Name: a rapid and quantitative fluorimetric method for protein-targeting small molecule drug screening
Uploaded: 2015-10-16T15:00:00
Description: Watch this Scientific Journal Video about A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening at JoVE.com

Y.N.T. would like to acknowledge the Agency for Science, Technology and Research (A*STAR), Singapore for the financial support under the JCO CDA grant 13302FG063.

Caution: Please consult the safety data sheets (SDS) of all involved chemicals before use. The drug screening experiment involves the synthesis and handling of nanomaterials, which may have additional hazards compared to their bulk counterpart. Please ensure all necessary control measures to be practiced throughout the experiment, including the use of engineering controls (fume hood) and personal protective equipment (PPE, e.g., safety length pants, closed-toe shoes, chemical resistant gloves, and safety goggles...

<ol>
	<li>Flarakos, J., Morand, K. L., Vouros, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Solution-Based+Medicinal+Library+Screening+against+Human+Serum+Albumin.">High-Throughput Solution-Based Medicinal Library Screening against Human Serum Albumin.</a> Anal. Chem. 77, 1345-1353 (2005).</li><li>Vuignier, K., Veuthey, J. -. L., Carrupt, P. -. A., Schappler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Analytical+Strategy+to+Measure+Drug&#8211;Plasma+Protein+Interactions:+From+High-Throughput+to+In-Depth+Analysis.">Global Analytical Strategy to Measure Drug&#8211;Plasma Protein Interactions: From High-Throughput to In-Depth Analysis.</a> Drug Discov. Toda. 18, 1030-1034 (2013).</li><li>Zsila, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subdomain+Ib+Is+The+Third+Major+Drug+Binding+Region+of+Human+Serum+Albumin:+Toward+The+Three-Sites+Model.">Subdomain Ib Is The Third Major Drug Binding Region of Human Serum Albumin: Toward The Three-Sites Model.</a> Mol. Pharm. 10, 1668-1682 (2013).</li><li>Dalvit, C., Fagerness, P. E., Hadden, D. T. A., Sarver, R. W., Stockman, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorine-NMR+Experiments+for+High-Throughput+Screening:+Theoretical+Aspects,+Practical+Considerations,+and+Range+of+Applicability.">Fluorine-NMR Experiments for High-Throughput Screening: Theoretical Aspects, Practical Considerations, and Range of Applicability.</a> J. Am. Chem. Soc. 125, 7696-7703 (2003).</li><li>Ghuman, J., Zunszain, P. A., Petitpas, I., Bhattacharya, A. A., Otagiri, M., Curry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Structural Basis of the Drug-binding Specificity of Human Serum. 353, 38-52 (2005).</li><li>Mao, H., Hajduk, P. J., Craig, R., Bell, R., Borre, T., Fesik, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+Design+of+Diflunisal+Analogues+with+Reduced+Affinity+for+Human+Serum+Albumin.">Rational Design of Diflunisal Analogues with Reduced Affinity for Human Serum Albumin.</a> J. Am. Chem. Soc. 123, 10429-10435 (2001).</li><li>Dalvit, C., 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=High-Throughput+NMR-Based+Screening+with+Competition+Binding+Experiments.">High-Throughput NMR-Based Screening with Competition Binding Experiments.</a> J. Am. Chem. Soc. 124, 7702-7709 (2002).</li><li>Krenzel, E. S., Chen, Z., Hamilton, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correspondence+of+Fatty+Acid+and+Drug+Binding+Sites+on+Human+Serum+Albumin:+A+Two-Dimensional+Nuclear+Magnetic+Resonance+Study.">Correspondence of Fatty Acid and Drug Binding Sites on Human Serum Albumin: A Two-Dimensional Nuclear Magnetic Resonance Study.</a> Biochemistr. 52, 1559-1567 (2013).</li><li>Lee, Y., Zeng, H., Ruedisser, S., Gossert, A. D., Hilty, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+Magnetic+Resonance+of+Hyperpolarized+Fluorine+for+Characterization+of+Protein&#8211;Ligand+Interactions.">Nuclear Magnetic Resonance of Hyperpolarized Fluorine for Characterization of Protein&#8211;Ligand Interactions.</a> J. Am. Chem. Soc. 134, 17448-17451 (2012).</li><li>Salvi, N., 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=Boosting+the+Sensitivity+of+Ligand&#8211;Protein+Screening+by+NMR+of+Long-Lived+States.">Boosting the Sensitivity of Ligand&#8211;Protein Screening by NMR of Long-Lived States.</a> J. Am. Chem. Soc. 134, 11076-11079 (2012).</li><li>Zsila, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+Dichroism+Spectroscopic+Detection+of+Ligand+Binding+Induced+Subdomain+IB+Specific+Structural+Adjustment+of+Human+Serum+Albumin.">Circular Dichroism Spectroscopic Detection of Ligand Binding Induced Subdomain IB Specific Structural Adjustment of Human Serum Albumin.</a> J. Phys. Chem. 117, 10798-10806 (2013).</li><li>Navratilova, I., Hopkins, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragment+Screening+by+Surface+Plasmon+Resonance.">Fragment Screening by Surface Plasmon Resonance.</a> ACS Med. Chem. Lett. 1, 44-48 (2010).</li><li>Wang, 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=Investigation+of+Phase+SPR+Biosensor+for+Efficient+Targeted+Drug+Screening+with+High+Sensitivity+and+Stability.">Investigation of Phase SPR Biosensor for Efficient Targeted Drug Screening with High Sensitivity and Stability.</a> Sensor. Actuat. B-Che. 209, 313-322 (2015).</li><li>Lu, Y., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-Nanometre+Sized+Metal+Clusters:+From+Synthetic+Challenges+to+The+Unique+Property+Discoveries.">Sub-Nanometre Sized Metal Clusters: From Synthetic Challenges to The Unique Property Discoveries.</a> Chem. Soc. Rev. 41, 3594-3623 (2012).</li><li>Yu, Y., Yao, Q., Luo, Z., Yuan, X., Lee, J. Y., Xie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precursor+Engineering+and+Controlled+Conversion+for+The+Synthesis+of+Monodisperse+Thiolate-Protected+Metal+Nanoclusters.">Precursor Engineering and Controlled Conversion for The Synthesis of Monodisperse Thiolate-Protected Metal Nanoclusters.</a> Nanoscal. 5, 4606-4620 (2013).</li><li>Jin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantum+Sized,+Thiolate-Protected+Gold+Nanoclusters.">Quantum Sized, Thiolate-Protected Gold Nanoclusters.</a> Nanoscal. 2, 343-362 (2010).</li><li>Jiang, D. -. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Expanding+Universe+of+Thiolated+Gold+Nanoclusters+and+Beyond.">The Expanding Universe of Thiolated Gold Nanoclusters and Beyond.</a> Nanoscal. 5, 7149-7160 (2013).</li><li>Aikens, 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=Electronic+Structure+of+Ligand-Passivated+Gold+and+Silver+Nanoclusters.">Electronic Structure of Ligand-Passivated Gold and Silver Nanoclusters.</a> J. Phys. Chem. Lett. 2, 99-104 (2010).</li><li>Gao, Y., Shao, N., Pei, Y., Chen, Z., Zeng, X. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+Activities+of+Subnanometer+Gold+Clusters+(Au16&#8211;Au18,+Au20,+and+Au27&#8211;Au35)+for+CO+Oxidation.+ACS.">Catalytic Activities of Subnanometer Gold Clusters (Au16&#8211;Au18, Au20, and Au27&#8211;Au35) for CO Oxidation. ACS.</a> ACS Nan. 5, 7818-7829 (2011).</li><li>Negishi, Y., Nobusada, K., Tsukuda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione-Protected+Gold+Clusters+Revisited:+Bridging+the+Gap+between+Gold(I)&#8722;Thiolate+Complexes+and+Thiolate-Protected+Gold+Nanocrystals.">Glutathione-Protected Gold Clusters Revisited: Bridging the Gap between Gold(I)&#8722;Thiolate Complexes and Thiolate-Protected Gold Nanocrystals.</a> J. Am. Chem. Soc. 127, 5261-5270 (2005).</li><li>Zhu, M., Aikens, C. M., Hollander, F. J., Schatz, G. C., Jin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlating+the+Crystal+Structure+of+A+Thiol-Protected+Au25+Cluster+and+Optical+Properties.">Correlating the Crystal Structure of A Thiol-Protected Au25 Cluster and Optical Properties.</a> J. Am. Chem. Soc. 130, 5883-5885 (2008).</li><li>Yu, 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=Identification+of+a+Highly+Luminescent+Au22(SG)18+Nanocluster.">Identification of a Highly Luminescent Au22(SG)18 Nanocluster.</a> J. Am. Chem. Soc. 136, 1246-1249 (2014).</li><li>Jiang, J., 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=Oxidation+at+the+Core&#8211;Ligand+Interface+of+Au+Lipoic+Acid+Nanoclusters+That+Enhances+the+Near-IR+Luminescence.">Oxidation at the Core&#8211;Ligand Interface of Au Lipoic Acid Nanoclusters That Enhances the Near-IR Luminescence.</a> J. Phys. Chem. 118, 20680-20687 (2014).</li><li>Zhu, Y., Qian, H., Jin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Atomic-Level+Strategy+for+Unraveling+Gold+Nanocatalysis+from+the+Perspective+of+Aun(SR)m+Nanoclusters.">An Atomic-Level Strategy for Unraveling Gold Nanocatalysis from the Perspective of Aun(SR)m Nanoclusters.</a> Chem. Eur. J. 16, 11455-11462 (2010).</li><li>Niesen, B., Rand, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin+Film+Metal+Nanocluster+Light-Emitting+Devices.">Thin Film Metal Nanocluster Light-Emitting Devices.</a> Adv. Mater. 26, 1446-1449 (2014).</li><li>Shang, L., Dong, S. J., Nienhaus, G. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-Small+Fluorescent+Metal+Nanoclusters:+Synthesis+and+Biological+Applications.">Ultra-Small Fluorescent Metal Nanoclusters: Synthesis and Biological Applications.</a> Nano Toda. 6, 401-418 (2011).</li><li>Wu, X., He, X., Wang, K., Xie, C., Zhou, B., Qing, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasmall+Near-Infrared+Gold+Nanoclusters+for+Tumor+Fluorescence+Imaging+in+Vivo.">Ultrasmall Near-Infrared Gold Nanoclusters for Tumor Fluorescence Imaging in Vivo.</a> Nanoscal. 2, 2244-2249 (2010).</li><li>Archana, R., 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=Molecular-Receptor-Specific,+Non-Toxic,+Near-Infrared-Emitting+Au+Cluster-Protein+Nanoconjugates+for+Targeted+Cancer+Imaging.">Molecular-Receptor-Specific, Non-Toxic, Near-Infrared-Emitting Au Cluster-Protein Nanoconjugates for Targeted Cancer Imaging.</a> Nanotechnolog. 21, 055103 (2010).</li><li>Yue, Y., Liu, T. Y., Li, H. W., Liu, Z. Y., Wu, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave-Assisted+Synthesis+of+BSA-Protected+Small+Gold+Nanoclusters+and+Their+Fluorescence-Enhanced+Sensing+of+Silver(I)+Ions.">Microwave-Assisted Synthesis of BSA-Protected Small Gold Nanoclusters and Their Fluorescence-Enhanced Sensing of Silver(I) Ions.</a> Nanoscal. 4, 2251-2254 (2012).</li><li>Liu, Y., Ai, K., Cheng, X., Huo, L., Lu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanocluster+Based+Fluorescent+Sensors+for+Highly+Sensitive+and+Selective+Detection+of+Cyanide+in+Water.">Gold Nanocluster Based Fluorescent Sensors for Highly Sensitive and Selective Detection of Cyanide in Water.</a> 20, 951-1907 (2010).</li><li>Liu, J., Yu, M., Zhou, C., Yang, S., Ning, X., Zheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+Tumor+Targeting+of+Renal-Clearable+Luminescent+Gold+Nanoparticles:+Long+Tumor+Retention+and+Fast+Normal+Tissue+Clearance.">Passive Tumor Targeting of Renal-Clearable Luminescent Gold Nanoparticles: Long Tumor Retention and Fast Normal Tissue Clearance.</a> J. Am. Chem. Soc. 135, 4978-4981 (2013).</li><li>Negishi, 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=Controlled+Loading+of+Small+Aun+Clusters+(n+=+10&#8211;39)+onto+BaLa4Ti4O15+Photocatalysts:+Toward+an+Understanding+of+Size+Effect+of+Co-Catalyst+on+Water+Splitting+Photocatalytic+Activity.">Controlled Loading of Small Aun Clusters (n = 10&#8211;39) onto BaLa4Ti4O15 Photocatalysts: Toward an Understanding of Size Effect of Co-Catalyst on Water Splitting Photocatalytic Activity.</a> J. Phys. Chem. C. , (2015).</li><li>Xie, J., Zheng, Y., Ying, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-Directed+Synthesis+of+Highly+Fluorescent+Gold+Nanoclusters.">Protein-Directed Synthesis of Highly Fluorescent Gold Nanoclusters.</a> J. Am. Chem. Soc. 131, 888-889 (2009).</li><li>Celej, M. S., Montich, G. G., Fidelio, G. 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+Stability+Induced+by+Ligand+Binding+Correlates+with+Changes+in+Protein+Flexibility.">Protein Stability Induced by Ligand Binding Correlates with Changes in Protein Flexibility.</a> Protein Sci. 12, 1496-1506 (2003).</li><li>Layton, C. J., Hellinga, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamic+Analysis+of+Ligand-Induced+Changes+in+Protein+Thermal+Unfolding+Applied+to+High-Throughput+Determination+of+Ligand+Affinities+with+Extrinsic+Fluorescent+Dyes.">Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes.</a> Biochemistr. 49, 10831-10841 (2010).</li><li>Yu, Y., New, S. Y., Xie, J., Su, X., Tan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-Based+Fluorescent+Metal+Nanoclusters+for+Small+Molecular+Drug+Screening.">Protein-Based Fluorescent Metal Nanoclusters for Small Molecular Drug Screening.</a> Chem. Commun. 50, 13805-13808 (2014).</li><li>Shortridge, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nuclear Magnetic Resonance Affinity Screening Methods for Functional Annotation of Proteins and Drug Discover. , (2010).</li></ol>

There are several critical steps that need to be highlighted in this method. In the protocol of screening the relative binding affinity of different small molecular drugs, Steps 3.1.2, 3.1.3, and 3.1.4 are critical to obtain good results showing consistent trend for the relative binding strength. In these steps, the actions of adding chemicals and drawing reaction solutions for measurement should be as quickly as possible to minimize the time lag effect and the same sequence of adding chemicals and drawing reaction solut...

Serum albumins such as human serum albumin (HSA) and bovine serum albumin (BSA) are the most abundant protein in plasma and play a vital role in maintaining the osmotic pressure of the blood compartment. They are also recognized as carrier proteins for small molecules of low water solubility, such as steroids, fatty acids, thyroid hormones, and a wide variety of drugs. The binding property (e.g., binding sites, binding affinity or strength) of these molecules to serum albumins forms an important topic in pharmacokinetics.1-4 Several analytical methods have been developed to study the binding properties of different drugs to serum albumins, such as X-ray crystallography,5,6 nuclear magnetic resonance (NMR),7-11 and surface plasmon resonance (SPR),12,13 etc. However, these methods are constrained by either a tedious and time-consuming analyzing process (e.g., growth of single crystal for X-ray crystallographic study), requirement of specialized and expensive equipment (SPR), or in need of costly isotope labeling (NMR) for detection. It is therefore highly desirable to develop alternative ways for small molecular drug screening in a fast, straight-forward, and cost-efficient manner.
Gold nanoclusters (Au NCs) are a special type of nanomaterial, which contain several to tens of metal atoms with sizes smaller than 2 nm.14-17 They have attracted extensive research interests due to their discrete and size-dependent electronic structure,18,19 and molecular-like absorptions and emissions.20-23 Such unique materials properties, in particular the strong fluorescence, have found diverse applications such as sensing and imaging in biological systems. 24-32 Ultrasmall fluorescent Au NCs can be synthesized using functional proteins, such as serum albumins, as template. 33 In a typical protein-templated synthesis of Au NCs, a certain amount of Au salts are first encapsulated inside the protein and subsequently reduced by the protein itself. The reducing ability of the protein is attributed to constituent functional amino acid residues (e.g., tyrosine) that can be activated by increasing the solution pH to alkaline. Unfolding of protein structure is considered as a critical step for the formation of Au NCs. This is because in an unfolded protein, more reducing functional groups can be exposed to the encapsulated Au salts. Protein unfolding can be achieved by heat treatment or exposure to denaturing agents. Introduction of small molecular drugs can also affect the unfolding process, i.e. modifying the midpoint denaturation temperature and the enthalpy of unfolding. 34,35 The effect of all these factors, in turn can be reflected by the formation kinetics of fluorescent Au NCs and manifested in the fluorescence intensity of resultant Au NCs.36
This video demonstrates the method of drug screening by synthesizing Au NCs in drug-loaded albumin proteins at a higher temperature (60 &#176;C) or in the presence of denaturing agents (e.g., urea). The fluorescence intensity of resultant Au NCs is the signal readout. First, Au NCs are synthesized in HSA and BSA templates treated at 60 &#176;C or in the presence of urea to show how protein unfolding (induced by heat treatment or denaturants) affects the formation kinetics of Au NCs. Second, Au NCs are synthesized in protein templates preloaded with different drugs, and the drug loading effect on the relative fluorescence intensities of resultant Au NCs is studied, which provide the measure of relative binding strength. Finally, the Au NC-drug screening protocol is modified for quantitative measurement of drug-protein binding constant (KD) by varying the drug content preloaded in the protein of a fixed concentration.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Gold (III) chloride solution, 30%</td>
			<td>Sigma-Aldrich</td>
			<td>484385</td>
			<td>Corrosive, irritant</td>
		</tr>
		<tr>
			<td>Human serum albumin, 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A1887</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum albumin, 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibuprofen, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>I4883&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>warfarin, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>A2250</td>
			<td></td>
		</tr>
		<tr>
			<td>phenytoin</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1139</td>
			<td></td>
		</tr>
		<tr>
			<td>sulphanilamide, 99%</td>
			<td>Sigma-Aldrich</td>
			<td>S9251</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5128</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>IKA</td>
			<td>RT5</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>Infinite M200</td>
			<td></td>
		</tr>
		<tr>
			<td>384-well plate</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5000 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL micro tube</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL glass vial with screw cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 mL glass vial with screw cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a rapid and quantitative fluorimetric method for protein-targeting small molecule drug screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

Protein unfolding is an important procedure for the formation of protein-templated Au NCs because more reactive functional groups (e.g., tyrosine residues) of a protein can be exposed to reduce the encapsulated Au ions and thus accelerate the formation rate of Au NCs. Heating and external denaturing agents are two common means to promote the protein unfolding process. Figure 1 demonstrates the effect of heating and adding external denaturing agents on the formation kinetics of Au NCs, using HSA ...

Watch this Scientific Journal Video about A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening at JoVE.com

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Research

JoVE Journal

Bioengineering

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...