Abstract
The successful management of tuberculosis (TB) requires efficient diagnosis and treatment. Further, the increasing prevalence of drug-resistant TB highlights the urgent need to develop novel inhibitors against both drug-susceptible and drug-resistant forms of disease. Malate synthase (MS), an enzyme of the glyoxylate pathway, plays a vital role in mycobacterial persistence, and therefore it is considered as an attractive target for novel anti-TB drug development. Recent studies have also ascribed an adhesin function to MS and established it as a potent diagnostic biomarker. In this study, a panel of Mycobacterium tuberculosis (Mtb) MS-specific single-stranded DNA aptamers was identified by Systematic Evolution of Ligands by EXponential enrichment (SELEX). The best-performing G-quadruplex-forming 44-mer aptamer, MS10, was optimized post-SELEX to generate an 11-mer aptamer, MS10-Trunc. This aptamer was characterized by various biochemical, biophysical, and in silico techniques. Its theranostic activity toward Mtb was established using enzyme inhibition, host cell binding, and invasion assays. MS10-Trunc aptamer exhibited high affinity for MS (equilibrium dissociation constant [KD] ∼19 pM) and displayed robust inhibition of MS enzyme activity with IC50 of 251.1 nM and inhibitor constant (Ki) of 230 nM. This aptamer blocked mycobacterial entry into host cells by binding to surface-associated MS. In addition, we have also demonstrated its application in the detection of tuberculous meningitis (TBM) in patients with sensitivity and specificity each of >97%.
Highlights
Active tuberculosis (TB) affected more than 10.4 million people in 2017, whereas an estimated 1.7 billion individuals are estimated to harbor a latent/persistent infection with Mycobacterium tuberculosis (Mtb).[1]
In the current study, high-affinity malate synthase (MS)-specific single-stranded DNA aptamers were identified from an 80-mer random DNA library (RDL) using Systematic Evolution of Ligands by EXponential enrichment (SELEX)-based screening
The best-performing aptamer candidate, MS10, was further optimized post-SELEX to generate MS10-Trunc.The theranostic potential of MS10-Trunc was established by its inhibitory activity against MS enzyme, interference of Mtb binding and invasion into host cells, and ability to detect MS in cerebrospinal fluid (CSF) specimens for the diagnosis of tuberculous meningitis (TBM)
Summary
Active tuberculosis (TB) affected more than 10.4 million people in 2017, whereas an estimated 1.7 billion individuals (about a quarter of the world’s population) are estimated to harbor a latent/persistent infection with Mycobacterium tuberculosis (Mtb).[1] The current treatment for TB is prolonged, and drug-resistant cases are increasing,[2] which together pose a significant threat to TB control in the community.[3,4] there is an urgent need to develop new inhibitory compounds or molecules that have the potential to work against both the drug-susceptible and resistant TB. Metabolic pathways of central metabolism have attracted attention in recent times as a source of new targets for novel TB drug development. The glyoxylate shunt pathway in particular is considered as a prominent target pathway owing to its role in mycobacterial survival and persistence, as demonstrated in cell and animal models of Mtb infection.[5,6] This two-enzyme pathway enables bypass of the decarboxylation steps in the tricarboxylic acid cycle and conserves carbon for subsequent gluconeogenesis.
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