Development of aptamer based inhibitor that blocks TB bacteria entry into host cells

An aptamer-based inhibitor targeting a vital protein, HupB, of Mycobacterium tuberculosis was developed through a multi-institutional team effort led by Dr. Jaya S. Tyagi and Dr. Hs. Krishna Prasad from AIIMS, Tarun K. Sharma from THSTI Faridabad and Dr. Amit Kumar from IIT Indore.

The HupB protein of the TB pathogen is anessentialmulti-functional protein that was discovered almost 20 years ago by Prof. H. Krishna Prasad’s group at AIIMS during immunological screening of TB patients¹. He found that inside the TB bacterial cell, this protein binds and protects bacterial DNA¹ and enables this bug to survive harsh stressful environments, such as during infection. Additionally, he found to his surprise that HupB protein was also present on the bacterial surface, where it interacted with host molecules and mediated TB bacterial entry into host cells. Because of its multifunctional nature and its essentiality, HupB protein is a powerful drug-target.

However, the C-terminal end of HupB is of disordered nature. As a result, the classical drug discovery route, which requires structural information, could not be used for designing inhibitors. Hence, we utilized aptamer technology to overcome this barrier, as aptamer generation does not require prior knowledge of target structure.

We screened candidate aptamers from random DNA libraries to target HupB protein and based on highest binding properties, out of 23 candidates, 2 aptamers were chosen. We found that both aptamers formed stable structures, and bound HupB protein with high selectivity and affinity. Moreover, both aptamers retained their stability in serum, which is a very important parameter while designing drugs or inhibitors. We found that both the aptamers robustly bound to the HupB protein on the surface of the TB bacteria and inhibited its interaction with host cells, thereby significantly blocking the entry of these bacteria into host cells. It is crucial to note that curtailing bacterial entry into host cell is of prime importance, as it is the first interaction of the TB bacteria with the host cell and is the first step in establishment of infection. The biggest advantage of targeting TB HupB protein is that because this protein is essential to the TB bacteria, the same aptamers can work alike on both drug-susceptible and resistant forms of this bug. This work was published in the Molecular Therapy Nucleic Acids journal^2,3 and was recently covered in the media by The Hindu ⁴ and Science chronicle ^5,6 (a Science and Technology blog).

In summary, in the present study we were able to inhibit the function of cell surface associated HupB protein, but our long-term plan is to inhibit the function of intracellular HupB as well. In the intracellular context, it is vital for HupB protein to bind and protect bacterial DNA to ensure bacterial survival. And through our work, we have found that one of the aptamers robustly blocks the HupB-DNA interaction. Thus, it is promising to target intracellular HupB and impede bacterial survival, and for that we are designing strategies to deliver these aptamers inside the TB bacterial cells.

Publications, Patent and Media Coverage:  

1. S. Prabhakar, P.S. Annapurna, N.K. Jain, A.B. Dey, J.S. Tyagi and H.K. Prasad. 1998. Identification of an immunogenic histone-like protein (HLPMt) of Mycobacterium tuberculosis. Tubercle and Lung Disease. 79(1): 43-53.

2. P. Kalra, S.K. Mishra, S. Kaur, A. Kumar, H.K. Prasad, T.K. Sharma and J.S. Tyagi. 2018. G-quadruplex forming DNA aptamers inhibit the DNA-binding function of HupB and Mycobacterium tuberculosis entry into host cells.Molecular Therapy Nucleic Acids. 13: 99-109. https://doi.org/10.1016/j.omtn.2018.08.011

3. P. Kalra, H.K. Prasad, T.K. Sharma and J.S. Tyagi. 2018. Novel DNA aptamers against nucleoid-associated protein HupB of Mycobacterium tuberculosis and uses thereof. Indian patent: 201711001246. http://thsti.res.in/patent_lic_dtl.php?id=54

4. Aptamer inhibits TB bacteria entry into cells. The Hindu. September 8, 2018. https://www.thehindu.com/sci-tech/science/aptamer-inhibits-tb-bacteria-entry-into-cells/article24903152.ece

5. Small DNA-based inhibitor blocks entry of TB bacteria into host cells. Science chronicle (a science and technology blog). Posted on September 8, 2018. https://journosdiary.com/2018/09/08/aptamer-inhibitor-tb-bacteria-into-cells/

6. Podcast: Using aptamers to block the entry of TB bacteria into host cells. Science chronicle (a science and technology blog). Posted on September 8, 2018. https://journosdiary.com/2018/09/08/podcast-tb-bacteria-aptamer/