of Endogenous Peptidylarginine Deiminases Activities Jing Zhao,† Lili Yang,† Yingying Tang,† Yucai Yang, ‡ Yongmei Yin*,‡ †

Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China

‡

Department of Oncology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, P. R. China

ABSTRACT: Peptidylarginine deiminase 4 (PAD4) is the only isoform of PADs located within the cell nucleus, which has been known to be related to several human diseases. In this work, we have proposed an electrochemical method for the assay of endogenous PAD4 activities as well as the studies of PAD4 inhibitors by making use of the supramolecular chemistry-assisted signal labeling. Specifically, peptide probes P1 and P2, separately containing cysteine residues and tripeptides FGG (Phe-Gly-Gly), can be self-assembled onto the surface of the gold electrode and silver nanoparticles, respectively. In the meantime, the peptide probes can be connected together through cucurbit[8]uril-mediated host-guest interaction. Nevertheless, after trypsin-catalyzed

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digestion, FGG at the N-terminal of P1 will be removed from the electrode surface, thereby inhibiting the connection of P1 and P2. Since PAD4 catalyzes the citrullination of arginine residue within P1, trypsin-catalyzed digestion of P1 can be prohibited by the addition of PAD4. Consequently, an obvious change of the electrochemical response can be obtained from the silver nanoparticles (AgNPs) immobilized on the electrode surface. Experimental results have shown that our method can display an improved sensitivity and specificity for both PAD4 assay and inhibitor screening, which may effectively trace endogenous PAD4 and the inhibitors in the cancer cells. Therefore, our method may have great potential for the diagnosis and treatment of PAD4-related diseases in the future.

KEYWORDS: Peptidylarginine deiminase 4, cucurbit[8]uril, silver nanoparticles, trypsin, inhibitor, electrochemical assay.


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1. INTRODUCTION Peptidylarginine deiminases (PADs) are a family of calcium ion-dependent post-translational enzymes, which catalyzes the conversion of peptidyl arginine to pepetidyl citrulline in vivo.1, 2 According to the subcellular localization and tissue distribution, PAD isoforms are designated as PAD 1, 2, 3, 4 and 6. Among them, PAD4 is the only isoform located within the nucleus, which is also recognized as the most extensively studied isoform.3, 4 PAD4 catalyzes the citrullination of arginine residues of the histones H2A, H3 and H4, thereby modulating the downstream gene transcription, differentiation and pluripotency.5,

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Recently, PAD4 has been found to be

associated with several human diseases, and has emerged as a potential target for the diagnosis and treatment of the human diseases. For example, the dysregulated PAD4 found in the joints of rheumatoid arthritis (RA) patients is believed to contribute to the onset of the disease, which may be associated with the production of disease-exacerbating autoantibodies through the protein citrullination process.7-10 Moreover, PAD4 has been found to be highly expressed in several human cancers (e.g. lung, breast and bone cancers), whose level may significantly decrease after the surgical removal of the tumors.11-13 Although the importance of protein citrullination has been gradually revealed by the researchers, there still are many PAD4-related problems worthy of probing into, such as the PAD4 substrates, its activation pattern and coregulators.5, 14 Because the precise assay of PAD4 activity may lay the foundation for the intensive studies of both physiological function of PAD4 and its potential inhibitors, it is extremely essential to develop some effective methods to monitor the PAD4 activities as well as screen the potent inhibitors. However, only a few analytical methods have been reported for the PAD assay, most of which are based on the use of the fluorescent techniques.15-19 So, these assays have suffered from several drawbacks that


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usually result from the relatively complex fluorescence labeling process. The detection sensitivity may also be easily interfered by the external environment. Therefore, more types of analytical methods should be developed for PAD4 assays as the alternatives to the fluorescent technique. Supramolecular chemistry is a sub-discipline of chemistry that studies the specific association of two or more molecules according to the principle of molecular complementarity.20-21 Hostguest interaction is a typical example in supramolecular chemistry.22-23 A host molecule with a large cavity can selectively bind to the guest molecule with a complementary shape, depending on the formation of the noncovalent bonds, including hydrogen bonding, electrostatic interaction, hydrophobic forces and van der Waals forces. Besides providing a new insight into the molecular self-assembly, the studies of host-guest interaction may also offer different choices for the signal labeling, signal amplification and even theranostics. Currently, the family of the host molecules cucurbit[n]urils (CB[n]) with rigid macrocyclic structures have attracted great attention for the potential application in the biological and pharmacological fields.24,

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Among them, CB[8],

unlike the smaller homologues CB[6] and CB[7], contains a larger cavity that is able to simultaneously bind to two guest molecules, forming 1:1:1 or 1:2 complexes.26, 27 Different types of guest molecules can bind to the host molecules CB[8], such as methyl viologen (MV), purpurine, naphthol, pyrene, cyclammonium and so on.28 Meanwhile, CB[8] has been reported to preferably bind with the aromatic amino acid residues (e.g. tryptophan, phenylalanine and tyrosine), and the binding is more favorable when these amino acids are located at the N-terminal of the oligopeptides.29 Moreover, CB[8] has been found to bind to the specific tripeptides (WGG or FGG) with the high binding constants, providing the possibility for the reversible dimerization of the oligopeptides and proteins.30, 31


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Electrochemical technique as an alternative to the fluorescent technique has become increasingly popular in the field of bioanalysis, due to the unique advantages of the rapid response, low cost, simple operation and high sensitivity.32-37 In this work, with the employment of the sensitive electrochemical technique, we have developed a facile method for the studies of PAD4 activities and the inhibitors by making use of trypsin-catalyzed cleavage and CB[8]assisted signal labeling. The use of trypsin-catalyzed cleavage that selectively recognizes arginine within the substrate peptide can be conductive to reveal PAD4-catalyzed citrullination, confirming the specificity of PAD4 activity assay.38 Meanwhile, the employment of CB[8]assisted signal labeling may avoid the steric hindrance in enzymatic digestion and arouse obvious electrochemical responses, leading to the high sensitivity of PAD4 assay. So, the proposed new method may have great potential use in the future.

2. EXPERIMENTAL SECTION 2.1. Materials. P1 (FGGGRGAC) and P2 (FGGGGC) were purchased from GL Biochem (Shanghai) Ltd. Silver nitrate (AgNO3), trisodium citrate, sodium borohydride (NaBH4), NaH2PO4, Na2HPO4, KCl, PAD4, bovine serum albumin (BSA), myoglobin(Mb), hemoglobin (Hb), ovalbumin (OVA), thrombin (TB), mercaptohexanol (MCH), tris(2-carboxyet-hyl) phosphine hydrochloride (TCEP), were purchased from Sigma-Aldrich. Cl-amidine was purchased from Cayman Chemical. Human Promyelocytic leukemia cells (HL-60) was purchased from Shanghai Genechem Co. Ltd. RPMI 1640 Medium was purchased from GIBCO. NE-PER nuclear and cytoplasmic extraction reagents from Pierce Biotechnology. Trans Retinoic acid, Dimethyl Sulfoxide (DMSO) and protease inhibitor cocktail were purchased from SigmaAldrich.


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Enzymatic reaction buffer for PAD4 is 50 mM Tris-HCl containing 1 mM CaCl2 (pH 7.6). Enzymatic reaction buffer for Trypsin is 50 mM Tris-HCl containing 20 mM CaCl2 (pH 8.4). The washing solution of Trypsin is 20 mM Tris-HCl containing 5 mM MgCl2, 0.1 M NaCl, 1.0% Tween-20 (pH 7.4). All solutions were prepared with double-distilled water, which was purified with a Milli-Q Plus 185 ultrapure water system to a specific resistance of 18 MΩ cm.

2.2. Preparation of P1 Modified Electrode. In order to obtain a mirror-like surface on the gold electrode, the electrode (Φ=3 mm) were carefully polished with 1.0 and 0.3 μm alumina slurry at first. After rinsing thoroughly with double-distilled water, the electrode was covered by the piranha solution (H2SO4: H2O2 = 3:1) for 5 min, and the residual was removed by sonicating the electrode separately in ethanol and double-distilled water for 5 min. Afterward, the polished electrode was electrochemically cleaned in 0.5 M H2SO4 by scanning from 0 V to 1.6 V. After drying in the nitrogen atmosphere, the cleaned electrode was coated with a solution containing 5 μM P1, 50 mM TCEP and 10 mM PBS (pH 7.4) for 16 h at 4 °C, which was then thoroughly rinsed by double-distilled water. After incubating with 1 mM MCH for another 30 min, P1 monolayer modified electrode was thoroughly rinsed by the washing buffer, which was then prepared for further use.

2.3. Synthesis of P2-functionalized Silver Nanoparticles. Silver nanoparticles were prepared based on the previous report.39 Briefly, 6 mL aqueous solution of NaBH4 (5 mM) was added drop-by-drop to 100 mL mixture of 0.25 mM AgNO3 and 0.25 mM trisodium citrate under vigorous stirring during a period of 30 min. When the color of the solution turned to light yellow, the obtained solution was kept in dark overnight. After then, the prepared silver nanoparticles


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were stocked at 4 oC for the further use (The morphology of the AgNPs was observed characterized by TEM as shown in Figure S1). For the functional modification, 0.2 μM P2 was incubated with the synthesized silver nanoparticles for 16 h at 4 oC, and the P2-functionalized silver nanoparticles was prepared through the silver-sulfur interaction. After the incubation, the excess peptides were removed by centrifuging at 15000 rpm for 20 min at 4 oC (The functionalization was characterized by FTIR as in the Figure S1).

2.4. PAD4 Assay on P1 Modified Electrode Surface. P1 modified electrode was firstly incubated with desired amount of PAD4 for 1 h at 37 oC, which catalyzed the conversion of arginine to citrulline within P1. Afterward, the modified electrode was thoroughly rinsed by the washing buffer and double-distilled water. After incubating with 10 mg/mL trypsin at 37 oC for another hour, P1 modified electrode reacted with 5 μM CB[8] and P2-functionalized AgNPs for 1 h and 2.5h, respectively (AgNPs attached on the gold electrode was charaterized by SEM in the Figure S1). In the control experiments, 1 μM BSA, Hb, Mb, OVA and thrombin have been added instead of PAD4. In the inhibition assay, different concentration of Cl-amidine were premixed with 100 nM PAD4 at 37 oC for 20 min.

2.5. Cell Culture. HL-60 human promyelocytic leukemia cells were incubated in RPMI 1640 at 37 ºC in a 5% CO2 incubator, which contains 10% fetal bovine serum (FBS) and is supplemented with gentamycin and kanamycin. For PAD4 expression, 1 μM trans retinoic acid was added in HL-60 cells (2.0×105 cells/mL), which were then incubated at 37 ºC for 72 h.40 Cytoplasmic and nuclear fractions were produced by using the NE-PER nuclear and cytoplasmic extraction reagents from Pierce Biotechnology with the addition of 1:50 protease inhibitor cocktail.40


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2.6. Electrochemical Measurements. All the electrochemical measurements were performed on CHI660c Electrochemical Analyzer (CH Instruments). The three-electrode system used in our experiments consisted of a working electrode, a reference electrode and a counter electrode, which were P1 modified electrode, a saturated calomel electrode (SCE) and a platinum wire electrode, respectively. For the electrochemical measurements, 1.0 M KCl was used as the electrolyte solution, and linear sweep voltammetry (LSV) was employed with scanning from -0.2 V to 0.2 V at a scan rate of 100 mV/s.

3. RESULTS AND DISCUSSION 3.1. The Principle of the Proposed Method. Figure 1 may illustrate the principle of the PAD4 assay and the inhibitor studies. P1 and P2 can be separately self-assembled onto the surface of the gold electrode and the silver nanoparticles through the free thiol group of cysteine at the C-terminal of the peptides. Because FGG tripeptide at N-terminals of both P1 and P2 can be connected together by CB[8]-mediated host-guest interaction, silver nanoparticles can be immobilized onto the electrode surface through the supramolecular polymerization. In the presence of trypsin that hydrolyses protein at the carboxyl terminal of lysine and arginine, P1 can be cleaved into two parts, and thus inhibit the connection of P1 and P2 on the electrode surface for the removal of FGG at the N-terminal of P1. Because PAD4 is able to catalyze the conversion of arginine to citrulline, the trypsin-catalyzed cleavage of P1 on the electrode can be inhibited by the addition of PAD4. Subsequently, P2-modified silver nanoparticles can be immobilized onto the electrode surface after CB[8]-mediated host-guest interaction, thereby leading to an obvious electrochemical response. Therefore, the assay of PAD4 activities can be easily realized by tracing the electrochemical responses of silver nanoparticles on the electrode


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surface in a “signal-on” manner. When the PAD4 inhibitors are added to inhibit PAD4-catalyzed citrullination, the electrochemical signals from the immobilized silver nanoparticles can be significantly reduced due to the trypsin-catalyzed cleavage of P1 on the electrode surface. Therefore, the studies of the inhibitory effect on PAD4 can also be realized by using the proposed method.

Figure 1. Schematic illustration of supramolecular chemistry-assisted electrochemical method for PAD4 assay and inhibitor studies. 3.2. The Electrochemical Studies of the Principle of Our Detection. We have used LSV to characterize the electrochemical responses of the immobilized silver nanoparticles as well as demonstrate the principle of our detection, which is a commonly-used electrochemical technique to monitor the electro-active species on the electrode surface.39 As shown in the Figure 2, nearly no electrochemical response can be observed at P1 modified electrode (curve a), while a quite high peak current can be obtained after CB[8]-mediated host-guest interaction, ascribing to the immobilized P2-functionalized silver nanoparticles on the electrode surface (curve b). However, when the immobilized P1 has been digested by the catalysis of trypsin, a low peak current that approaches to the background signal can be obtained, suggesting the inhibition of CB[8]-


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mediated signal labeling as a result of the removal of FGG from the electrode surface (curve c). In the presence of PAD4, because PAD4 can catalyze the citrullination of arginine within P1 to inhibit the trypsin-catalyzed digestion, a high electrochemical response can be obtained after CB[8]-mediated signal labeling of P2-functionalized silver nanoparticles (curve d). Therefore, LSV studies have demonstrated the feasibility of our method. We have then optimized the experimental conditions for the detection. Firstly, we have studied the influencing factors of trypsin-catalyzed digestion. The efficiency of the trypsin digestion increases with the increase of both the trypsin concentration and the digestion time. As shown in Figure S1, the optimized concentration for trypsin is 10 mg/mL, and the optimized reaction time is 1 h. Then, we have studied the optimized condition for the interaction of CB[8] and P1 on the electrode surface. As shown in the Figure S2, the optimized CB[8] concentration is 5 μM, and the optimized reaction time is 1 h. Finally, the optimized reaction time for CB[8] and P2-functionalized silver nanoparticles has been found to be 2.5 h. (Figure S3).

Figure 2. LSV responses obtained at P1 modified electrode (curve a, black), the modified electrode after CB[8]-mediated signal labeling (curve b, blue) and that after trypsin-catalyzed digestion without or with 100 nM PAD4 (curve c, red; curve d, green).


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3.3. The Sensitivity and the Specificity of Our Method. In order to demonstrate the sensitivity of our method, we have studied the electrochemical responses obtained in the presence of different concentrations of PAD4. As shown in Figure 3A, the electrochemical response increases along with the addition of the PAD4 concentration. The results are reasonably in consistence with our assumption. The increased PAD4 concentration indicates the higher PAD4 activity, which may accelerate the citrullination of arginine residue within P1 as well as inhibit the trypsin-catalyzed digestion of P1. Subsequently, increased amounts of P2-functionalized silver nanoparticles can be captured onto the electrode surface, resulting in the enhanced electrochemical responses. Figure 3B has shown the relationship between the absolute value of the peak current and PAD4 concentration, and Figure 3C has shown a linear relationship between the absolute value of the peak current and the logarithm values of PAD4 concentration in the range from 0.005 nM to 100 nM. The regression equation is |I| (10-5A) =2.924+1.113lgCPAD4 (nM), R2=0.991. The detection limit of our method, defined as 3σ (where σ is the standard deviation of the zero standards), is calculated to be 0.78 pM, which is more sensitive than that in the previous reports.17, 18 Furthermore, the average of the relative standard deviation (RSD) for three independent repeated measurements are all within 10% and the average of the RSDs are calculated to be 2.61%, well confirming the satisfactory reproduction of our method.


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Figure 3. (A) LSV responses obtained with different PAD4 concentrations (from a to l: 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 200 nM). (B) The relationship between the absolute values of the peak current and PAD4 concentration from 0.005 to 200 nM. (C) The linear relationship between the absolute value of the peak current and the logarithm of PAD4 concentration in the range from 0.005 nM to 100 nM. Error bar demonstrates standard deviations for three independent measurements. Moreover, we have conducted the control experiments to demonstrate the selectivity of our method by using some other proteins (BSA, Hb, Mb, OVA and thrombin) as the controls. Figure 4 has displayed the peak currents obtained with the addition of different proteins. A significantly high peak current can be obtained with the presence of PAD4, while quite low peak currents can be observed with the addition of the control proteins even when the concentrations of the control proteins are much higher than that of PAD4. The comparisons of the electrochemical signals


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have clearly shown that the control proteins have no obvious effect on our detection, confirming the high specificity of our method in PAD4 assay.

Figure 4. The peak current obtained with the addition of different proteins. The concentration of PAD4 is 100 nM, and the concentration of the control protein is 1 μM. 3.4. The Studies of the Potent Inhibitors. In order to demonstrate the potential use of our method in the inhibitor screening, we have also studied the inhibition effect on PAD4 activities by using Cl-amidine as a model inhibitor.16, 41 Cl-amidine especially binds to cys645 of PAD4, which may induce an irreversible inactivation of PAD4 activities in both calcium- and substratedependent manner. Figure 5 has shown the electrochemical responses obtained with the addition of the different concentrations of Cl-amidine. The presence of Cl-amidine inhibits the PAD4catalyzed citrullination of arginine residue within P1, so the reduced amount of P2functionalized silver nanoparticles will be immobilized onto the electrode surface after the trypsin-catalyzed digestion of P1. As a result, the decrease of peak currents has been observed with the addition of the inhibitors due to the reduced amount of the immobilized silver nanoparticles, which is also found to aggravate with the increase of the concentration of the inhibitors. The Figure 5B has displayed the relationship between the inhibitory ratio and the


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concentration of Cl-amidine. The half maximal inhibitory concentration (IC50) was calculated to be 5.10, which is in good agreement with that from the previous literatures.42, 43 Therefore, the inhibition studies has well indicated the great potential of our method for PAD4 inhibitor screening, which might provide technical support for the treatment of PAD4-related diseases in the future.

Figure 5. (A) The electrochemical responses obtained with different concentration of Cl-amidine (from a to f: 0, 2.5, 5, 10, 25 and 50 μM). (B) The relationship between the inhibitory ratio and Cl-amidine concentration. 3.5. The Detection of the Endogenous PAD4 Activities. Based on the above studies of our method, we have examined the availability of our method in the detection of endogenous PAD4 activity by using HL-60 cell lysates as the real samples.44, 45 Firstly, we have assayed PAD4 activity in the nuclear extraction and cytoplasmic extraction. As shown in Figure 6, a comparatively high peak current can be obtained in the nuclear extraction, while only a low peak current can be obtained in the cytoplasmic extraction. The comparison is consistent with the previous report that PAD4 can be found primarily in the cell nucleus.4 Then, we have confirmed the presence of PAD4 in the nuclear extraction by removing calcium ions or adding PAD4


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inhibitors. Figure 6 has shown that the low peak currents can be obtained in the nuclear extraction with the absence of calcium ions or in the presence of the PAD4 inhibitors (Clamidine and minocycline), which are also in accordance with the previous reports.40 On the one hand, Ca2+-binding is able to induce conformational changes of the protein and generate the catalytic site for the substrate,46-48 so reduced PAD4 activity can be found without the addition of calcium ions. On the other hand, the addition of potent inhibitors Cl-amidine and minocycline inactivates PAD4 through the specific binding with the active site, so reduced PAD4 activity as well as the electrochemical responses can be found with the addition of both inhibitors.41, 49 Therefore, the detection of endogenous PAD4 in the cell lysates have not only reconfirmed the high selectivity of our method, but also clearly proven its potential use in the complex samples.

Figure 6. The peak currents obtained in HL-60 cell nuclear and cytoplasmic extraction, and that in the HL-60 nuclear extraction without the addition of Ca2+ or with the addition of 50 μM Cl-amidine and minocycline.

4. CONCLUSIONS In this paper, we have proposed an electrochemical method to assay PAD4 activity based on selective digestion and supramolecular chemistry. Because arginine is the substrate of both


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trypsin and PAD4, the introduction of trypsin-catalyzed digestion reaction helps to reveal the conversion of arginine to the citrulline within the substrate peptide, thereby ensuring the high specificity of our detection. Meanwhile, the signal labeling by making use of CB[8]-assisted host-guest interaction not only avoids the steric hindrance in the enzymatic reaction,50, 51 but also improves the flexibility of schematic design in the detection, thereby leading to the high sensitivity of PAD4 assay. Therefore, our methods have been proven to have a great potential in the clinical use due to the available use in the detection of endogenous PAD4 activity and the studies of PAD4 inhibitors in HL-60 cell lysates, which may be of great importance in the diagnosis and treatment of the related diseases. Moreover, considering the universality of hostguest interaction, our method may be extendedly applied in the more types of enzyme assays by changing the sequences of the substrate peptides in the future.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. TEM characterization of AgNPs and SEM images of AgNPs attached to the Au electrode and the FTIR of P2 and P2-functionalized AgNPs, the optimization of the trypsin concentration and reaction time, the CB [8] concentration and reaction time with P1 modified on the electrode, the reaction time for the immobilization of P2-functionalized silver nanoparticles. AUTHOR INFORMATION Corresponding Author *E-mail address: [email protected]; Tel.: +86 25 68136043 (Y. Yin).


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Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (Grant Nos. 81671781, 31200745) and the Innovation Program of Shanghai Municipal Education Commission (Grant No. 14YZ026).

ABBREVIATIONS PAD4, Peptidylarginine deiminase 4; CB [8], cucurbit [8] urils. REFERENCES (1) Jamali, H.; Khan, H. A.; Tjin, C. C.; Ellman, J. A. Cellular Activity of New Small Molecule Protein Arginine Deiminase 3 (PAD3) Inhibitors. ACS Med. Chem. Lett. 2016, 7, 847-851. (2) Subramanian, V.; Knight, J. S.; Parelkar, S.; Anguish, L.; Coonrod, S. A.; Kaplan, M. J.; Thompson, P. R. Design, synthesis, and biological evaluation of tetrazole analogs of Clamidine as protein arginine deiminase inhibitors. J. Med. Chem. 2015, 58, 1337-1344. (3) Slade, D. J.; Fang, P.; Dreyton, C. J.; Zhang, Y.; Fuhrmann, J.; Rempel, D.; Gross, M. L. Protein arginine deiminase 2 binds calcium in an ordered fashion: implications for inhibitor design. ACS Chem. Biol. 2015, 10, 1043-1053. (4) Wang, S.; Wang, Y. Peptidylarginine deiminases in citrullination, gene regulation, health and pathogenesis. BBA-Gene. Regul. Mech. 2013, 1829, 1126-1135.


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(5) Slack, J. L.; Causey, C. P.; Luo, Y.; Thompson, P. R. Development and use of clickable activity based protein profiling agents for protein arginine deiminase 4. ACS Chem. Biol. 2011, 6, 466-476. (6) Fuhrmann, J.; Clancy, K. W.; Thompson, P. R. Chemical biology of protein arginine modifications in epigenetic regulation. Chem. Rev. 2015, 115, 5413-5461. (7) Bicker, K. L.; Anguish, L.; Chumanevich, A. A.; Cameron, M. D.; Cui, X.; Witalison, E.; Coonrod, S. A. D-Amino acid-based protein arginine deiminase inhibitors: synthesis, pharmacokinetics, and in cellulo efficacy. ACS Med. Chem. Lett. 2012, 3, 1081-1085. (8) Tutturen, A. E.; Fleckenstein, B.; de Souza, G. A. Assessing the citrullinome in rheumatoid arthritis synovial fluid with and without enrichment of citrullinated peptides. J. Proteome Res. 2014, 13, 2867-2873. (9) Tanikawa, C.; Espinosa, M.; Suzuki, A.; Masuda, K.; Yamamoto, K.; Tsuchiya, E.; Matsuda, K. Regulation of histone modification and chromatin structure by the p53–PADI4 pathway. Nat. Commun. 2012, 3, 676. (10) Lewallen, D. M.; Bicker, K. L.; Subramanian, V.; Clancy, K. W.; Slade, D. J.; Martell, J.; Thompson, P. R. Chemical proteomic platform to identify citrullinated proteins. ACS Chem. Biol. 2015, 10, 2520-2528. (11) Bello, A. M.; Wasilewski, E.; Wei, L.; Moscarello, M. A.; Kotra, L. P. Interrogation of the Active Sites of Protein Arginine Deiminases (PAD1,-2, and-4) Using Designer Probes. ACS Med. Chem. Lett. 2013, 4, 249-253.


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For Table of Contents Only


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Schematic illustration of supramolecular chemistry-assisted electrochemical method for PAD4 assay and inhibitor studies. 21x13mm (300 x 300 DPI)


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LSV responses obtained at P1 modified electrode (curve a, black), the modified electrode after CB[8]mediated signal labeling (curve b, blue) and that after trypsin-catalyzed digestion without or with 100 nM PAD4 (curve c, red; curve d, green). 88x68mm (300 x 300 DPI)



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(A) LSV responses obtained with different PAD4 concentrations (from a to l: 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 200 nM). (B) The relationship between the absolute values of the peak current and PAD4 concentration from 0.005 to 200 nM. (C) The linear relationship between the absolute value of the peak current and the logarithm of PAD4 concentration in the range from 0.005 nM to 100 nM. Error bar demonstrates standard deviations for three independent measurements. 95x82mm (300 x 300 DPI)


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The peak current obtained with the addition of different proteins. The concentration of PAD4 is 100 nM, and the concentration of the control protein is 1 µM. 89x65mm (300 x 300 DPI)



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(A) The electrochemical responses obtained with different concentration of Cl-amidine (from a to f: 0, 2.5, 5, 10, 25 and 50 µM). (B) The relationship between the inhibitory ratio and Cl-amidine concentration. 102x49mm (300 x 300 DPI)


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The peak currents obtained in HL-60 cell nuclear and cytoplasmic extraction, and that in the HL-60 nuclear extraction without the addition of Ca2+ or with the addition of 50 µM Cl-amidine and minocycline.

62x45mm (600 x 600 DPI)


Supramolecular Chemistry-Assisted Electrochemical Method for the

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