Researchers at the Free University of Berlin and the FMP find a new way to keep intruders out of cells

Infectious diseases begin when viruses, bacteria, or parasites enter cells; many other health conditions are caused by defects in cells, for example when they receive faulty signals that change their fates. Both processes depend on a complex transport system that acts as a cellular post office, moving molecules and substances to their proper sites. Volker Haucke of the Free University of Berlin, in collaboration with researchers in Australia and at the FMP, has just developed a new method of sealing off cells to prevent the entry of both pathogens such as HIV and some undesirable signals. The groups, which collaborate under a program called the NeuroCure Cluster of Excellence, hope that the method can be adapted into new therapies that could eventually protect people from a wide range of infections, cancer, neurological disorders, and other diseases. The study appears in the Aug. 5 edition of the journal Cell.
The plasma membrane, which is the outermost border of the cell, acts as a first line of defense when things go wrong, and it can bar entry to most unwanted parasites or substances. Some make it through, though, by taking advantage of an intricate system that usually transports healthy molecules through cells. Their delivery relies on envelopes made of membranes, address labels made of proteins, motor molecules that drive the packages around, and many other players.
"Without this sophisticated system, working just as it does," Volker says, "pathogens can't enter the cell and cause havoc. So we wondered what would happen, if we made the cellular postal system go on strike. This might buy the cell time to detect problems before they get out of hand."
Disrupting a single vital component could close down the entire system. Volker and his colleagues focused on a protein called clathrin, which made a good candidate because it carries out several important tasks. First, it picks the site where a package will be assembled, by creating a pit-like depression in the membrane. The well forms a bubble-like shape containing the cargo which clathrin now pinches off, creating a compartment called a vesicle that is released and sent on its way.
Stopping this process required finding something that could block clathrin. In a first round of testing,  Jens von Kries and the FMP's Small Molecule Screening Unit began looking for such a substance in 384-well plate  experiments. The Screening Unit has amassed an impressive "library" over 50,000 drugs and substances, partly through donations from academic chemists and cooperative agreements with pharmaceutical companies, that could be tested for an impact on clathrin. They bombarded the purified molecule with the substances in hopes of finding something that would disrupt clathrin's ability to bind to other proteins and carry out its jobs. Similar experiments were carried out by the FU and a group in Australia.
The work produced a list of "hits" to focus on in further experiments with living cells. This posed a challenge because some of the most interesting candidates from the initial screen with purified clathrin couldn't pass through the cell membrane, meaning they couldn't reach clathrin. The institutes' chemists began tinkering with the substances to enable them to do so and to make them more potent.
The result, Volker says, is the discovery of two compounds that he calls "pitstops," because of their effect on clathrin's ability to form pits in the membrane. Within minutes of their introduction into cells, the substances caused a general "strike" in the transport system. Since this is the mechanism that HIV, undesirable signaling molecules, and other intruders take advantage of, the intervention successfully blocked their entry into cells.
Additional experiments revealed the mechanism by which the pitstops work. The scientists obtained clathrin molecules bound to the substances in crystal form and exposed them to X-rays, a method which provides three-dimensional maps of the structures of molecules. This revealed the means by which the two pitstops bind to clathrin. Interestingly, although the two substances are chemically unrelated to each other, they snap into the same small cavity in the clathrin protein. This alters its structure and prevents it from binding to some of its normal partners – interactions which are necessary for the formation of pits and vesicles.
Volker says the study suggests that it might be possible to develop drugs from the two substances. "Exposure to the pitstops blocked the entry of HIV and disease-causing signals without damaging or killing the cells," he says. "Even after long exposures, the cells survived. This suggests that the substances act in a selective way, interfering with some harmful processes without disrupting some necessary healthy ones. And that is often one of the most difficult hurdles to overcome in the search for effective drugs."
 - Russ Hodge

 
 


von Kleist L, Stahlschmidt W, Bulut H, Gromova K, Puchkov D, Robertson MJ, Macgregor KA, Tomlin N, Pechstein A, Chau N, Chircop M, Sakoff J, von Kries JP, Saenger W, Kräusslich HG, Shupliakov O, Robinson PJ, McCluskey A, Haucke V.

Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition.

Cell. 2011 Aug 5;146(3):471-84.

 

Link to the Screening Unit