title: Development of ssDNA aptamers as potent inhibitors of Mycobacterium tuberculosis acetohydroxyacid synthase.
abstract: Acetohydroxyacid synthase (AHAS) from Mycobacterium tuberculosis (Mtb) is a promising potential drug target for an emerging class of new anti-tuberculosis agents. In this study, we identify short (30-mer) single-stranded DNA aptamers as a novel class of potent inhibitors of Mtb-AHAS through an in vitro DNA-SELEX method. Among all tested aptamers, two candidate aptamers (Mtb-Apt1 and Mtb-Apt6) demonstrated the greatest inhibitory potential against Mtb-AHAS activity with IC50 values in the low nanomolar range (28.94±0.002 and 22.35±0.001 nM respectively). Interestingly, inhibition kinetics analysis of these aptamers showed different modes of enzyme inhibition (competitive and mixed type of inhibition respectively). Secondary structure-guided mutational modification analysis of Mtb-Apt1 and Mtb-Apt6 identified the minimal region responsible for their inhibitory action and consequently led to 17-mer and 20-mer shortened aptamers that retained equivalent or greater inhibitory potential. Notably, a modeling and docking exercise investigated 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 the DNA-protein interactions that lead to its potent inhibition. Importantly, these two short candidate aptamers, Mtb-Apt1 (17-mer) and Mtb-Apt6 (20-mer), also demonstrated significant growth inhibition against multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of tuberculosis with very low MIC of 5.36 μg/ml and 6.24 μg/ml, respectively and no significant cytotoxicity against mammalian cell line. This is the first report of functional inhibitory aptamers against Mtb-AHAS and provides the basis for development of these aptamers as novel and strong anti-tuberculosis agents.
authors.affiliation: Department of Chemistry, Institute of Natural Science, Hanyang University, Seoul 133-791, Republic of Korea.
authors.forename: Irshad Ahmed
authors.initials: IA
authors.lastname: Baig
authors.affiliation: Department of Chemistry, Institute of Natural Science, Hanyang University, Seoul 133-791, Republic of Korea.
authors.forename: Ji-Young
authors.initials: JY
authors.lastname: Moon
authors.affiliation: Department of Chemistry, Institute of Natural Science, Hanyang University, Seoul 133-791, Republic of Korea.
authors.forename: Sang-Choon
authors.initials: SC
authors.lastname: Lee
authors.affiliation: The Korean Institute of Tuberculosis, Korean National Tuberculosis Association, Cheongju 361-954, Republic of Korea.
authors.forename: Sung-Weon
authors.initials: SW
authors.lastname: Ryoo
authors.affiliation: Department of Chemistry, Institute of Natural Science, Hanyang University, Seoul 133-791, Republic of Korea. Electronic address: myyoon@hanyang.ac.kr.
authors.forename: Moon-Young
authors.initials: MY
authors.lastname: Yoon
mesh_terms: D000095:Acetolactate Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D000818:Animals; D000995:Antitubercular Agents / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D052157:Aptamers, Nucleotide / Q000096:biosynthesis / Q000737:chemistry* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002470:Cell Survival / Q000187:drug effects; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000737:chemistry* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D008264:Macrophages / Q000166:cytology / Q000187:drug effects; D051379:Mice; D008826:Microbial Sensitivity Tests; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000187:drug effects* / Q000201:enzymology / Q000254:growth & development; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D052156:SELEX Aptamer Technique
chemical_list: D000995:Antitubercular Agents; D052157:Aptamers, Nucleotide; D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D000095:Acetolactate Synthase
keywords: Acetohydroxyacid synthase; DNA aptamer; Inhibitor; SELEX; Tuberculosis