Monday, 9 March 2015

Towards antiviral drugs for Ebola infection?

While news coverage may have died down, the West African Ebola outbreak continues. Fortunately, there are signs that the number of cases may be levelling off, suggesting that the outbreak is starting to be controlled. At the time of writing, there have been, 24202 cases and 9936 deaths, giving a case fatality rate of around 41%. The previous largest Ebola outbreak only had 425 cases. One of the biggest issues in tackling Ebola is the lack of any proper treatment for the viral disease. There are currently no approved vaccines, nor drugs to combat Ebola virus (EBOV). However, a study, recently published in the journal Science, has presented very interesting data regarding a potential therapeutic agent able to block EBOV infection of cells. But before getting to the study, I’d like to give some background into how EBOV infects cells. 
A simplified view of the lipid bilayer that surrounds cells

Cells are encased in a double layer of lipid molecules that make them impenetrable to all but the tiniest of molecules. This lipid bilayer is necessary to keep the inside of a cell distinct from its environment, allowing the cell to control its composition. However, a cell must interact with its environment, and for this receptor molecules are incorporated into the lipid bilayer. To regulate this interaction with the external environment, a process called endocytosis has evolved (endo- being from the Greek for within and -cytosis referring to a cell). 

One of the best studied endocytic mechanisms is the response of cells to a protein known as epidermal growth factor, without which, cells can die. To interact with the growth factor in the external environment, cells have epidermal growth factor receptors in the lipid bilayer. When the growth factor binds to the receptors, signalling events trigger within the cell that stimulate growth and division. However, too much growth and division is damaging to an organism; cancer being the best known consequence. To regulate the response to growth factors cells use endocytosis to destroy the receptors once they have bound to growth factor. This destruction pathway has three steps: firstly, the receptor is removed from the lipid bilayer by folding this in on itself and
The first step of the endocytic pathway, internalisation from the cell surface
pinching off a small
bubble which becomes an endosome (an endocytic body). In the second step, the cell modifies the contents of the endosome, causing it to become acidic. Finally, this acidified endosome will fuse with compartments of the cell known as lysosomes which contain enzymes, that only work in acidic conditions, which destroy the receptor, and turn off the growth signal.

Endocytosis is a natural mechanism allowing a cell to control interactions with the environment. Viruses are obligate, intracellular parasites, they must infect a cell and access its replication machinery to produce new virus particles. The lipid bilayer stands between a virus and the replication machinery, so it must be crossed - to do this many viruses hijack the endocytic system. EBOV is one of these viruses. EBOV will bind to the cell surface, it is then taken into the cell by an endocytic mechanism known as macropinocytosis, which places the virus in an endosome. Here the virus cannot access the replication machinery of the cell because a lipid bilayer is still in the way - the cell is not infected at this point. When the endosome becomes acidified, this triggers fusion of EBOV with the endosome lipid bilayer, allowing deposition of the viral genetic material into the cell, before it can be destroyed in the lysosome. Once the genetic material has been released, it can access the replication machinery, and produce new viruses, the cell is infected. 
Ebola entry - taken from Grove and Marsh 2011. JCB 195 (7): 1071-1082

With that background in mind, we can now move on to look at the drug discovery work published in Science. In endosomes, along with changes to pH, there is also controlled movement of other ions, such as calcium. The group, from the lab of Robert Davey in Texas, previously showed that calcium is important for EBOV infection, but had not known why. To address this, cells were treated with a range of chemical compounds that block calcium signalling in the cell. Only a subset of these chemicals could protect cells from EBOV infection, the most potent being a chemical know as Tetrandrine, which was originally isolated from herbs that grow in Japan and China. 

Treatement of cells with Tetrandrine protected them from EBOV infection, and it was shown that this chemical was blocking calcium signalling from cellular proteins know as two-pore channels (TPCs). This suggests that the inhibition of TPCs was causing alterations to the normal cellular pathways that the virus hijacks for entry, and because of these changes the virus cannot enter the cells to infection them. Indeed, the group also demonstrated that Tetrandrine treatment disrupted the normal degradation of epidermal growth factor, further arguing for a disruption to this cellular pathway, leading to protection of cells from EBOV. Precisely what changes the inhibition of TPCs is causing to the cell remains unknown (and will no doubt be the source of much further study). 

Tetrandrine is able to block the functions of TPCs and inhibit EBOV infection of cells. The team then moved on to test whether Tetrandrine would be of any use in an animal, not just cells. Mice were infected with EBOV and then treated with Tetrandrine. Those that were not given Tetrandrine all died from the dose of virus, while the vast majority of those given the drug survived. The drug treated mice had improved clinical signs and had a reduced amount of virus in their blood, suggesting the infection was being controlled. 
A false coloured electron microscope image of Ebola virus

Whether Tetrandrine will be useful for humans remains unclear. A high dose was used in the mice to give the protective effect, which may have dangerous side effects in humans. However, Tetrandrine represents a starting point from which to look for more effective compounds. It could be that derivatives of the chemical could be made that would be more potent, and therefore require smaller doses, or chemicals that function in a similar way could be produced. On top of the potential direct clinical relevance, Tetrandrine has shown a previously unappreciated aspect of EBOV entry into cells, the need for TPCs. Having a better understanding of EBOV entry will improve the search for other chemicals to block infection, boosting chances for developing effective antiviral therapy. Moreover, a huge number of other viruses hijack the endosomal system, it will be interesting to see if any of these have dependence on the function of TPCs to infect cells. If so, TPCs might represent a new target for developing broad-spectrum antiviral therapeutics, which could be used to tackle a range of human pathogens.

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