Genetic Selection of Protective Antigen Heptamerisation Inhibitors

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.

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).