Development of Chemical and DNA Aptamer Based Potent Anti-tuberculosis Agents targeting Mycobacterium tuberculosis Acetohydroxyacid Synthase

Abstract

Acetohydroxyacid synthase (AHAS) from Mycobacterium tuberculosis (Mtb) is a promising and potential drug target for the emerging class of new anti-tuberculosis agents. With the absence of the crystal structure of Mtb-AHAS, we selected few highly conserved residues and evaluated for their possible role by site-directed mutagenesis. In silico analyses of AHAS amino acid sequences and homology model have suggested that Proline126 (Pro126), Histidine84 (His84), Glutamate85 (Glu85) and Glutamine86 (Gln86) are highly conserved among many spp and lies in the catalytic dimer interface of Mtb-AHAS. The mutational analyses of these invariants led to significant reduction in their catalytic activity with altered kinetic parameters towards substrate and cofactor ThDP. Further, molecular dynamics simulation studies suggested that these residues are likely to play a key role in maintaining the Glu85 side chain in the required geometry with N1’ atom of ThDP during catalysis. Collectively, these results suggest that these conserved residues might play a crucial role in the ThDP binding of Mtb-AHAS. Secondly, in order to find some new potent chemical inhibitor scaffolds, a high-throughput screening of chemical library was performed. Among the tested 6800 compounds, total 15 compounds were identified as hit-compounds, exhibiting more than 80-90% inhibition of in vitro Mtb-AHAS activity at the fixed 20µM concentration. Importantly, 5 compounds belonging to a structural class of triazolylpyrimidine derivative showed higher inhibition potency with an IC50 in the low micro molar range (0.4–1.24μM). Furthermore, potent inhibitors demonstrated non-competitive, un-competitive or mixed competitive inhibition. The molecular docking exercise of these potent chemicals with an homology model of Mtb-AHAS, indicated hydrophobic and hydrogen bond interactions with some of the key herbicide binding site resdiues with binding energies (ΔG) of -8.5 ~ -8.9kcal/mol respectively. The binding modes were consistent with inhibition mechanisms, as both chemicals oriented outside the active site. Importantly, these potent inhibitors demonstrated significant growth inhibition of various MDR-TB and XDR-TB strains of tuberculosis with an MIC ranging between 0.2 μg/ml~0.8 μg/ml, which is close to MIC of conventional drug for tuberculosis (INH: 0.1 μg/ml, RIF : 0.4 μg/ml). Thus, the identified potent inhibitor scaffolds in this study might provide an impetus for the development of a strong anti-tuberculosis agents targeting Mtb-AHAS. Lastly, we also report for the first time, identification of short (30-mer) single stranded DNA aptamers as novel class of potent inhibitors of Mtb-AHAS, through in vitro selection method, systematic evolution of ligands by exponential enrichment (SELEX). Importantly, among all the tested aptamers, two candidate aptamers (Mtb-Apt1, and Mtb-Apt6) demonstrated greater inhibitory potential against Mtb-AHAS activity with IC50 in the low nanomolar range as 28.94 and 22.35 nM respectively. Interestingly, the inhibition kinetics analysis of these aptamers showed diverse mode of enzyme inhibition as competitive and mixed type of inhibition, respectively. In comparison with other homologous enzymes, these two candidate aptamers exhibited higher target specificity and binding affinity towards Mtb-AHAS with dissociation constant (KD) in the low micro molar to nanomolar range as evaluated by ELISA method. The secondary structure guided, mutational and modification analysis of these two most potent aptamers, Mtb-Apt1 and Mtb-Apt6, identified the minimal region responsible for their inhibitory action and consequently led to 17-mer and 22-mer shortened aptamers retaining equivalent or greater inhibitory potential. Notably, the modelling and docking exercise investigates the binding site of these two potent inhibitory aptamers on the target protein and showed possible involvement of some key catalytic-dimer interface residues of AHAS in DNA-protein interactions, leading to its potent inhibition. Importantly, these two short candidate aptamers, Mtb-Apt1 (17-mer) and Mtb-Apt6 (20-mer) also demonstrated significant rate of growth inhibition against multi and extensively drug resistant strains of tuberculosis with very low MIC of 5.27 µg/ml and 6.16 µg/ml respectively and no significant cytotoxicity against mammalian cell line. Taken together, this is the first report to confirm functional inhibitory aptamers against Mtb-AHAS which would provide the basis to develop these aptamers as novel and strong anti-tuberculosis agents.