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Cite This: J. Am. Chem. Soc. 2018, 140, 16925−16928
Metal-Ion-Responsive Bionanocomposite for Selective and Reversible Enzyme Inhibition Junqiu Zhai,†,‡ Muhua Zhao,† Xiangjian Cao,† Mengyuan Li,*,† and Meiping Zhao*,† †
Beijing National Laboratory for Molecular Sciences and MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China ‡ School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong 510006, China J. Am. Chem. Soc. 2018.140:16925-16928. Downloaded from pubs.acs.org by EASTERN KENTUCKY UNIV on 01/10/19. For personal use only.
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structure of their targets in size and shape, thus providing high specificity and high affinity.11,12 Inspired by these features, we envision that in situ fabrication of a tailor-made contact surface surrounding the APE1-binding domain of AVD preassembled on the SiMNPs would have great potential to obtain an artificial binding pocket for the target enzyme (Figure 1A).
ABSTRACT: A bionanocomposite with artificial binding pockets for a DNA repair enzyme has been developed by in situ assembly of an affinity protein with a surrounding contact surface of polydopamine on the surface of silica coated magnetic nanoparticles via molecular imprinting reactions. The obtained nanoparticles exhibited antibodylike binding behavior toward the target enzyme with highly specific and efficient inhibition effect. Moreover, the binding and inhibition could be flexibly tuned by the addition of metal ions such as Mn2+ and Mg2+, which provided a convenient tool to regulate enzyme activity with artificially engineered nanoinhibitors.
P
rotein−protein interactions are ubiquitous in biological systems. Despite the significant increase of the number of identified protein−protein interactions in recent years, it remains a big challenge to create a protein-based enzyme inhibitor by modification of the sequence of the protein.1−3 Human apurinic/apyrimidinic endonuclease/redox effector factor 1 (APE1) is a multifunctional human enzyme which plays an essential role in the base excision repair (BER) pathway of DNA damage to keep the stability of genome. It also regulates the redox condition of cells.4 Due to its relationship to the occurrence of tumors, especially nonsmall cell lung cancer (NSCLC) and ovarian and breast cancers,5,6 specific binding to this enzyme and inhibition of its activity have been regarded as a potential targeted therapy against the related cancers.7−9 Very recently, we have disclosed an unexpected strong interaction between APE1 and avidin (AVD) with 1:1 stoichiometry.10 The dissociation constant value (Kd) of APE1−AVD complex was as low as 3.2 nM, providing AVD great potentials as an affinity ligand for APE1. Moreover, the avidin-oriented assembly of biotin-labeled DNA probes on the surface of silica coated magnetic nanoparticles (SiMNP) enabled selective response to APE1 and resistance to the digestion by other nucleases. However, direct binding of APE1 to the avidin modified nanoparticles (SiMNP@AVD) does not inhibit the enzyme activity, suggesting that further modifications were needed to obtain a specific inhibitor for the enzyme. Natural antibodies, receptors, or enzymes tend to bind their target molecules with a binding pocket which combines multiple noncovalent interactions. The structural flexibility of these binding pockets allows proteins to adapt to the specific © 2018 American Chemical Society
Figure 1. (A) Schematic illustration of the synthetic approach for the metal-ion-responsive bionanocomposite with artificial binding pockets for APE1. (B) XPS spectra of SiMNP@AVD-2 and MIP-2. (C) Zeta potential values of different nanoparticles.
Molecular imprinting has been extensively used for generating specific binding sites against various targets.13−18 Using small-molecular inhibitor benzamidine as one of the functional monomers, Haupt and co-workers have developed the first MIP-based enzyme inhibitor.17 However, few investigations have been reported on generation of artificial binding pockets for inhibition of the enzyme’s activity without the need of a natural inhibitor. Using AVD as an affinity ligand for APE1, here, we chose dopamine as the functional monomer to perform the surface imprinting reactions (Figure 1A). Dopamine tends to undergo oxidative polymerization at room temperature and deposit on the surface of substrates without the need of other cross-linkers or initiators.19−21 Moreover, the thickness of the polydopamine (PDA) layer can be controlled Received: October 8, 2018 Published: November 28, 2018 16925
DOI: 10.1021/jacs.8b10848 J. Am. Chem. Soc. 2018, 140, 16925−16928
Communication
Journal of the American Chemical Society
as high as that on SiMNP@AVD-2, while the nonspecific adsorption of other tested nucleases on MIP-2 was as low as that on NIP-2. These results substantially proved the specificity of the obtained binding pockets in MIP-2 toward APE1, which should be attributed to both the APE1-binding domain of AVD and the surrounding PDA. Then, we further tested whether MIP-2 might serve as a nanoinhibitor of APE1. Before adding the APE1-probe to measure the activity of APE1 bound on MIP-2, we first blocked the surface of MIP-2 with irrespective unlabeled DNA strands (UL-DNA) (Figure S3) to avoid nonspecific adsorption of the fluorescent probes on PDA.16 After washing, we redispersed the MIP-2 NPs containing APE1 in 1× Buffer 1.1 with 0.04% Triton-100 and added APE1-probe to measure the activity of APE1 bound on MIP-2. Figure 2D demonstrated that, after washing with 1× Buffer 1.1 + 0.04% Triton-100, about 52% of the originally added APE1 was retained on MIP2, while the relative activity of the bound APE1 was only about 12%. In contrast, the SiMNP@AVD-2 retained about 74% of the added APE1, while the relative activity was as high as 90%, most likely due to the enhancing effect of AVD on the APE1 activity.10 NIP-2 only retained about 10% of the added enzyme. On the basis of these data, neither SiMNP@AVD-2 nor PDA (on NIP) alone could inhibit the activity of the bound APE1, but when these two types of interactions were combined together via molecular imprinting in the presence of the target enzyme, the resultant MIP-2 successfully sequestered the enzyme and effectively inhibited its activity. To evaluate the respective contribution of the AVD molecule and PDA layer in the construction of the specific binding pockets, we further synthesized MIP-0 and NIP-0 by replacing the SiMNP@AVD-2 with SiMNP-COOH in the APE1-imprinting reaction (Figure S4). Both MIP-0 and NIP-0 showed much lower binding capacity than SiMNP-COOH itself. This confirmed the formation of the PDA surface layer with reduced nonspecific adsorption of APE1. However, the binding amount of APE1 on MIP-0 was only slightly higher than that on NIP-0 and much smaller than that on MIP-2. These data proved that, in the absence of the APE1-binding domain of AVD, the PDA formed under the reaction conditions (10 nM free APE1, Tris buffer, pH 8.0) only provided very weak interactions with APE1. From the kinetic study results, more than 80% of APE1 was captured by MIP-2 within 5 min (Figure S5). This further confirmed that, with APE1 preassembled on the surface of AVD modified SiMNP, the functional groups of PDA and the APE1-binding domain of AVD were well organized during the imprinting process and the generated binding pockets were easily accessible for the target APE1. Next, we investigated the dissociation behavior of APE1 from MIP-2. Ten mM Tris buffer (pH 8.0) could only release about 12% of the APE1 bound on MIP-2. Since 1× Buffer 1.1 (10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2, 100 μg/mL BSA, pH 7.0) is the most commonly used buffer for the APendonucleolytic reactions of APE1,25 we tested the use of 5× or 10× Buffer 1.1 to release the APE1 bound on MIP-2 according to our previous work.26,27 As shown in Figure 3A, 5× Buffer 1.1 released about 80% of the bound APE1, and the dissociation efficiency of 10× Buffer 1.1 was close to 100%. Different from the results of MIP-2, 10× Buffer 1.1 could only release about 50% of the bound APE1 from SiMNP@AVD-2, suggesting different interactions between APE1 and the two different NPs (Figure S6). For MIP-2, though APE1 was
by the reaction conditions and deposition time, which is beneficial to obtain a monomolecular layer of binding pockets for the template enzyme.22 By fixing the concentrations of APE1 and SiMNPs, we performed surface imprinting around the APE1 bound by SiMNP@AVD. The binding properties of the resultant surfaceimprinted nanoparticles (MIPs) with different amounts of avidin preimmobilized on the surface of SiMNPs were compared in Figure 2A. As a control, nonimprinted polymers
Figure 2. (A) Binding amount of APE1 on MIPs and NIPs obtained with different amounts of avidin preimmobilized on the surface of SiMNPs. (B) Binding amount of APE1 on MIP and NIP obtained at different polymerization times. (C) Comparison of the binding selectivity of the obtained MIP-2, NIP-2, and SiMNP@AVD-2 for APE1 and other reference enzymes. APE1: 10 nM; Exo III: 10 nM; DNase I: 30 nM; T5: 10 nM; Lambda exo: 10 nM. (D) Measurement of the activity of APE1 bound on different NPs. The amount of the bound-APE1 was also shown for comparison. Control: original APE1 solution.
(NIPs) were also prepared in the absence of the template enzyme and tested. The amount of avidin covalently conjugated to the surface of SiMNPs was optimized to be 200 nmol/g (SiMNP@AVD-2). Using SiMNP@AVD-2, we investigated the influences of dopamine polymerization time on the binding property of the resultant composites (Figure 2B), which was found to be significantly affected by the presence of different proteins on the surface of the SiMNPs (Figure S1). Such features could be very useful for the control of the thickness of the PDA layer.23 From the transmission electron microscopy imaging (Figure S2), MIP-2 had an overall size of 50 ± 5 nm in diameter with a PDA layer of 4−6 nm thickness. The XPS data (Figure 1B, Tables S3 and S4) showed that the nitrogen-to-carbon signal ratio (N/C) changed from 0.163 for SiMNP@AVD-2 to 0.114 for MIP-2, close to the theoretical value for dopamine (N/C = 0.125),24 which strongly confirmed the formation of PDA layer around the nanoparticles. Figure 1C displayed the zeta potential values of different nanoparticles. MIP-2 clearly had more positive charges than both SiMNP@AVD-2 and NIP-2, which further verified the presence of both PDA and avidin (pI = 10.0) in the surface layer of MIP-2. Figure 2C demonstrated the binding capacity and selectivity of MIP-2 to the target enzyme APE1. Compared to SiMNP@ AVD-2, NIP-2 showed much lower adsorption capacity of all the tested nucleases due to the effective protection by the PDA layer. In contrast, the binding amount of APE1 on MIP-2 was 16926
DOI: 10.1021/jacs.8b10848 J. Am. Chem. Soc. 2018, 140, 16925−16928
Communication
Journal of the American Chemical Society
three water molecules and, upon metal binding, E96 and D70 shift 1.2 and 1.9 Å, respectively.32 Thus, the significant conformational change of APE1 induced by the metal ions may have dominated the dissociation of the enzyme from the binding pockets on MIP-2. Besides, metal ions such as Mn2+, Zn2+, and Cu2+ could also chelate with the functional groups on PDA, which might help break the interactions between PDA and APE1 and promote the dissociation.33 These offer the bionanocomposite a unique metal-ion-responsive property which would be very useful for tuning its interactions with the target enzyme. We also tested the performance of MIP-2 in a complex biological background. From Figure 3B, APE1 was successfully extracted from the serum with a recovery rate as high as 82 ± 5% (n = 3). The bionanocomposite shows great potential for further application in an intracellular environment as an effective inhibitor of APE1. In summary, employing APE1 as a target enzyme and AVD as a bioaffinity ligand, we have successfully prepared a bionanocomposite with artificial binding pockets for the enzyme via surface molecular imprinting. The APE1-binding domain of AVD was well preserved and exposed in the binding pocket, with a surrounding contact surface of polydopamine in the vicinity. The obtained bionanocomposites were proved to bind APE1 via multiple noncovalent interactions like the natural antibodies. The bound enzyme was effectively inhibited most likely because of conformational change, which could not be achieved by either the SiMNP@AVD or polydopamine alone. Moreover, a unique metal-ion-responsive property was observed for the bionanocomposites, and the bound enzyme could be reversibly released from the binding pockets with high activity under very mild conditions. With these features, the artificial nanoinhibitors have great potential for targeted cancer therapy and other biomedical applications. The strategy is also applicable to generate tailor-made binding pockets for other proteins.
Figure 3. (A) The desorption efficiency of different solutions for dissociating APE1 from MIP-2. 1: 5× Buffer 1.1; 2: 10× Buffer 1.1; 3: 20 mM MgCl2; 4: 100 mM MgCl2; 5: 10 mM MgCl2 and 10 mM MnCl2; 6: 100 mM MgCl2 and 100 mM MnCl2; 7: 100 mM MgCl2 and 1.0 mg/mL BSA; 8: 100 mM MgCl2, 100 mM MnCl2, and 1.0 mg/mL BSA; 9: 0.5 mg/mL BSA; 10: 1.0 mg/mL BSA. (B) Fluorescence responses of APE1-probe (100 nM) to the APE1 isolated from human serum samples (400 μL) by using MIP-2 (0.2 mg/mL) or NIP-2 (0.2 mg/mL). For the recovery test, human serum samples spiked with APE1 standard solution were also measured under the same conditions.
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preimmobilized on the SiMNPs by forming a stable complex with AVD before the surface imprinting reaction, the interface between APE1 and AVD most likely had been modified during the in situ polymerization of dopamine. The PDA formed surrounding APE1 not only introduced new interactions to the binding pockets but also adjusted the original interactions between APE1 and AVD. Thereby, the APE1 bound on MIP-2 could be reversibly recovered by mild buffer solutions. The Kd values of MIP-2 and NIP-2 for APE1 were measured to be 3.8 and 291 nM, respectively (Figure S7). The binding affinity of MIP-2 toward APE1 was comparable to those of the natural antibodies.28 The detailed results in Figure 3A clearly showed that the combination of metal ions, BSA, and Tris-HCl enabled the highly efficient release of APE1 from MIP-2. Considering that APE1 is a metal-dependent enzyme and Mn 2+ is also an effective metal-ion cofactor of the enzyme,4,29−32 we further tested whether Mn2+ can facilitate the release of bound APE1 from MIP-2. Surprisingly, a combination of 10 mM MgCl2 and 10 mM MnCl2 achieved a recovery rate as high as 80% of the bound protein (Figure 3A), indicating unusual roles of Mn2+ and Mg2+ in disrupting the interactions between the enzyme and the binding pocket. Previous structural study on the metal-binding site(s) of APE1 has proved that the metal ions coordinate D308, E96, D70, and
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b10848. Chemical reagents and materials, experimental section, sequences of the oligonucleotides, composition and pH of the reaction buffers, and supplemental results and discussions, including percentage of the free dopamine in the Tris buffer reaction solution, TEM images, XPS analysis results, fluorescence responses and relative reaction rates, binding amount and kinetics, recovery rate, and Scatchard plot analysis (PDF)
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AUTHOR INFORMATION
Corresponding Authors
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[email protected] ORCID
Meiping Zhao: 0000-0002-8696-8738 Notes
The authors declare no competing financial interest. 16927
DOI: 10.1021/jacs.8b10848 J. Am. Chem. Soc. 2018, 140, 16925−16928
Communication
Journal of the American Chemical Society
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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 21775009, 81571130100, 21575008, and 31471671) and the Beijing Municipal Natural Science Foundation (No. 2184103).
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DOI: 10.1021/jacs.8b10848 J. Am. Chem. Soc. 2018, 140, 16925−16928
