More than 40 years ago, doctors in the United States began treating tuberculosis (TB) with the drug rifampin, but they only recently began understanding how rifampin could stop the spread of TB on a structural level. Earlier this month, a team from Rutgers—New Brunswick made a major breakthrough on this front.
Rutgers researchers collaborated with a group from the Rutgers New Jersey Medical School in Newark published a paper outlining the structure of Mycobacterium tuberculosis ribonucleic acid polymerase (Mtb RNAP), which rifampin binds to in order to inhibit the bacteria from replicating, said senior author Richard Ebright.
“The drug rifampin is critically important, it’s a cornerstone of anti-(tuberculosis) therapy,” he said. “It has the property that it can kill not only replicating bacteria but also dormant, non-replicating bacteria, and TB infection involves a substantial component of dormant bacteria. The only way to clear the infection is to have a compound that can kill those bacteria.”
When rifampin binds to Mtb RNAP, it prevents the enzyme from acting — it literally blocks the action — which halts bacteria replication.
The Board of Governors Professor of Chemistry and Chemical Biology Edward Arnold said the paper’s other purpose was to explain alternate binding sites for new drugs, due to the increasing number of drug-resistant strains of tuberculosis.
These mutated strains are as dangerous as any other type of tuberculosis bacterium, but are immune to rifampin because their binding site is different, he said. As a result, they can continue to replicate, complicating treatment efforts.
“Therefore, there’s an urgent need to find new classes of compounds that can inhibit the same enzyme but can do so with different binding sites,” he said. “Around a decade ago we began working on the project. We knew this was the rate-limiting step, the critical essential step, to finding new compounds which inhibit this enzyme.”
By finding new binding sites, other drugs can be created to treat tuberculosis which is resistant to rifampin, he said. The reason they could not be created before is because researchers did not know how tuberculosis looked on a structural level.
“In order to (create new medicines) one needs to know the structure of the enzyme,” he said. “You can’t use rational, structure-based drug design methods if you don’t know the structure of the target that you’re addressing, and until now, there has not been structural information available on that target.”
By understanding how the enzyme works, researchers can see more clearly how rifampin binds to it and prevents tuberculosis bacteria from replicating.
To determine how rifampin works, researchers first needed large amounts of the tuberculosis bacterium’s enzyme, he said. They placed Mtb RNAP genes into E. coli bacteria, which allowed them to grow and assemble the enzyme.
The enzymes were then extracted from the bacteria and tested, he said.
“We developed assays that allowed us to demonstrate the activity of the enzyme and we began efforts to try to determine its structure using a technique called x-ray crystallography, which requires that you first crystallize the structure so it diffracts well," he said.
By seeing how the rifampin binds to Mtb RNAP, researchers can also see how the protein changes, which would indicate drug-resistant bacteria, he said. This is also how the research team was able to discover new binding sites.
By using new binding sites, researchers can make sure to avoid overriding rifampin’s binding site, he said.
It took nearly the full decade to discover the enzyme’s structure, Ebright said.
“We finally succeeded in obtaining its structure … and have spent the last two years performing additional experiments validating the structure and verifying the structural inferences we drew,” he said. “It represents a 10-year effort, and it feels very good to have had that effort succeed and know that the results are now available and can be used both by us and others in the scientific community.”
Ebright said he plans to continue researching ways to kill the bacterium which causes TB. Discovering different sites on the bacterium which can be used to inhibit it should help in developing a new medicine.
“From our perspective, the most important avenue to pursue is the new class of compounds that bind to a different site on Mtb RNA Polymerase, and are therefore able to inhibit versions of the enzyme that are resistant to rifampin,” he said.
The research project was performed by a team of researchers from Rutgers—New Brunswick and the New Jersey Medical School at Rutgers—Newark, Ebright said.
The 2013 merger between Rutgers and the University of Medicine and Dentistry of New Jersey made working with the New Jersey Medical School an easier process, he said.
“Just for the half of the period before the merger, we were working with researchers from another university,” he said. “And after the merger, we were working with colleagues in the same university, which greatly simplified the bureaucratic process of collaboration.”
Nikhilesh De is a correspondent for The Daily Targum. He is a School of Arts and Sciences senior. Follow him on Twitter @nikhileshde for more.