In a pathbreaking study addressing our fight against mycobacterial infections like Tuberculosis, researchers from the Central University of Punjab have identified a new drug target that aims at the defense mechanism used by the bacteria. Their study, published in the journal PLOS One, could lay out a path to finding a cure for mycobacterial infections.
We live in a ‘post-antibiotic’ era, where bacteria around us have quickly built a strong and versatile armory against many antibiotics. Mycobacterium tuberculosis, the causative agent of Tuberculosis (TB) is one such bacterium that has proven to be hard to kill. Just like the zombie ant fungus that hijack the ants for their own growth and spreading, M. tuberculosis hijacks our macrophages, a type of white blood cells that fight infections, and use them for their own growth and reproduction. Hence fighting TB is a challenge.
“Most anti TB drugs in the market and our own macrophages try to kill the bacteria by producing reactive oxygen species (ROS)”, explained Dr. Mahesh Kulharia from the School of Basic and Applied Science, Central University of Punjab. Any chemical species that have one or more unpaired electron is called a radical and ROS refers collectively to such oxygen containing radicals.
“The Mycobacterium can withstand these ROS with the help of a gene called mel2, which encodes proteins that protect against ROS. Finding drugs that target this machinery could help increase the susceptibility of the bacteria to anti-TB drugs”, added Dr. Kulharia.
The protein that helps the bacteria fight against ROS is MelF, an enzyme that is made of flavin -- an organic molecule capable of undergoing oxidation-reduction reactions. The researchers believed that modeling the structure of this enzyme using data from a protein database may hold key to identifying a suitable target.
“2WKG, an enzyme that adds a -OH group to the substrates it reacts with, of the soil bacterium Pseudomonas putida has a 30-40% sequence identity with the MelF protein and we used it to model the MelF structure and used Molecular Dynamics, a computer simulation method to refine it”, said Dr. Kulharia, explaining the process of the study.
Although there many drugs available on various drug databases that could potentially inhibit the protein MelF, screeing all of them manually is tedious. Hence, the researchers used a computer program to screen through such a database and picked 1000 small drug like molecules that could potentially act against Mycobacteria. “This entire process of choosing from the top 1000 cannot be done just by the program used. A combination of both human intuition and mathematical analysis is required to obtain the best of these compounds based on their interaction with the protein model” said Dr. Kulharia.
After selecting 20 most promising compounds that act on the enzyme MelF, they tested these compounds on MeIF extracted and purified from a surrogate bacteria. They found that those compounds that inhibited the activity of the enzyme in a whole cell lysate (WCL) -- a concoction of cellular proteins -- did not do so when used on purified MelF. The researchers concluded that this was because of the presence of other oxidoreductases (oxidizing and reducing enzymes) in WCL.
Out of the 20, six compounds were found to kill or weaken Mycobacteria in a test tube and only two eventually showed promising results with no toxic effects on mammalian cells at inhibitory concentration.
Though these findings are the first steps in identifying processes in the Mycobacteria that could be drug targets, there is still a long way to go. Obtaining the structure of the MelF protein could reveal how accurate the computer model of the protein is and the conclusions from the binding studies actually are. “The results published here are just a small part of a bigger picture that is yet to come. Once we obtain substantial results from present and future experiments, we will know the true capabilities of some of the compounds studied”, signs off Dr. Kulharia.