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

Summary

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.

Abstract

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 °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 (K_D) 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.

Introduction

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 ....

Protocol

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).

1. Preparation of Chemical Reagents for Drug Screening

1. Precursors for Au NCs synthesis
   1. Dissolve....

Results

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 .......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gold (III) chloride solution, 30%	Sigma-Aldrich	484385	Corrosive, irritant
Human serum albumin, 96%	Sigma-Aldrich	A1887
Bovine Serum albumin, 96%	Sigma-Aldrich	A2153
Ibuprofen, 98%	Sigma-Aldrich	I4883
warfarin, 98%	Sigma-Aldrich	A2250
phenytoin	Sigma-Aldrich	PHR1139
sulphanilamide, 99%	Sigma-Aldrich	S9251
dimethyl sulfoxide	Sigma-Aldrich	D8418
urea	Sigma-Aldrich	U5128
Sodium hydroxide	Sigma-Aldrich	221465
Magnetic stirrer	IKA	RT5
Microplate reader	Tecan	Infinite M200
384-well plate	Corning
5 ml air displacement pipette	Eppendorf
1,000 μl air displacement pipette	Eppendorf
100 μl air displacement pipette	Eppendorf
5,000 μl Eppendorf tips
1,000 μl Eppendorf tips
100 μl Eppendorf tips
1.5 ml micro tube	Eppendorf
20 ml glass vial with screw cap
4 ml glass vial with screw cap