The proof-of-concept study opens up new approaches to intervention; but better molecules are needed to take it beyond the lab

A novel approach adopted by a team of researchers based in Bangalore has opened a window of opportunities to design new antimicrobials that could potentially be used to kill the tuberculosis-causing bacteria.

The nine-member team comprised of Ph.D students and faculty/professors. The new antimicrobials have been tested only in drug-sensitive TB. But in principle, drug-resistant TB bacteria should be equally vulnerable.

The results of the study were published recently in the journal Nature Communications. The team was led by Prof. Valakunja Nagaraja, Department of Microbiology and Cell Biology, Indian Institute of Science (IISc), Bangalore.

“We targeted a class of proteins never targeted anywhere [in the world] and in any bacteria [as an antimicrobial],” said Prof. Nagaraja, highlighting the importance of the study.

The protein in question is HU, which is a histone-like protein that binds to DNA and compacts it. (The DNA is a long thread and has to be compacted and condensed into a ball-like structure.) By binding and compacting the DNA, the HU protein is able to regulate the DNA expression.

“HU plays a role in the expression of several genes (a few hundred genes). So it is called a global regulator of gene expression,” said Prof. Suryanarayanarao Ramakumar, Department of Physics, IISc, Bangalore and a co-author of the paper.

Considering the predominant role played by the gene encoding for HU protein, knocking the gene would in effect kill the TB causing bacterium — Mycobacterium tuberculosis. And that was precisely what the team ended up doing.

Since the HU protein binds to DNA and compacts it, they identified small molecules that can bind to the surface of the HU protein and inhibit the binding.

“In effect, the small molecules prevent HU-DNA interaction by binding to the protein. As a result, DNA compaction is disrupted and gene expression goes haywire,” explained Prof. Nagaraja. “Since gene expression is the hallmark of life, the small molecule causes the bacteria’s death.”

To identify the small molecules that can bind to the HU protein, the researchers first cloned the HU-encoding gene. The protein that got expressed in large quantities was then purified. Prof. Ramakumar’s team went ahead and derived the 3D structure of the protein using X-ray crystallography. The next step involved the identification of the core of interaction in the HU protein. “This was done for the first time,” said Prof. Ramakumar.

The DNA-binding protein has a large interface for interaction with the DNA. So in the large interface, the researchers had to identify the core of interaction region — the major part that interacts with the DNA. Prof. Nagaraja likens the core of interaction to a cavity where something can go and fit in well. “Only one cavity which plays a role in the DNA binding was identified,” said Prof. Nagaraja.

Following the identification of the core region, the researchers had to identify molecules that inhibit the binding of HU protein with DNA. This was done using computational method. Two small (with respect to proteins) chemical entities — trans-stilbene derivatives — were identified. “Subsequently, did further studies to confirm the inhibitory action of stilbene derivatives,” explained Prof. Ramakumar.

The basis

In the case of E. coli, there are 12 genes encoding for histone-like proteins that play a key role in DNA compaction, and there are many genes that can act as a backup in case one gene is compromised. “Many of these genes in E. coli serve a redundant or backup function for HU. So HU protein is not essential for cell survival in the case of E. coli,” Prof. Nagaraja explained.

“In contrast, in Mtb, only five histone-like proteins have been identified. But there seems to be no additional gene that can do a function similar to that of HU protein.” Hence, HU protein is vital for Mtb’s survival.

“We realised that the under-representation of histone-like protein in Mtb means there is no backup for the HU protein when it is knocked off. We realised the new approach to target the TB pathogen,” Prof. Nagaraja explained. “Had a hunch … wanted to study the HU protein in greater detail.”

Quite surprisingly, while HU has been studied extensively in other organisms, it has not been studied thoroughly in Mycobacterium tuberculosis — the bacterium that causes TB.

Miles to go

Prof. Nagaraja is extremely cautious of the new molecules’ clinical potential. “This kind of basic research opens up new approaches to intervention. But clearly more studies are required to take it to the next level,” he noted.

“This is an important, breakthrough research, but there are caveats for translation [into clinical use]. We need better molecules and need to take it beyond the lab,” he emphasised, snuffing out any false hope of these molecules becoming the magic bullet to get rid of TB bacteria right away.

“Inhibition [of binding] is inefficient … The potency is not sufficient to go in for animal studies. Need to find better molecules … [there is] no way you can use these molecules directly but can aim to develop better molecules.”

In the end, it’s a proof-of-concept study, and a novel way of targeting TB bacteria. “That’s the key point,” Prof. Nagaraja concluded.