Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron

Summary

We describe a fluorescent assay that can quickly and inexpensively confirm the ability of novel compounds to chelate iron. The assay measures the ability of compounds to outcompete the iron binding activity of the weak iron chelating fluorescent probe Calcein, resulting in a quantifiable increase in fluorescence when chelation occurs.

Abstract

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target for anti-cancer approaches. The development of novel compounds and the identification of leads for further modification requires that proof of mechanism assays be carried out. There are many assays to evaluate the impact on proliferation; however, the ability to chelate iron is an important and sometimes overlooked end-point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron. Our assay relies on the commercially available inexpensive fluorescent dye Calcein, whose fluorescence can be quantified on most fluorescent microtiter plate readers. Calcein is a weak iron chelator, and its fluorescence is quenched when it binds Fe^2+/3+; fluorescence is restored when a novel chelator outcompetes Calcein for bound Fe^2+/3+. The removal of fluorescent quenching and the resulting increase in fluorescence allows the chelation ability of a novel putative chelator to be determined. Therefore, we offer an inexpensive, high-throughput assay that allows the rapid screening of novel candidate chelator compounds.

Introduction

Phenotypic changes to cells that relate to the development of cancer through a common set of altered biological capabilities are now commonly referred to as the hallmarks of cancer. Amongst them are changes resulting from the reprogramming of energy metabolism, which are widespread in cancer cell biology¹. Such metabolic reprogramming includes an increased requirement for iron to support rapid proliferation and tumor growth². This thirst for iron leads to dysregulated iron metabolism, which in and of itself is considered a hallmark of cancer^3,4, with dysregulatio....

Protocol

1. Stock solution preparation

1. When preparing Calcein stocks solutions, prevent photo degradation by keeping solutions in the dark. Prepare a 1 mM solution of Calcein in Dulbecco's PBS, without magnesium or calcium¹⁴. For 5 mL of 1 mM Calcein solution, add 3.1 mg of Calcein (622.5 g/M) to 5 mL of Dulbecco's PBS. Using the 1 mM sock of Calcein, prepare 1 mL aliquots of secondary stock solutions in microfuge tube as detailed in Table 1.
2. .......

Discussion

The over reliance of cancers on iron to fuel their metabolism makes iron chelation a potential addition to therapeutic regimes⁴. However, there is a limited ability to quickly screen novel metal ion chelators for their ability to bind iron ions. The commonly used and widely available fluorescent probe Calcein is known to act as a weak iron chelator and binding by iron ions quenches Calcein fluorescence. Fluorescence can then be recovered by competing for binding to iron using a stronger iron chela.......

Materials

Name	Company	Catalog Number	Comments
Ammonium iron(II) sulfate hexahydrate	Sigma-Aldrich	215406	other wise known as FAS
Calcein	Sigma-Aldrich	C0875
Deferiprone	Sigma-Aldrich	379409
Dulbecco′s Phosphate Buffered Saline	Sigma-Aldrich	D5652	magnesium and calcium free
Greiner CELLSTAR 96 well plates	Sigma-Aldrich	M0812	any optically transparent 96 well plate will work