Assay:Molecules that Inhibit Anthrax Intoxication(RMI)

Christensen, Kenneth

Abstract

This proposal details the development of a fluorescence resonance energy transfer (FRET) assay for use in molecular screening of small molecule libraries for identification of anti-anthrax and anti-angiogenic therapies. As part of the process of anthrax intoxification, protective antigen (PA), an otherwise non-toxic component of the anthrax toxin complex, binds to the mammalian cell surface receptors CMG2 and/or ATR. Receptor binding prompts assembly of the complete anthrax toxin, which is subsequently delivered to the cytosol. Since receptor binding is the first step in anthrax toxin formation and delivery, small molecules or peptides that block the interaction(s) of PA with receptor can be effective anthrax toxin therapies. Importantly, we have discovered that the binding of PA to anthrax receptor has potent anti-angiogenic properties and significantly inhibits tumor growth. Hence, small molecules that mimic PA and bind to anthrax receptor(s) could be used as cancer therapeutics as well as anti-anthrax agents. We propose a robust, sensitive, mix and measure high-throughput FRET screening assay that can identify molecules that compete with PA and bind to anthrax toxin receptor. An existing FRET assay used to quantify anthrax toxin binding to a soluble CMG2 receptor will be converted from cuvette to well-plate format. Experimental conditions will be optimized to maximize robustness, reproducibility, sensitivity, and stability, while minimizing variation across the well-plate. The developed assay will be tested using chemical libraries at the NSRB screening facility at the New England Research Center of Excellence (NERCE). Results of these initial screens can be evaluated more stringently with respect to both binding affinity and kinetics and/or their biological activity can be assessed using toxicity or adapted FRET cell-based assays. Experiments proposed here describe the first adaptation of FRET technology to high throughput screens for anthrax receptor binding. As such, they represent the development of a highly sensitive and robust technique for generation of leads for both cancer and anthrax toxin therapies. Resulting compounds would circumvent a key problem with the testing of inhibitors of anthrax intoxication because they can be tested for their safety in the cancer context, and issues regarding testing for a rare indication (anthrax intoxication) are avoided. Strong inhibitors of receptor binding could be used as a probe of the (otherwise undetermined) cellular function of the anthrax receptor(s), while a comparison of the structures of identified compound structures will provide information into the molecular recognition processes operating at the receptor.