3D Molecular Models, Humanitarian Role Models

Adding to the effects of poverty and limited access to medical care, civil unrest and movement of large populations exacerbate public health problems in the developing world. The parasitic disease leishmaniasis has decimated refugee populations in Africa more than once.  Although neglected, for the most part, by drug developers, researchers are seeking more effective treatments, using computer simulations to reveal the structure of individual proteins that they hope to make the parasite’s Achilles heel.

Adding to the effects of poverty and limited access to medical care, civil unrest and movement of large populations exacerbate public health problems in the developing world. The parasitic disease leishmaniasis has decimated refugee populations in Africa more than once.  Although neglected, for the most part, by drug developers, researchers are seeking more effective treatments, using computer simulations to reveal the structure of individual proteins that they hope to make the parasite’s Achilles heel.

 

CDC/ Frank Collins
Mosquito

Just over one year ago, anti-government riots in Libya marked the beginning of a period of major upheaval in the country, resulting, finally, in a new era of democracy.  During the fighting, tens of thousands fled to Tunisia, living in refugee camps at the border.  Even after the return to peace, this movement of the population may have had a negative impact on public health.  An article published last summer on the Tunisia Live news website suggested that, in addition to burgeoning democracy, another consequence of the war could prove to be an epidemic of leishmaniasis.  This disease, caused by the parasitic leishmania protozoan, comes in different forms, with varying degrees of severity.  In northwest Libya, the predominant species leads to a cutaneous leishmaniasis that causes skin lesions, but can clear up without treatment in a few months.  The feared public health risk is that Libyans returning home could bring with them, and pass on, a new species of leishmania found in southern Tunisia where they have been living.  This variety produces a chronic form of the disease—disfiguring lesions may last for years—to which the Libyan population is not immune.

According to Doctors Without Borders/Médecins Sans Frontières (MSF), “Leishmaniasis is prone to epidemics, especially when previously unexposed populations are forced by war and famine to move into endemic areas.”  This was seen in Sudan when civil war pushed refugees to move into new regions of the country.  A 10-year epidemic (1984-94) of visceral leishmaniasis took hold, killing 100,000 people—one-third of the population in the affected provinces.

MSF laments that, on the international level, there is a lack of cooperation in addressing these epidemics.  This is regrettable, but unsurprising, as the disease has been largely neglected across the board in terms of diagnosis and vaccine and drug development.  No methods for preventing infection exist, and the most common treatments today were developed in the 1930s.  These drugs, derived from the highly toxic element antimony, are far from perfect; the compounds themselves can kill a patient, and, even in the best case, require 30 days of hospitalization and painful, daily injections that don’t come cheap.

Leishmaniasis, caused by a parasitic microorganism, is transmitted by the bite of the female sand fly.  When the parasite enters the body, phagocytic cells of the person’s immune system, such as macrophages, engulf the invader in an attempt to neutralize it.  Leishmania, though, is able to survive inside these cells and reproduce, eventually bursting out and infecting other cells.  The immune system is weakened in the process, leaving patients more susceptible to other infections.  The species of parasite and interactions with the individual host determine whether cutaneous or visceral disease results.  If left untreated, visceral leishmaniasis, the most severe form, is fatal.

The World Health Organization believes that leishmaniasis is highly underreported, in part due to limited access to medical care by the very poor populations often affected.  Treatment availability can be irregular, and resistance to anti-leishmania drugs has already been reported in India.  Emna Harigua is one scientist working to improve the treatments currently available for this neglected, but major, health problem for the developing world.  In her doctoral work at the Institut Pasteur in Tunis, Tunisia, she uses computer simulations to study, in minute detail, a single protein of the parasitic organism responsible for visceral leishmaniasis.

Trained initially in chemical engineering, she now uses computer modeling to study the three-dimensional shape of the protein.  Her goal is to identify locations on the molecule that could act as “landing pads” for an anti-leishmania drug, blocking the action of the parasite’s protein.  The molecule in question, called LeIF (leishmania eukaryotic initiation factor), is involved in the translation of the protozoan’s RNA into protein.  Researchers believe this could be a good drug target because the human version is already used in some cancer treatments.  But will it also prove useable in leishmaniasis?

The first step in Emna Harigua’s method, known as homology modeling, involves searching a protein databank for similar molecules whose 3D structure has already been determined through crystallography or NMR (nuclear magnetic resonance spectroscopy).  These then serve as templates for building a likely model of LeIF.  When identifying matches, she accepts a similarity of 80 percent.  The proteins that make the cut are usually other initiation factors with a similar function in other organisms.

Having determined a potential structure for the leishmania protein in question, Emna’s team next performed a molecular dynamic simulation.  In this test, the software models how the protein behaves in solution.  It applies a force field to the virtual solution, which calculates the interaction between all the atoms. “It calculates the vibration of the atoms—of the protein, the water—and tries to minimize the energy of the system.  This is a basic principle of physics, that a system will lose energy to become more stable.  If our system stabilizes quickly, it’s probably naturally relevant.  We found that our structure was quite stable.”

To understand better what is going on while the protein vibrates, Emna looked at the trajectory, or the “film” of the protein in motion produced by the computer model. She examined 200 still shots from the video, which allowed her to identify the main positions that the protein takes. Analyzing these conformations, the team was able to identify cavities and spaces in the structure that were not occupied by protein atoms.  These are the spots that could potentially serve as docking sites for a drug against leishmania.

Another level of screening determines the movement of the cavities as the whole protein moves, describing these minuscule sites in even greater detail.  Analysis showed that many of the cavities were too small or too open: a docking site really needs to be a pocket of at least 30 cubic angstroms (Å3). Having identified the two most promising sites, Emna Harigua performed virtual screenings of chemical compound databanks to see what molecules could potentially bind to these sites.  Her search was fruitful, to say the least, yielding nearly 85,000 possible compounds.  Docking software allowed her test every compound, one by one, in each pocket.  “We ended up with a lot of data: the coordinates of the atoms, the energy interaction between them…  The challenge is to get down to the 100 best molecules.”

To do so, Emna had to devise a way to filter all the data.  She eliminated all unstable, high energy molecules.  She considered only compounds that were “comfortable” in the pocket and able to dock in it in 20 different ways.  Three more rounds of complex simulations gave her the last criteria she needed: the nature and intensity of the chemical bond between the protein and each candidate compound; the chemical structure of the compound itself; and the docking score, measuring the interaction energy between compound and protein.

From 85,000 possibilities to the top 100 most likely drug candidates for each pocket under study, Emna Harigua has waded through an ocean of data, looking for a way to target the microscopic but destructive leishmania parasite.  From here, she will pass the torch to her lab mates for the biological validation step.  In vitro tests will determine if the compounds and the LeIF protein interact as predicted by Emna’s models.  As a recipient of the L’Oréal-UNESCO For Women in Science fellowship, she will also explore other avenues at the Institut Pasteur in Paris.  “Our protein doesn’t act alone. It forms complexes with other initiation factors.  The next step is to see if we can block this interaction.”

Doctors Without Borders/Médecins Sans Frontières, declares on its website that in the fight against leishmaniasis,“the best of efforts are dwarfed by the limitations of existing treatments and the world's lack of interest in this forgotten disease.”  At the same time, the US Centers for Disease Control and Prevention believes that climate change threatens to expand the geographic range of the sand fly and leishmaniasis transmission.  If this is so, then the world had better take notice of this disease.  The good news is that researchers like Emna Harigua are already paying attention and that, once treated, a leishmaniasis patient remains immune for life.  As MSF Health Advisor Koert Ritmeijer puts it, every case treated is a life saved.