Detection of Tumor Invasive Biomarker using a Peptamer of Signal

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Detection of Tumor Invasive Biomarker using Peptamer of Signal Conversion and Signal Amplification Hao Li, Weiwei Li, Fengzhen Liu, Zhaoxia Wang, Genxi Li, and Yannis Karamanos Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04423 • Publication Date (Web): 03 Mar 2016 Downloaded from http://pubs.acs.org on March 9, 2016

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Analytical Chemistry

Scheme 1. (a) Design of the peptamer. (b) The peptamer conformation and affinity for cofactor ion can be regulated variously by the interactions with ligands. 140x138mm (300 x 300 DPI)

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Fig. 1 Isothermal titration calorimetry (ITC) results corresponding to the different types of peptamer-ligand interactions designed in Scheme 1. In each of the above results, the top row is the raw data of power versus time (in minutes), while the bottom row is the corresponding integrated enthalpy versus the molar ratio of titrant vs titrand. The titrants vs titrands are respetively: (a1\') Cucurbituril (Q8) vs Peptamer (Pep), (a1) Q8 vs Pep, (b1\') Pep-2Q8 vs Integrin (Int), (b1) Pep-Q8 vs Int, (b2) Pep vs Int, (c1\') Cu (II) vs Pep-2Q8Int, (c1) Cu (II) vs Pep-Q8-Int, (c2) Cu (II) vs Pep-Int, (c3) Cu (II) vs Pep. 259x260mm (300 x 300 DPI)


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Scheme 2. Integrin bioassay developed based on the peptamer. (a) assay procedure, (b) scheme of the reaction of catalytic signal amplification. 116x97mm (600 x 600 DPI)



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Fig. 2 Quantitative analysis of integrin using the peptamer-based bioassay. (a) Square wave voltammograms (SWVs) of DAP showing the gradual increase of signal response with integrin concentration. (b) Peak currents in (a) plotted as a function of integrin concentration. Inset is the linear range and the corresponding formula obtained via regression analysis. The error bars represent standard deviation from average (n=3). 53x22mm (600 x 600 DPI)


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Fig. 3 SWVs of DAP showing the specificity of the assay. All control species are of 100 nM. 53x41mm (600 x 600 DPI)



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Fig. 4 Box chart to show the distribution of the detected concentration of integrin in patients of thyroid carcinoma, grouped by (a) T stage, (b) N stage and (c) M stage. For each sample, the detected integrin in cancerous tissue is normalized as the fold of increase compared with the adjacent normal tissue. 50x14mm (600 x 600 DPI)


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Detection of Tumor Invasive Biomarker using Peptamer of Signal Conversion and Signal Amplification Hao Li†, Weiwei Li†, Fengzhen Liu‡, Zhaoxia Wang‡, Genxi Li†, §,*, Yannis Karamanos∥ †. State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Biochemistry, Nanjing University, Nanjing 210093, China. ‡. Department of Oncology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, China. §. Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai, China. ∥. Laboratoire de la Barrière Hémato-encéphalique, Faculté des Sciences, Université d'Artois, rue Souvraz SP18, 62307 Lens Cedex, France. ABSTRACT: Inspired by the structural and functional feature of proteins in cell signaling, a switchable peptide is designed in this work. This switchable peptide is named as a “peptamer” and it can react to ligand binding with conformational change and activation/deactivation of catalytic ability. The peptamer is constructed by elaborately integrating several different peptide motifs with targeting and catalytic abilities. Thus targeted binding of the peptamer to integrin can be regulated by a synthetic ligand. Moreover, the conformational re-arrangement of the peptamer induced by both integrin and the synthetic ligand can resolve in altered affinity of the peptamer for a catalytic cofactor, cupric ion. This leads to greatly contrasted efficiency of catalysis in the presence/absence of integrin. This distinct switching on/off of catalytic activity also enables a bioassay of tissue integrin expression in clinical samples of thyroid carcinoma. Experimental results reveal that the detected integrin level shows parallel with the state of lymph node metastasis. So, this simple peptide model may help to understand the structural reconfiguration of proteins involved in cellular signal transduction, as well as to provide a new means to assess protein activity under pathological conditions such as cancer.

both a structural component and a regulatory factor of the cell skeleton17, while the pentameric recognition sequence can specifically block integrin from interactions with the tumor stroma, thereby therapeutically modulating or halting tumor invasion. This pentameric sequence shows the highest affinity when constrained in a loop18, so in our design the affinity is designed to be controllable through the formation/deformation of the loop. This formation/deformation of loop is in turn controlled by the interaction with an artificial regulatory factor, cucurbituril (Q8) (Scheme 1b), and both the concentration of Q8 and the sequence of interactions are designed to affect formation/deformation of the loop. Such spatiotemporally distinct aspects of protein/ligand interactions are molecular mechanisms usually employed by the cell signaling processes for pinpoint regulation of protein functions19. Moreover, each of these sequential interactions with the regulatory factors of different concentrations can either stabilize or destabilize the loop, while the final conformation adopted depends on the sum effect of all such factors. This is a process to integrate signal20, or the mechanism enabling the crosstalk of various signaling pathways and eventually leading to the formation of cell signaling network. Finally, as a result of conformational rearrangement, the peptide may show

Introduction Investigation of the functional structures of proteins is of fundamental importance to the current understanding of protein pathological functions1-6. For example, in the development of cancer, tumor promoting effects can be realized through structural rearrangements induced by various cancerous and pro-metastatic cellular signals7-10. Specifically, the interactions with messengers or protein ligands force the active site to adopt a different conformation, leading to effects such as altered catalytic activity or changed affinity towards effectors11-12. So, it can be said that the process of signal transduction is mainly realized by the flexible motifs in the protein active centers, such as structurally reconfigurable peptide loops1314-16. To apply such “smart” peptide loops in protein detection, we propose in this work to model the principal bioactivity of proteins in cell signaling (Scheme 1) on a “peptamer” that can change conformation in response to the binding of analytes. “Peptamer” is here defined as a synthetic peptide sequence that can recognize certain targets and can change its conformation to bind with the target, similar to the recently developed nucleic acid-based “aptamer”. The major functional motif of this peptamer is a pentameric recognition sequence of integrin. Integrin is

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enhanced or hindered ability of cofactor (cupric ion) binding (Scheme 1b), resulting in correspondingly augmented or diminished catalytic activity. In this designed process of peptide/ligand interactions, the relative abundance of each ligand, their affinity towards the peptamer, and most importantly, the interaction with a first ligand resulting in changed affinity of the peptamer for a subsequent ligand, are all properties21-23 manipulated by the cell signaling network to adapt protein functions to specific biochemical niches. Using ligand binding to finetune the targeting and catalytic abilities of peptides, the development of this peptamer may help to improve the protein-targeted bioanalytical strategies.

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was buffered with 4 mL 10 mM PBS pH 7.4. Signal response of the generated 2,3-diaminophenazine (DPA) was then recorded in the buffered reaction solution. These electrochemical measurements were carried out on a CHI660D Potentiostat (CH Instruments) with a conventional three-electrode system: the electrode immobilized with peptide as the working electrode, a saturated calomel electrode (SCE) as the reference electrode and a platinum wire as the counter electrode. Isothermal titration calorimetry (ITC) measurements were conducted using a MicroCal ITC200 System (GE healthcare life sciences). The titration was conducted at 25 °C. The titration schedule consisted of 38 consecutive injections of 1 μL with at least a 120 s interval between injections. Heats of dilution, measured by titrating beyond saturation, were subtracted from each data set. All solutions were degassed prior to titration. The data were analyzed using Origin 7.0 software.

Method Chemicals and Reagents Peptide probes (NH2-GH-GK(-F-NH2)-GGG-VRGDF-GGG-K(-F-NH2)-HK-CONH2, NH2-GH-G-K(-MUA)-G-K(-F-NH2)-GGG-VRGDF-GGGK(-F-NH2)-HK-CONH2, Figure S1) were manufactured by Shanghai Science Peptide as lyophilized powder, purity>90%, For the interface probe (shown as the second sequence), 11-mercaptoundecanol was incorporated into their sequences for surface immobilization of these probes. Human recombinant integrin αVβ6 was purchased from R&D Systems. Powder of the peptides was dissolved with 10 mM phosphate buffer solution (PBS) (pH 7.4). The target integrin was reconstituted with 10 mM PBS, pH 7.4 Redistilled water for the preparation of all the solutions was produced with a Milli-Q purification system, the resistance of 18 MΩ·cm was achieved to guarantee the purity. Cancerous tissue samples of patients of thyroid carcinoma were obtained from Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University, after elected consent by the local ethical committee. Upon receiving the samples, they were directly sliced on ice to 1 mm3, followed by fractioned using a nuclear extraction kit of Abnova, the cellular fraction thus obtained was 100× diluted and immediately brought to detection procedures.

Results and discussion The detailed design of the peptamer is shown in Scheme 1. The peptamer is designed to have three distinct motifs (Scheme 1a): the integrin affinity motif is at the center, flanked by two cucurbituril (Q8) affinity motifs, and at the two ends is the Cu(II) binding and catalytic motif split in two halves. The peptamer may have a variety of interactions with the ligands (Scheme 1b). Conformational change of the peptamer could be directed by Q8 concentration and facilitated by integrin binding. If Q8 is abundant (leftmost column of Scheme 1b), each of the two Q8 affinity motifs can be saturated by Q8, then the peptamer could remain in a linear conformation. The linear form will have greatly reduced affinity towards integrin. In the meantime, even if the loop form is induced by integrin binding, the peptamer will still have the two halves of the cupric ion binding motif too separated to effectively act as a single motif, leading to reduced affinity towards the cofactor Cu (II). On the contrary, if Q8 is of low abundance (the column second to the left), the two Q8 affinity motifs of the peptamer could be zipped together by one molecule of Q8. The integrin affinity motif located between will then be constrained into a loop, the most appropriate conformation for integrin binding. Binding of integrin may further stabilize this constrained conformation, then the two halves could be in close

Electrode Treatment and Modification These steps were essentially the same as previously reported24. Briefly, the electrode was reacted with 5 μM peptide in 10 mM PBS (pH 7.4) at 4 °C for 16 h, followed by being dipped in 1 μM 9-mercaptononanol for 3 h. To the surface of this electrode was added a solution of proper amount of Q8, after incubation, the electrode was then gently washed with ddH2O and ready for protein detection. Protein Detection Firstly, standard or clinical samples were incubated with the peptide modified electrodes at ambient temperature for proper time. After that, the electrode was gently rinsed sequentially with Q8 and ddH2O. To generate readout signal, the electrode was first dipped in 60 μM CuCl2 briefly, after gentle rinsing with ddH2O, it was then transferred into a 1 mM HCl solution (1 mL) containing 0.1 mg/ml ο-phenylenediamine (OPD). The solution was then placed in 50 °C water bath for 30 min. After being cooled to room temperature, this solution

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Scheme 1. (a) Design of the peptamer. (b) The peptamer conformation and affinity for cofactor ion can be regulated variously by the interactions with ligands.

most telling features showing the regulatory effect of each ligand on the final peptamer conformation and the ability of cofactor binding. First, using proper amount of Q8 to enhance peptamer binding with integrin, as well as the

vicinity of each other, and could act as an effective binding site of the cofactor Cu (II). In these processes, the affinity for both integrin and the cofactor ion can be regulated by the abundance of Q8. If the peptamer is allowed to interact with them without the pre-treatment with Q8 (the third column), both interactions can be expected to be in a different state. The final formation of the effective Cu (II) binding motif is also the cumulative effect of both Q8 and integrin binding, whether they tend to enhance or offset the conformational change induced by each other. Without the interactions with these two ligands, the peptamer will remain in a linear form very difficult to recognize the cofactor ion (the rightmost column). So, the ligand binding-induced conformational change may lead to altered affinity towards the cofactor cupric ion and the corresponding activation/de-activation of catalytic ability.

final Cu (II) binding (Fig. 1, a1~c1), highest affinity towards integrin and cupric ion can be observed (Table S1, subtable b1, c1). Specifically, 1:1 binding between Q8 and peptamer is first observed on Figure 1, a1, corresponding to two Q8 affinity motifs of the peptamer locked together by one molecule of Q8. Meanwhile, negative entropy accompanies this interaction (Sub-table a1), indicating a enthalpy-driven process with conformational change of the peptamer which might be assigned to the forming of a loop for subsequent integrin binding. This binding shows high affinity not too far away from that of the 5-mer therapeutic cyclic peptide “cilengitide” (2.5 nM of cilengitide as compared with 7.3 nM of the proposed peptide),25 with almost no entropy penalty, suggesting an optimized looped structure of the targeting sequence already formed via Q8 binding. Second, if surplus amount of cucurbituril is employed to keep the peptamer in a linear form upon integrin binding, reduced affinity towards both integrin and Cu (II) can be observed (Fig. 1,

The above designed peptamer and its conformational response to the ligands can be examined by investigating the thermodynamic aspects of the peptamer-ligand interactions (Fig. 1). For each interaction, a characteristic thermo-titration plot can be obtained. Among essential thermodynamic parameters that can be deduced from the plot (Table S1), the affinity and entropy of binding are

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Fig. 1 Isothermal titration calorimetry (ITC) results corresponding to the different types of peptamer-ligand interactions designed in Scheme 1. In each of the above results, the top row is the raw data of power versus time (in minutes), while the bottom row is the corresponding integrated enthalpy versus the molar ratio of titrant vs titrand. The titrants vs titrands are respetively: (a1') Cucurbituril (Q8) vs Peptamer (Pep), (a1) Q8 vs Pep, (b1') Pep-2Q8 vs Integrin (Int), (b1) Pep-Q8 vs Int, (b2) Pep vs Int, (c1') Cu (II) vs Pep-2Q8-Int, (c1) Cu (II) vs Pep-Q8-Int, (c2) Cu (II) vs Pep-Int, (c3) Cu (II) vs Pep.

a1’~c1’, Table S1, sub-table b1’, c1’). Specifically, surplus amount of Q8 can first result in a 2:1 binding between Q8 and peptamer with positive entropy consistent with the fact that this interaction is mainly driven by hydrophobic force (sub table a1’). Upon integrin binding, the bending of the peptamer put two bulky Q8 molecules in proximity, resulting in structural hindrance and negative entropy, as well as reduced affinity (sub table b1’). As the control, if

the interaction with Q8 is omitted, direct interactions with integrin and Cu (II) ion both have very slight increase of binding strength compared with the case of surplus Q8 (Fig. 1, b2~c2, Table S1, sub-table b2, c2), while the unstructured peptamer, without any interactions with Q8 or integrin, show almost no affinity towards the cupric ion (Fig. 1, c3, Table S1, sub-table c3). According to these results, the design of this peptamer is successful, thus its

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Scheme 2. Integrin bioassay developed based on the peptamer. (a) assay procedure, (b) scheme of the reaction of catalytic signal amplification.

surplus Q8 is incubated with the sensing interface to open all the integrin-free peptamers, while the integrin-bound peptamers will resist this effect via the strong peptamerintegrin binding and these peptamers can bind with Cu (II). The cupric ion complexed by these integrin-bound peptamers can then be used for catalytic signal amplification to generate readout signal positively related to integrin concentration in the samples. To better realize this design, the essential step for signal conversion, i.e., the blocking of peptide loop re-opening by integrin binding, is first investigated by ITC. As shown in Fig. S1a, the locked-up peptamer, in the absence of captured integrin, can interact with Q8 at a 1:1 ratio, in an entropy-driven manner, indicating that these peptamers can be relieved by surplus Q8 from the entropically unfavorable locked form. In the presence of captured integrin (Fig. S2b), no evident interaction is observed, indicating that integrin binding with the Q8-locked peptamer can effectively stabilize this structure to negate the trend of the re-opening induced by excessive Q8. Meanwhile, the interaction of Cu (II) with Pep-Q8 in the absence of integrin has also been investigated (Fig. S3). The open Pep-Q8 has no evident interaction with Cu (II) (Fig. S3a), leading to negligible background (curve A in Fig. S3c). On the other hand, the closed Pep-Q8 can have interaction with Cu (II) (Fig. S3b and curve B of Fig. S3c), but it can be opened by surplus Q8 as shown in Fig. S2a. So following the detection procedure in Scheme 2, low background can be obtained.

Fig. 2 Quantitative analysis of integrin using the peptamerbased bioassay. (a) Square wave voltammograms (SWVs) of DAP showing the gradual increase of signal response with integrin concentration. (b) Peak currents in (a) plotted as a function of integrin concentration. Inset is the linear range and the corresponding formula obtained via regression analysis. The error bars represent standard deviation from average (n=3).

final conformation and affinity for cofactor ion can reliably represent the upstream interactions with protein and other ligands. Employing this peptamer, an integrin assay with high sensitivity suitable for tissue sample analysis can be developed (Scheme 2). The peptamer pre-immobilized on the electrode surface can be locked-up in the looped form by binding with proper concentration of Q8. This asprepared sensing interface can then be used for capturing target integrin in standard or biological samples, while the strong binding strength of this looped form can greatly facilitate the sensitivity in diluted samples. To quantitatively convert integrin binding to signal readout,

For a balanced best performance of the proposed assay,

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Also, as shown in Fig. S8c, free Cu ion has no evident interference to the signal readout. Under these optimal conditions, the catalytically generated signal response increases proportionally to the logarithm of integrin concentration from 0.01 nM to around 0.5 μM (Fig. 2). The average standard deviation of all repetitive measurements is within 5%, showing an acceptable reproducibility. Besides, several control species have been employed to examine the specificity of this method. As can be seen in Fig. 3, only background level of response can be observed for these species, so the specificity is satisfactory.

Fig. 3 SWVs of DAP showing the specificity of the assay. All control species are of 100 nM.

As an essential component of cell skeleton, integrin connects the framework of the cells to the extra-cellular matrix (ECM). In the process of tumor invasion, as well as other tissue remodeling processes such as embryonic development, the expression and activity of integrin are frequently regulated to direct and facilitate cell migration26. In a range of cancers, it has been reported that integrin over-expression is connected with tumor invasiveness and integrin may serve as a biomarker of malignant transformation26-28. Here, employing the above designed peptamer and the developed biosensing method, integrin abundance in several clinical samples of thyroid carcinoma is assayed, with the results being grouped respectively according to the T (tumor), N (node) and M (distant metastasis) stages of the cases examined (Fig. 4). Under the grouping based on T stage and M stage, the samples show no evident connection with the advancing of cancer (Fig. 4a,c), but the expression seems to be, to some extent, correlated with N stage, or the situation of lymph node metastasis (Fig. 4b). This can be explained considering the biological function of integrin in regulating cell connection with ECM. As a matter of fact, breaking through the tissue barriers such as the ECM and the basal membrane is the necessary step of tumor filtration and metastasis. In this process, the up-regulation of integrin can indicate a frequent reconfiguration of cellular connection with the ECM, so integrin overexpression can usually be detected on the front edge of invasion, as well as sites of early metastasis located closely to the primary tumor, such as the sentinel lymph nodes.

other important steps of the assay method have been optimized. First, the proper concentration of Q8 to induceloop-formation of the peptamer is investigated. As is shown in Fig. S4, the amount of looped peptamer formed by different concentrations of Q8 is evaluated by the signal readout of cupric ion complexed by the looped peptamers. While insufficient Q8 cannot turn all the surface-immobilized peptamers into the looped form, excessive Q8 may keep the peptamer in a linearform, resulting in reduced signal readout. For the accuracy of detection, the optimized Q8 concentration (60 μM) is selected for all the following experiments. The incubation time for this step has also been optimized (Fig. S5), and 40 min incubation time is known to required to convert all the surface immobilized peptamers to the closed form. Since the catalytic activity of cupric ion is exploitable for signal amplification, the response of the product of catalytic signal amplification is recorded for all the following experiments as the final signal readout. Using this type of signal readout, as well as the biosensing interface prepared under the above optimized conditions, the interaction with the target integrin is found to approach saturation in 40 min (Fig. S6). Finally, the concentration of Q8 required to re-open the surplus peptamers is studied, and 40 μM is known to be sufficient to open most of the surplus peptamers on the electrode surface (Fig. S7). In addition, the concentration of Cu (II) has also been optimized (60 μM), the experimental result of which has been shown in Fig. S8a, b.

Fig. 4 Box chart to show the distribution of the detected concentration of integrin in patients of thyroid carcinoma, grouped by (a) T stage, (b) N stage and (c) M stage. For each sample, the detected integrin in cancerous tissue is normalized as the fold of increase compared with the adjacent normal tissue.

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Conclusion In this work, a peptamer is designed to model the structural feature of protein observed in cell signaling on a simplified peptide sequence. This peptamer can react to the presence and the concentration of the target protein as well as other ligands by the formation/de-formation of a specific conformation. In the meantime, its temporally defined interaction with a series of ligands can stabilize this conformation for the forming of a stable peptide-Cu (II) complex that can show catalytic activity. Using this peptamer, an interface electrochemical bioassay method is established for the analysis of integrin expression. Moreover, this method can also be successfully applied in the detection of integrin in clinical tissue samples of thyroid carcinoma. The detected integrin level has a parallel with the state of lymph node metastasis, indicating the active role of integrin in promoting tumor invasion. So, these results may point to the prospective use of the proposed peptamer in clinical analysis of biomarker in the future.

ASSOCIATED CONTENT Supporting Information Supporting data on the design principle as well as optimization of various experimental conditions. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Corresponding author at State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing 210093, China. Fax: +86 25 83592510. E-mail address: [email protected].

ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (Grant Nos. 21235003, 21327902, J1103512, J1210026).

REFERENCES

(10) Monecke, T.; Dickmanns, A.; Ficner, R., Febs Journal 2014, 281, 4179-4194. (11) Fontecilla-Camps, J. C.; Amara, P.; Cavazza, C.; Nicolet, Y.; Volbeda, A., Nature 2009, 460, 814-822. (12) Jordan, F.; Nemeria, N. S.; Sergienko, E., Acc. Chem. Res. 2005, 38 (9), 755-763. (13) Shearer, J., Acc. Chem. Res. 2014, 47, 2332-2341. (14) Balakumar, P.; Jagadeesh, G., J. Mol. Endocrinol. 2014, 53, R71-R92. (15) Richard, J. P.; Amyes, T. L.; Goryanova, B.; Zhai, X., Curr. Opin. Chem. Biol. 2014, 21, 1-10. (16) Skala, W.; Utzschneider, D. T.; Magdolen, V.; Debela, M.; Guo, S.; Craik, C. S.; Brandstetter, H.; Goettig, P., J. Biol. Chem. 2014, 289, 34267-34283. (17) Shattil, S. J.; Kim, C.; Ginsberg, M. H., Nat. Rev. Mol. Cell Biol. 2010, 11, 288-300. (18) Mas-Moruno, C.; Rechenmacher, F.; Kessler, H., AntiCancer Agent. Med. Chem. 2010, 10, 753-768. (19) Dehmelt, L.; Bastiaens, P. I. H., Nat. Rev. Mol. Cell Biol. 2010, 11, 440-452. (20) Eijkelenboom, A.; Burgering, B. M. T., Nat. Rev. Mol. Cell Biol. 2013, 14, 83-97. (21) Borodinsky, L. N.; Belgacem, Y. H.; Swapna, I.; Visina, O.; Balashova, O. A.; Sequerra, E. B.; Tu, M. K.; Levin, J. B.; Spencer, K. A.; Castro, P. A.; Hamilton, A. M.; Shim, S., Develop. Neurobiol. 2015, 75, 349-359. (22) Brown, N. E.; Goswami, D.; Branch, M. R.; Ramineni, S.; Ortlund, E. A.; Griffin, P. R.; Hepler, J. R., J. Biol. Chem. 2015, 290, 9037-49. (23) Filteau, M.; Diss, G.; Torres-Quiroz, F.; Dube, A. K.; Schraffl, A.; Bachmann, V. A.; Gagnon-Arsenault, I.; Chretien, A.E.; Steunou, A.-L.; Dionne, U.; Cote, J.; Bisson, N.; Stefan, E.; Landry, C. R., Proc. Natl. Acad. Sci. USA 2015, 112, 4501-6. (24) Li, H.; Huang, Y.; Zhang, B.; Pan, X.; Zhu, X.; Li, G., Anal. Chem. 2014, 86, 12138-12142. (25) Dechantsreiter, M. A.; Planker, E.; Matha, B.; Lohof, E.; Holzemann, G.; Jonczyk, A.; Goodman, S. L.; Kessler, H., J. Med. Chem. 1999, 42, 3033-3040. (26) Zhang, Z. Y.; Xu, K. S.; Wang, J. S.; Yang, G. Y.; Wang, W.; Wang, J. Y.; Niu, W. B.; Liu, E. Y.; Mi, Y. T.; Niu, J., Clin. Oncol. 2008, 20, 61-66. (27) Bendas, G.; Borsig, L., Inter. J. Cell Biol. 2012, 2012, 676731676731. (28) Oxboel, J.; Binderup, T.; Knigge, U.; Kjaer, A., Oncol. Rep. 2009, 21, 769-775.

(1) Consonni, S. V.; Maurice, M. M.; Bos, J. L., Nat. Rev. Mol. Cell Biol. 2014, 15, 357-362. (2) Derbyshire, E. R.; Marletta, M. A., Annu. Rev. Biochem., 2012, 81, 533-559. (3) Endicott, J. A.; Noble, M. E. M.; Johnson, L. N., Annu. Rev. Biochem., 2012, 81, 587-613. (4) Frame, M. C.; Patel, H.; Serrels, B.; Lietha, D.; Eck, M. J., Nat. Rev. Mol. Cell Biol. 2010, 11, 802-814. (5) Joerger, A. C.; Fersht, A. R., Structural biology of the tumor suppressor p53. Annu. Rev. Biochem., 2008; 77, 557-582. (6) Reinhardt, H. C.; Yaffe, M. B., Nat. Rev. Mol. Cell Biol. 2013, 14, 563-580. (7) Boehmer, F.; Szedlacsek, S.; Tabernero, L.; Ostman, A.; den Hertog, J., Febs Journal 2013, 280, 413-431. (8) Gherardi, E.; Birchmeier, W.; Birchmeier, C.; Woude, G. V., Nat. Rev. Cancer 2012, 12, 89-103. (9) He, R.; Zeng, L.-F.; He, Y.; Zhang, S.; Zhang, Z.-Y., Febs Journal 2013, 280 (2), 731-750.



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