Genetic Selection of Protective Antigen Heptamerisation Inhibitors

Lead Research Organisation: University of Southampton
Department Name: School of Chemistry

Abstract

The anthrax spore attacks along the eastern united states in autumn 2001 focused attention on the lack of effective treatments for this disease. Pulmonary anthrax results from inhalation of airborne spores and is highly fatal. Anthrax acts by releasing two toxins into the body that are transported into cells by a third protein. These toxins, called lethal factor and edema factor, cause cell death and when present at high levels are fatal. The disease is without symptoms for several weeks, progressing to mild fever, aches and cough. As bacteria reach high levels in the circulation, the disease progresses rapidly, causing respiratory disease, shock and widespread haemorage. Death usually occurs within 24 hours and antibiotics are without benefit at this point due to the accumulation of the bacterial toxins.
Our research aims to develop a new line of compounds for use in anthrax infection. These compounds will act by preventing the anthrax toxins being transported into human cells. This is achieved by targeting the assembly of a key anthrax protein complex, which forms pores in the cell membrane allowing toxin entry.
The compounds developed in this project will prevent cellular entry of anthrax toxins, therefore allowing anthrax infections to become treatable by a combination of therapy approach with antibiotics.

Technical Summary

The toxicity and disease causing ability of Bacillus anthracis depends on two secreted virulence proteins, termed lethal factor (LF) and edema factor (EF). Along with these two toxins, a third protein is secreted, a receptor binding moiety called protective antigen, which is responsible for the cellular transport of the toxins. Recent molecular studies have revealed the intricate mechanism that operates; monomeric PA binds and is cleaved (to a 63 kDa protein termed PA63) at the surface of receptor-bearing eukaryotic cells, which triggers its heptamerisation. The PA heptamer forms a pore in the plasma membrane, through which the anthrax toxins are actively transported.
There is a need for new approaches to treating anthrax infections. Pulmonary anthrax is asymptomatic for several weeks, followed by flu-like symptoms which progress rapidly as bacteria reach high levels in the circulation, causing fulminant disease characterised by respiratory disease, shock and widespread haemorage. Death usually occurs within 24 hours of reaching the fulminant stage, and antibiotics are without therapeutic benefit from this point onwards due to the accumulation of the bacterial toxins.
In this study we will use genetic selection methodology develop to uncover compounds that selectively inhibit the heptamerisation of PA from cyclic peptide libraries of up to a hundred million members. The resulting compounds will inhibit the protein-protein interaction between activated PA monomers, thus preventing the transport of the anthrax toxins into the cell. This will eliminate or greatly reduce the toxicity of athrax, thus allowing treatment with antibiotics to clear the infection. The proposed work will contribute to and improve on the approaches taken by others, as we are targeting the process of toxin internalisation at an earlier stage (PA heptamerisation), therefore targeting internalisation of both toxic proteins (LF and EF).
 
Title High throughput fluorescence assay 
Description we have developed a high throughput assay to monitor protein protein interactions in vivo, based ona fluorescent reporter. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact generally applicable method. we are preparing publication. 
 
Description Interleukin signalling in asthma 
Organisation University of Southampton
Department Faculty of Health Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution identified a new interleukin interaction that seems to modulate signalling in asthma attacks.
Collaborator Contribution knowledge exchange, one publication, another one being prepared, grant application being prepared.
Impact one paper (19614610) another one in preparation and a grant proposal in preparation.
Start Year 2008
 
Description Targeting the phage shock system of bacteria with cyclic peptides 
Organisation Defence Science & Technology Laboratory (DSTL)
Country United Kingdom 
Sector Public 
PI Contribution we are currently using the SICLOPPS methodology to screen for cyclic peptide inhibitors that disrupt the protein-protein interaction between components of the bacterial phage shock system as a means to developing new antibiotics.
Impact we have been awarded a MOD grant with DSTL.
Start Year 2010