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Updated: Jun 11, 2026
A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening
Published on: October 16, 2015
Simultaneous detection of intracellular target and off-target binding of small molecule cancer drugs at nanomolar
Heike Glauner1, Ivo R Ruttekolk, Kerrin Hansen
1Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Background And Purpose:
In vitro assays that determine activities of drug candidates with isolated targets have only limited predictive value for activities in cellular assays. Poor membrane permeability and off-target binding are major reasons for such discrepancies. However, it still difficult to directly analyse off-target binding at the same time as target binding, on a subcellular level. Here, we present a combination of fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) as a solution to this problem.
Experimental Approach:
The well-established dihydrofolate reductase inhibitor methotrexate and the kinase inhibitors PD173956 and purvalanol B were conjugated via polyethylene glycol linkers with the fluorophore Cy5. The cellular uptake and subcellular distribution of these compounds in single human cancer-derived cells were investigated by confocal laser scanning microscopy. In addition, molecular interactions inside the cell with the respective target proteins and off-target binding were detected simultaneously in the nanomolar range by FCCS and FCS, respectively, using cells expressing green fluorescent protein fusion proteins of dihydrofolate reductase and Abelson kinase 1.
Key Results:
Large differences in the interaction patterns were found for these compounds. For methotrexate-Cy5, drug-target interactions could be detected and dissociation constants determined. In contrast, PD173956-Cy5 showed strong interactions with intracellular high-molecular weight structures, other than its target.
Conclusions And Implications:
The combination of FCS and FCCS provides a powerful means to assess subcellular pharmacokinetics and dynamics of drug candidates at nanomolar concentrations.
Insights
This study introduces fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) to simultaneously analyze drug-target interactions and off-target binding within cells. This method reveals crucial pharmacokinetic and pharmacodynamic differences for drug candidates at the subcellular level.
Area of Science:
- Pharmacology
- Biophysics
- Cell Biology
Background:
- In vitro drug assays have limited predictive power for cellular activity due to issues like poor membrane permeability and off-target binding.
- Analyzing simultaneous target and off-target binding at the subcellular level remains challenging.
- Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) offer a potential solution.
Purpose of the Study:
- To develop and validate a method combining FCS and FCCS for simultaneous analysis of drug-target and off-target interactions within living cells.
- To investigate the subcellular pharmacokinetics and pharmacodynamics of drug candidates.
- To overcome limitations of traditional in vitro assays in predicting cellular drug activity.
Main Methods:
- Drug candidates (methotrexate, PD173956, purvalanol B) were labeled with Cy5 via polyethylene glycol linkers.
- Confocal laser scanning microscopy was used to track cellular uptake and distribution.
- FCS and FCCS were employed to detect molecular interactions with target proteins and off-target binding in real-time within cells expressing fluorescently tagged proteins.
Main Results:
- Significant differences in drug-target and off-target interaction patterns were observed.
- Methotrexate-Cy5 showed measurable drug-target interactions and allowed for dissociation constant determination.
- PD173956-Cy5 exhibited substantial binding to non-target intracellular structures, indicating off-target effects.
Conclusions:
- The combined FCS and FCCS approach provides a powerful tool for assessing drug behavior within cells.
- This method enables simultaneous evaluation of pharmacokinetics and pharmacodynamics at nanomolar concentrations.
- It offers a more accurate prediction of drug efficacy and potential side effects by analyzing subcellular interactions.
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