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

1. Synthesis of the &#39;Chemical Transducer&#39;
<ol>
	<li>Preliminary Preparations
	<ol>
		<li>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.</li>
		<li>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 ...

<ol>
	<li>Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling—2000+and+Beyond.">Signaling—2000 and Beyond.</a> Cell. 100, 113-127 (2000).</li><li>Levitzki, A., Klein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+therapy+of+cancer.">Signal transduction therapy of cancer.</a> Mol Aspects Med. 31, 287-329 (2010).</li><li>Peri-Naor, R., Motiei, L., Margulies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+signal+transduction+therapy:+a+futuristic+approach+to+disease+treatment.">Artificial signal transduction therapy: a futuristic approach to disease treatment.</a> Future Med. Chem. 7, 2091-2093 (2015).</li><li>Peri-Naor, R., Ilani, T., Motiei, L., Margulies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-Protein+Communication+and+Enzyme+Activation+Mediated+by+a+Synthetic+Chemical+Transducer.">Protein-Protein Communication and Enzyme Activation Mediated by a Synthetic Chemical Transducer.</a> J. Am. Chem. Soc. 137, 9507-9510 (2015).</li><li>Corson, T. W., Aberle, N., Crews, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+Applications+of+Bifunctional+Small+Molecules:+Why+Two+Heads+Are+Better+Than+One.">Design and Applications of Bifunctional Small Molecules: Why Two Heads Are Better Than One.</a> ACS Chem. Biol. 3, 677-692 (2008).</li><li>Rutkowska, A., Schultz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Tango:+The+Toolbox+to+Capture+Interacting+Partners.">Protein Tango: The Toolbox to Capture Interacting Partners.</a> Angew. Chem. Int. Ed. 51, 8166-8176 (2012).</li><li>Meyer, C., K&#246;hn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Molecular+T&#234;te-&#224;-T&#234;te+Arranged+by+a+Designed+Adaptor+Protein.">A Molecular T&#234;te-&#224;-T&#234;te Arranged by a Designed Adaptor Protein.</a> Angew. Chem. Int. Ed. 51, 8160-8162 (2012).</li><li>Klemm, J. D., Schreiber, S. L., Crabtree, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimerization+as+a+Regulatory+Mechanism+in+Signal+Transduction.">Dimerization as a Regulatory Mechanism in Signal Transduction.</a> Annu. Rev. Immunol. 16, 569-592 (1998).</li><li>DeRose, R., Miyamoto, T., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+signaling+at+will:+chemically-inducible+dimerization+(CID)+techniques+resolve+problems+in+cell+biology.">Manipulating signaling at will: chemically-inducible dimerization (CID) techniques resolve problems in cell biology.</a> Pflugers Arch. 465, 409-417 (2013).</li><li>Gestwicki, J. E., Marinec, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+control+over+protein-protein+interactions:+beyond+inhibitors.">Chemical control over protein-protein interactions: beyond inhibitors.</a> Comb. Chem. High Throughput. Screen. 10, 667-675 (2007).</li><li>Battle, C., Chu, X., Jayawickramarajah, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligonucleotide-based+systems+for+input-controlled+and+non-covalently+regulated+protein+binding.">Oligonucleotide-based systems for input-controlled and non-covalently regulated protein binding.</a> Supramol. Chem. 25, 848-862 (2013).</li><li>Diezmann, F., Seitz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-guided+display+of+proteins+and+protein+ligands+for+the+interrogation+of+biology.">DNA-guided display of proteins and protein ligands for the interrogation of biology.</a> Chem. Soc. Rev. 40, 5789-5801 (2011).</li><li>R&#246;glin, L., Ahmadian, M. R., Seitz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-Controlled+Reversible+Switching+of+Peptide+Conformation+and+Bioactivity.">DNA-Controlled Reversible Switching of Peptide Conformation and Bioactivity.</a> Angew. Chem. Int. Ed. 46, 2704-2707 (2007).</li><li>R&#246;glin, L., Altenbrunn, F., Seitz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+and+RNA-Controlled+Switching+of+Protein+Kinase+Activity.">DNA and RNA-Controlled Switching of Protein Kinase Activity.</a> ChemBioChem. 10, 758-765 (2009).</li><li>Harris, D. C., Chu, X., Jayawickramarajah, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-Small+Molecule+Chimera+with+Responsive+Protein-Binding+Ability.">DNA-Small Molecule Chimera with Responsive Protein-Binding Ability.</a> J. Am. Chem. Soc. 130, 14950-14951 (2008).</li><li>Harris, D. C., Saks, B. R., Jayawickramarajah, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-Binding+Molecular+Switches+via+Host-Guest+Stabilized+DNA+Hairpins.">Protein-Binding Molecular Switches via Host-Guest Stabilized DNA Hairpins.</a> J. Am. Chem. Soc. 133, 7676-7679 (2011).</li><li>Kim, Y., Cao, Z., Tan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+assembly+for+high-performance+bivalent+nucleic+acid+inhibitor.">Molecular assembly for high-performance bivalent nucleic acid inhibitor.</a> Proc. Nat. Acad. Sci. U.S.A. 105, 5664-5669 (2008).</li><li>Han, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Logical+Molecular+Circuit+for+Programmable+and+Autonomous+Regulation+of+Protein+Activity+Using+DNA+Aptamer-Protein+Interactions.">A Logical Molecular Circuit for Programmable and Autonomous Regulation of Protein Activity Using DNA Aptamer-Protein Interactions.</a> J. Am. Chem. Soc. 134, 20797-20804 (2012).</li><li>Motiei, L., Pode, Z., Koganitsky, A., Margulies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+Protein+Surface+Sensors+as+a+Tool+for+Analyzing+Small+Populations+of+Proteins+in+Biological+Mixtures.">Targeted Protein Surface Sensors as a Tool for Analyzing Small Populations of Proteins in Biological Mixtures.</a> Angew. Chem. Int. Ed. 53, 9289-9293 (2014).</li><li>Ranallo, S., Rossetti, M., Plaxco, K. W., Vall&#233;e-B&#233;lisle, A., Ricci, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Modular,+DNA-Based+Beacon+for+Single-Step+Fluorescence+Detection+of+Antibodies+and+Other+Proteins.">A Modular, DNA-Based Beacon for Single-Step Fluorescence Detection of Antibodies and Other Proteins.</a> Angew. Chem. Int. Ed. 54, 13214-13218 (2015).</li><li>Franzini, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Structure-Activity+Relationships+from+Screening+a+Structurally+Compact+DNA-Encoded+Chemical+Library.">Identification of Structure-Activity Relationships from Screening a Structurally Compact DNA-Encoded Chemical Library.</a> Angew. Chem. Int. Ed. 54, 3927-3931 (2015).</li><li>Unger-Angel, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+recognition+by+bivalent,+'turn-on'+fluorescent+molecular+probes.">Protein recognition by bivalent, 'turn-on' fluorescent molecular probes.</a> Chem. Sci. 6, 5419-5425 (2015).</li><li>Nissinkorn, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+Protein+Surfaces+with+Targeted+Fluorescent+Receptors.">Sensing Protein Surfaces with Targeted Fluorescent Receptors.</a> Chem. Eur. J. 21, 15981-15987 (2015).</li><li>Huber, C. G., Oefner, P. J., Bonn, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Liquid+Chromatography+of+Oligonucleotides+on+Nonporous+Alkylated+Styrene-Divinylbenzene+Copolymers.">High-Resolution Liquid Chromatography of Oligonucleotides on Nonporous Alkylated Styrene-Divinylbenzene Copolymers.</a> Anal. Biochem. 212, 351-358 (1993).</li><li>Lyon, R. P., Hill, J. J., Atkins, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+class+of+bivalent+glutathione+S-transferase+inhibitors.">Novel class of bivalent glutathione S-transferase inhibitors.</a> Biochemistry. 42, 10418-10428 (2003).</li><li>Battle, C., Chu, X., Jayawickramarajah, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligonucleotide-based+systems+for+input-controlled+and+non-covalently+regulated+protein+binding.">Oligonucleotide-based systems for input-controlled and non-covalently regulated protein binding.</a> Supramol. Chem. , 1-16 (2013).</li></ol>

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...

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 &#39;signal transduction therapy of cancer&#39;,2 whereby drugs are designed to disrupt malignant signaling pathways. We have recently proposed an alternative approach to signal transduction therapy that relies on the ability of drugs to generate unnatural signal transduction pathways.3 In particular, we believe that by designing synthetic agents that mimic the function of signaling proteins, it would be possible to modulate the cell&#39;s function indirectly. For example, these artificial networks may enable protein biomarkers to activate enzymes that cleave prodrugs. Alternatively, these signaling protein mimetics might be able to activate unnatural cell signaling pathways, resulting in therapeutic effects.
To demonstrate the feasibility of this approach, we have recently created a synthetic &#39;chemical transducer&#39;4 that enables platelet-derived growth factor (PDGF) to trigger the cleavage of an anticancer prodrug by activating glutathione-s-transferase (GST), which is not its natural binding partner. The structure of this &#39;transducer&#39; consists of an anti-PDGF DNA aptamer that is modified with a bivalent inhibitor for GST. Hence, this synthetic agent belongs to a family of molecules with binding sites to different proteins,5-7 such as chemical inducers of dimerization (CIDs)8-10 and also to the group of protein-binders based on oligonucleotide-synthetic molecule conjugates.11-21
The general principles underlying the design of such systems is described herein and detailed protocols for synthesizing and testing the function of this &#39;transducer&#39; with conventional enzymatic assays are provided. This work is intended to facilitate developing additional &#39;transducers&#39; of this class, which may be used to mediate intracellular protein-protein communication and consequently, to induce artificial cell signaling pathways.
Figure 1 schematically describes the operating principles of synthetic &#39;chemical transducers&#39; that can mediate unnatural protein-protein communications. In this illustration, a &#39;chemical transducer&#39;, which integrates synthetic binders for proteins I and II (binders I and II), enables protein II to trigger the catalytic activity of protein I, which is not its natural binding partner. In the absence of protein II, the transducer binds the catalytic site of the enzyme (protein I) and inhibits its activity (Figure 1, state ii). The binding of the &#39;transducer&#39; to protein II, however, promotes interactions between binder I and the surface of protein II (Figure 1, state iii), which reduces its affinity toward protein I. As a result, the effective concentration of the &#39;free&#39; transducer in the solution is reduced, which leads to dissociation of the transducer-protein I complex and to reactivation of protein I (Figure 1, state iv). Taken together, these steps highlight three fundamental principles underlying the design of efficient &#39;transducers&#39;: (1) a &#39;transducer&#39; should have a specific binder for each of the protein targets, (2) the interaction between binder II and protein II should be stronger than the interaction between binder I and protein I, and (3) binder I must be able to interact with the surface of protein II. This last principle does not necessarily require that binder I alone would have a high affinity and selectivity toward protein II. Instead, it is based on our recent studies which showed that bringing a synthetic molecule in proximity to a protein is likely to promote interactions between this molecule and the surface of the protein.19,22,23
<img alt="Figure 1" src="/files/ftp_upload/54396/54396fig1.jpg" />
Figure 1: Operating principles of &#39;chemical transducers&#39;. When the &#39;chemical transducer&#39; is added to an active protein I (state i), it binds to its active site through binder I and inhibits its activity (state ii). In the presence of protein II, however, the unbound &#39;chemical transducer&#39; interacts with protein II through binder II, which promotes interactions between binder I and the surface of protein II. This induced binder I-protein II interaction reduces the effective concentration of binder I, which leads to dissociation of the &#39;transducer&#39;-protein I complex and to protein I reactivation (state iv). <a href="https://www.jove.com/files/ftp_upload/54396/54396fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="1144">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">1-chloro-2,4-dinitrobenzene</td>
			<td style="width:344px;">Sigma-Aldrich</td>
			<td style="width:397px;">237329</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Acetic acid</td>
			<td style="width:344px;">Bio Lab</td>
			<td style="width:397px;">01070521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Acetnitrile</td>
			<td style="width:344px;">J.T.Baker</td>
			<td style="width:397px;">9017-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Ascorbic acid</td>
			<td style="width:344px;">Sigma-Aldrich</td>
			<td style="width:397px;">A4544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Copper(II) Sulfate pentahydrate</td>
			<td style="width:344px;">Merck-Millipore</td>
			<td style="width:397px;">102790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Dimethyl sulfoxide</td>
			<td style="width:344px;">Merck-Millipore</td>
			<td style="width:397px;">802912</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:236px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:344px;">Biological Industries</td>
			<td style="width:397px;">02-023-5A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Ethacrynic acid</td>
			<td style="width:344px;">Tokyo Chemical Industry Co. Ltd</td>
			<td style="width:397px;">E0526</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:236px;">Glutathione-s-transferase M1-1</td>
			<td style="width:344px;">Israel Structural Proteomics Center (Weizmann Institute of Science, Rehovot, Israel)</td>
			<td style="width:397px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">JS-K</td>
			<td style="width:344px;">Sigma-Aldrich</td>
			<td style="width:397px;">J4137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">L-glutathione reduced</td>
			<td style="width:344px;">Sigma-Aldrich</td>
			<td style="width:397px;">G4251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Magnesium Chloride</td>
			<td style="width:344px;">J.T.Baker</td>
			<td style="width:397px;">0162</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:236px;">nitrate/nitrite colorimetric assay kit</td>
			<td style="width:344px;">Cayman Chemical</td>
			<td style="width:397px;">780001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:236px;">Oligonucleotides</td>
			<td style="width:344px;">W. M. Keck Foundation Biotechnology at Yale University</td>
			<td style="width:397px;"></td>
			<td>custom order</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:236px;">PDGF-BB</td>
			<td style="width:344px;">Israel Structural Proteomics Center (Weizmann Institute of Science, Rehovot, Israel)</td>
			<td style="width:397px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">TBTA</td>
			<td style="width:344px;">Sigma-Aldrich</td>
			<td style="width:397px;">678937</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Triethylamine</td>
			<td style="width:344px;">Sigma-Aldrich</td>
			<td style="width:397px;">T0886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Desalting column</td>
			<td style="width:344px;">GE Healthcare</td>
			<td style="width:397px;">illustra MicroSpin G-25 Columns</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">HPLC</td>
			<td style="width:344px;">Waters&#160;</td>
			<td>2695 separation module</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">HPLC column</td>
			<td style="width:344px;">Waters</td>
			<td style="width:397px;">XBridgeTM OST C18 column (2.5 &#956;M, 4.6 mm &#215; 50 mm)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">HPLC column</td>
			<td style="width:344px;">Waters&#160;</td>
			<td style="width:397px;">XBridgeTM OST C18 column (2.5&#956;M, 10 mm &#215; 50 mm)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Plate reader</td>
			<td style="width:344px;">BioTek</td>
			<td style="width:397px;">synergy H4 hybrid</td>
			<td></td>
		</tr>
	</tbody>
</table>

mimicking the function of signaling proteins: toward artificial signal transduction therapy

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.

The design, synthesis, and mechanism of action of a &#39;chemical transducer&#39; that can induce artificial communication between PDGF and GST are presented in Figure 2. The structure of the &#39;transducer&#39; integrates a PDGF DNA aptamer and a bis-ethacrynic amide (bEA), which is a known GST inhibitor (Figure 2a).19 These binders enable the &#39;transducer&#39; to bind both PDGF and GST with different affinities, namely, with dissociation ...

Watch this Scientific Journal Video about Mimicking the Function of Signaling Proteins: Toward Artificial Signal Transduction Therapy at JoVE.com

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

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 ...

The overall goal of this experiment is to spectroscopically follow the unnatural communication between two proteins induced by a synthetic chemical transducer. The will demonstrate the possibility of developing synthetic analogs of signalling proteins that will be able to carry out signal transduction pathways not found in nature. The ability to follow unnatural protein to protein communication by common spectrophotometer could help develop and improve the function of artificial chemical transducers and bring us closer to achieving artificial signal transduction therapy.The chemical transducer is composed of a platelet derived growth factor, or PDGF aptamer. A bivalent ethacrynic amide GST inhibitor, a fluorophore and a quencher. The synthesis of the chemical transducer will not be demonstrated in this video.But is described in the text protocol. The operating principles of the chemical transducer are illustrated here. The chemical transducer inhibits the enzymatic activity of GST, when the two EA groups bind the enzymes active sites.Addition of PDGF leads to the formation of the PDGF chemical transducer complex. Which disrupts the transducer-GST interaction, therefore restoring GST enzymatic activity. The following addition of an unmodified PDGF aptamer releases the chemical transducer and allows it to re-inhibit the enzyme.Prior to starting the experiments for examining control of GST activity by PDGF, set up an experimental procedure in the plate reader for kinetic measurement. Create a new experiment as a standard protocol. Select Procedure to open the Procedure Settings window.In the pop-up list on the upper side of the window, choose the 384 Plate Type according to the plate manufacturer. Select Read on the left menu. Choose Absorbance for the Detection Method.And Endpoint for the Read Type. In the Wavelength window, type 340 nanometers. Select Full Plate, and choose the wells to be measured.Then click OK to close the Read window. On the left menu, choose Start Kinetic. Set the Run Time as 10 minutes.Select the Minimum Intervals option. Then click OK to close the Kinetic window. Drag the Read Line into the Kinetic Measurement.Select the Validate button, followed by the OK button. Save the experiment. Select the Play button.A dialog box will appear, but do not select the OK button until ready to start the kinetic measurement. Once the plate reader has been set up, the next step is to prepare the samples for measuring GST activity in the presence of the chemical transducer and PDGF. In order to test four concentrations of PDGF in triplicate, prepare four samples where each contains 3.25 microliters of the chemical transducer, and 3.25 microliters of GST M-1.Add to each sample, zero, 1.2, 2.4 or 4.9 microliters of PDGF respectively. Then add the appropriate volume of the assay buffer to each sample to bring the total volume to 130 microliters. Incubate the samples at room temperature for 10 minutes.After 10 minutes, transfer 40 microliters of each sample to the wells of a 384 transparent well plate. Transfer each of the four samples to either the odd or the even wells in the same row only. To allow the use of a multi-pipetter for substrate addition.Next, use a 12-channel multi-pipetter to quickly add to each sample 10 microliters of each substrate previously prepared in a 96 well plate. Mix quickly, but gently, to avoid bubbles. Immediately insert the plate into the plate reader, and start the kinetic measurement by selecting the OK button in the dialog box.To examine GST activation inhibition cycles mediated by the chemical transducer, prepare five samples, each containing 84.5 microliters of assay buffer. 3.25 microliters of chemical transducer, and 3.25 microliters of GST M1-1. Incubate at room temperature for three minutes.Add 3.65 microliters of assay buffer to sample one, and 3.65 microliters of PDGF to samples two, three, four and five. Incubate at room temperature for three minutes. Add 3.12 microliters of assay buffer to samples one and two, and 3.12 microliters of PDGF aptamer to samples three, four and five.Incubate at room temperature for three minutes. Add 24.4 microliters of assay buffer to samples one, two and three. And 24.4 microliters of PDGF to samples four and five.Incubate at room temperature for three minutes. Add 7.8 microliters of assay buffer to samples one to four, and 7.8 microliters of PDGF aptamer to sample five. Incubate at room temperature for five minutes.Transfer the samples to a 384 transparent well plate. To perform the experiment in triplicate, and quickly add the substrate to the samples. Insert the plate into the reader, and start the kinetic measurement.To begin this assay, set up an experimental procedure in the plate reader for kinetic measurement as described in the protocol text. Prepare two samples in two wells of a 384 transparent well plate. For each well, mix one microliter of GST M1-1 and one microliter of the chemical transducer in 38 microliters of assay buffer.Leave an empty well between the two wells. Using a 12-channel multi-pipetter, quickly add 10 microliters of substrate to each well. And mix gently and quickly to avoid bubbles.Immediately insert the plate into the reader and start the kinetic measurement. When the plate reader opens up after 3.5 minutes, quickly add 1.125 microliters of PDGF to one of the wells, mix gently, and allow the plate to re-enter the reader for the remaining kinetic measurements. Prior to preparing the samples for this experiment, set up an experimental procedure in the plate reader for kinetic measurement as described in the protocol text.A nitrite/nitrate colorimetric kit will be used for measuring nitric oxide production. In a 96 well plate, dispense 50 microliters of assay buffer per well into the first row. 70 microliters of Griess One Reagent per well into the second row.And 70 microliters of Griess Two Reagent per well into the third row. Prepare four samples, each containing 4.8 microliters of the chemical transducer. To sample one, add 155.2 microliters of assay buffer.To sample two, add 3.2 microliters of GST M1-1, and 152 microliters of assay buffer. To sample three, add 9.6 microliters of PDGF, and 145.6 microliters of assay buffer. And to sample four, add 3.2 microliters of GST M1-1, 9.6 microliters of PDGF, and 142.4 microliters of assay buffer.Incubate the samples at room temperature for 10 minutes. Dispense 50 microliters of sample per well in a 384 transparent well plate to perform the experiment in triplicate. Dispense samples only to the odd or even wells in the same line to allow the use of a multi-pipetter for substrate addition.Add 0.54 microliters of JS-K prodrug to each sample. Quickly add to each sample 10 microliters of glutathione solution and mix gently and quickly to avoid bubbles. Insert the plate into the reader, and start the kinetic measurement.Immediately after the kinetic measurement, transfer 50 microliters of each sample to each well of the first row of the 96 well plate with assay buffer. Then transfer 50 microliters from each well of the second row of the plate into the wells of the first row of the plate. And 50 microliters per well from the third row of the plate into the wells of the first row of the plate.Incubate while protecting from light for 10 minutes at room temperature. Before measuring the absorbance at 550 nanometers. Measuring GST activity without the chemical transducer, and with the chemical transducer showed that the chemical transducer inhibits GST activity.GST is reactivated by the addition of increasing concentrations of PDGF to the GST-chemical transducer complex. Sequential additions of PDGF, and an unmodified PDGF aptamer to the GST-chemical transducer complex revealed that the communication between PDGF and GST was reversible. The addition of PDGF to the GST-transducer complex resulted in a rapid increase in GST activity, 3.5 minutes after adding substrates.While the addition of a PDGF aptamer to a mixture of GST, PDGF and the chemical transducer resulted in a decrease in GST activity. These results indicate that the system responds in real-time to changes in its environment. Subsequently, the ability of this system to control prodrug activation was investigated using JS-K, an anti-cancer prodrug that is activated by GST to release toxic nitric oxide.Measurements of nitric oxide amounts released upon the addition of JS-K to the chemical transducer and different combinations of GST and PDGF confirmed that only the presence of both GST and PDGF activates JS-K. This all shows how a synthetic agent can enable a growth factor to trigger the catalytic activity of Glutathione S-Transferase, which is not its natural enzyme partner. Future biomimetics of this class may therefore be used to alter the response of cells to environmental signals or provide them with new properties.

mimicking-function-signaling-proteins-toward-artificial-signal

Research

JoVE Journal

Biochemistry

6.9K ビュー.  Weizmann Institute of Science. The overall goal of this experiment is to spectroscopically follow the unnatural communication between two proteins induced by a synthetic chemical transducer. The will demonstrate the possibility of developing synthetic analogs of signalling proteins that will be able to carry out signal transduction pathways not found in nature. The ability to follow unnatural protein to protein communication by common spectrophotometer could help develop and improve the function of artificial chemical tran...