Fluorescence Polarization-Based Measurement of Protein-Ligand Interaction in Fungal Cell Lysates

Abstract

Fungi infect over a billion people worldwide and contribute substantially to human morbidity and mortality despite all available therapies. New antifungal drugs are urgently needed. Decades of study have revealed numerous protein targets of potential therapeutic interest for which potent, fungal-selective ligands remain to be discovered and developed. To measure the binding of diverse small molecule ligands to their larger protein targets, fluorescence polarization (FP) can provide a robust, inexpensive approach. The protocols in this article provide detailed guidance for developing FP-based assays capable of measuring binding affinity in whole cell lysates without the need for purification of the target protein. Applications include screening of libraries to identify novel ligands and the definition of structure-activity relationships to aid development of compounds with improved target affinity and fungal selectivity. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Use of saturation binding curves to optimize tracer and lysate protein concentrations Basic Protocol 2: Establishment of competition binding experiments Support Protocol 1: Preparation of fungal cell lysates Support Protocol 2: Preparation of human HepG2 cell lysate.

Keywords: binding affinity; fluorescence polarization; fungi; whole cell lysate.

Figure 1.. A. Schematic of the basic principle of FP applied to the measurement of ligand binding.

After passing through an excitation polarizer, plane-polarized light excites the fluorescent tracer. Emitted light intensity is measured after passing through emission polarizers parallel (I ∥) and perpendicular (I⊥) to the excitation light’s plane of polarization. The low molecular weight free tracer exhibits relatively high rotational diffusion during the excited state lifetime (τ) resulting in the emission of depolarized light. The much larger protein-bound tracer complex exhibits reduced rotational diffusion during τ and thus more of the emitted light remains polarized in the same plane as the excitation light. B. FP assay for the assessment of a test compound’s relative binding affinity to a protein target in whole cell lysate. A fluorescently labeled small molecule (tracer) tumbling in solution results in a low measured FP. When the tracer binds with high affinity to a larger protein target, the rotational reorientation is minimized, and the measured FP is high. Addition of a test compound which competes for binding to the same target displaces the tracer in a concentration- dependent manner, reducing the measured FP. ^a Lysates are prepared following Support Protocols 1 and 2. Lysate protein concentration and optimal incubation time are determined by defining the saturation level of polarization as described in Basic Protocol 1. ^b Relative binding affinity of an investigational compound is established by generating competitive displacement curves as described in Basic Protocol 2.

Figure 2.. Saturation binding of the tracer, Cy3B-geldanamycin, to the target protein, Hsp90, in HepG2 and C. albicans lysates.

The average fluorescence polarization (mP) is plotted against increasing lysate protein concentration with three fixed tracer concentrations, either 0.2, 0.1, or 0.05 nM. In this assay, the highest concentration of lysate was 20 μg/well, as binding saturation was observed at this concentration. The dotted line indicates 75% tracer binding at a tracer concentration of 0.1 nM.

Figure 3.. Competitive tracer displacement by unlabeled geldanamycin in C. albicans lysate.

The average fluorescence polarization (mP) is plotted against increasing geldanamycin concentration on a log10 scale. The concentration of tracer was 0.1 nM and the lysate protein concentration was 3.5 μg/well.

Figure 4.. Recommended plate setup for competition-binding experiment.

To assess candidate inhibitors, a control compound (blue wells, ideally the unlabeled tracer molecule) should be used in addition to the investigational compounds (yellow wells) in technical duplicate. Compounds should be titrated in two-fold dilutions across twelve wells to which the pre-determined lysate-tracer mix is added. Duplicate wells of FP assay buffer only serve as blanks (green wells).

Figure 5.. Identification of key components of the French Press used in the preparation of whole cell lysate.

A. The piston was inserted in the cell to the appropriate volume marking and the cell was placed upside down on the tripod where the sample was loaded. The flow valve was inserted, and the closure plug sealed the assembled unit. B. The assembled cell was then loaded right-side up onto the French Press platform, ensuring that the piston arms were perpendicular to the support rods. Lysate was collected on ice in blue-capped tube to left.