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Biochemistry

Mimicking the Function of Signaling Proteins: Toward Artificial Signal Transduction Therapy

Published: September 29th, 2016

DOI:

10.3791/54396

1Department of Organic Chemistry, Weizmann Institute of Science

We present guidelines for developing synthetic 'chemical transducers' that can induce communication between naturally unrelated proteins. In addition, detailed protocols are presented for synthesizing and testing a specific 'transducer' that enables a growth factor to activate a detoxifying enzyme and consequently, to regulate the cleavage of an anticancer prodrug.

Signal transduction pathways, which control the response of cells to various environmental signals, are mediated by the function of signaling proteins that interact with each other and activate one other with high specificity. Synthetic agents that mimic the function of these proteins might therefore be used to generate unnatural signal transduction steps and consequently, alter the cell's function. We present guidelines for designing 'chemical transducers' that can induce artificial communication between native proteins. In addition, we present detailed protocols for synthesizing and testing a specific 'transducer', which can induce communication between two unrelated proteins: platelet-derived growth-factor (PDGF) and glutathione-S-transferase (GST). The way by which this unnatural PDGF-GST communication could be used to control the cleavage of an anticancer prodrug is also presented, indicating the potential for using such systems in 'artificial signal transduction therapy'. This work is intended to facilitate developing additional 'transducers' of this class, which may be used to mediate intracellular protein-protein communication and consequently, to induce artificial cell signaling pathways.

Signal transduction pathways play a significant role in virtually every cellular process and allow the cell to rapidly respond to environmental signals.1 These pathways are often triggered by the binding of a signaling molecule to an extracellular receptor, which results in activation of intracellular enzymes. Amplification and propagation of this signal within the cell is mediated by the function of signaling proteins that form a network of protein-protein interactions in which enzymes are reversibly activated with high specificity. Because dysregulation of these networks frequently leads to cancer development, there has been much interest in establishing ....

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1. Synthesis of the 'Chemical Transducer'

  1. Preliminary Preparations
    1. Prepare 2 M triethylammonium acetate (TEAA) buffer by mixing 278 ml of triethylamine with 114 ml of acetic acid and 400 ml of ultrapure water. Adjust the pH to 7 and add water to a final volume of 1 L. Keep it in a dark bottle.
      Note: This solution is stable for years.
    2. Prepare a 5 mM ascorbic acid solution by dissolving 18 mg of ascorbic acid in 20 ml of ultrapure water. Use a fresh solution; the solution .......

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The design, synthesis, and mechanism of action of a 'chemical transducer' that can induce artificial communication between PDGF and GST are presented in Figure 2. The structure of the 'transducer' integrates a PDGF DNA aptamer and a bis-ethacrynic amide (bEA), which is a known GST inhibitor (Figure 2a).19 These binders enable the 'transducer' to bind both PDGF and GST with different affinities, namely, with dissociation .......

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We presented a method for designing and testing of a 'chemical transducer' that can induce artificial communication between two naturally unrelated proteins, GST and PDGF, without modifying the native proteins. The unnatural GST-PDGF communications could be detected in real time by using enzymatic assays that follow the changes in the activity of GST in the presence of the 'chemical transducer' and increasing the concentrations of PDGF. In addition to detecting the activation of GST by PDGF, these assays were used to fol.......

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This research was supported by the Minerva Foundation, the HFSP Organization, and a European Research Council Grant (Starting Grant 338265).

....

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Name Company Catalog Number Comments
1-chloro-2,4-dinitrobenzene Sigma-Aldrich 237329
Acetic acid Bio Lab 01070521
Acetnitrile J.T.Baker 9017-03
Ascorbic acid Sigma-Aldrich A4544
Copper(II) Sulfate pentahydrate Merck-Millipore 102790
Dimethyl sulfoxide Merck-Millipore 802912
Dulbecco's Phosphate Buffered Saline Biological Industries 02-023-5A
Ethacrynic acid Tokyo Chemical Industry Co. Ltd E0526
Glutathione-s-transferase M1-1 Israel Structural Proteomics Center (Weizmann Institute of Science, Rehovot, Israel)
JS-K Sigma-Aldrich J4137
L-glutathione reduced Sigma-Aldrich G4251
Magnesium Chloride J.T.Baker 0162
nitrate/nitrite colorimetric assay kit Cayman Chemical 780001
Oligonucleotides W. M. Keck Foundation Biotechnology at Yale University custom order
PDGF-BB Israel Structural Proteomics Center (Weizmann Institute of Science, Rehovot, Israel)
TBTA Sigma-Aldrich 678937
Triethylamine Sigma-Aldrich T0886
Desalting column GE Healthcare illustra MicroSpin G-25 Columns
HPLC Waters  2695 separation module
HPLC column Waters XBridgeTM OST C18 column (2.5 μM, 4.6 mm × 50 mm)
HPLC column Waters  XBridgeTM OST C18 column (2.5μM, 10 mm × 50 mm)
Plate reader BioTek synergy H4 hybrid

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