Identification and validation of l-asparaginase as a potential metabolic target against Mycobacterium tuberculosis.

Multidrug-resistant Mycobacterium tuberculosis (Mtb) has emerged as a major health challenge, necessitating the search for new molecular targets. A secretory amidohydrolase, l-asparaginase of Mtb (MtA), originally implicated in nitrogen assimilation and neutralization of acidic microenvironment inside human alveolar macrophages, has been proposed as a crucial metabolic enzyme. To investigate whether this enzyme could serve as a potential drug target, it was studied for structural details and active site-specific inhibitors were tested on cultured Mycobacterial strain. The structural details of MtA obtained through comparative modeling and molecular dynamics simulations provided insights about the orchestration of an alternate reaction mechanism at the active site. This was contrary to the critical Tyr flipping mechanism reported in other asparaginases. We report the novel finding of Tyr to Val replacement in catalytic triad I along with the structural reorganization of a ß-hairpin loop upon substrate binding in MtA active site. Further, 5 MtA-specific, active-site-based inhibitors were obtained by following a rigorous differential screening protocol. When tested on Mycobacterium culture, 3 of these, M3 (ZINC 4740895), M26 (ZINC 33535), and doxorubicin showed promising results with inhibitory concentrations (IC 50 ) of 431, 100, and 56 µM, respectively. Based on our findings and considering stark differences with human asparaginase, we project MtA as a promising molecular target against which the selected inhibitors may be used to counteract Mtb infection effectively.

Systematic computational strategies for identifying protein targets and lead discovery.

Computational algorithms and tools have retrenched the drug discovery and development timeline. The applicability of computational approaches has gained immense relevance owing to the dramatic surge in the structural information of biomacromolecules and their heteromolecular complexes. Computational methods are now extensively used in identifying new protein targets, druggability assessment, pharmacophore mapping, molecular docking, the virtual screening of lead molecules, bioactivity prediction, molecular dynamics of protein-ligand complexes, affinity prediction, and for designing better ligands. Herein, we provide an overview of salient components of recently reported computational drug-discovery workflows that includes algorithms, tools, and databases for protein target identification and optimized ligand selection.

Distinct functional properties of secretory l-asparaginase Rv1538c involved in phagosomal survival of Mycobacterium tuberculosis.

The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) stains has escalated the need for developing more efficient drugs and therapeutic strategies against tuberculosis. Here we functionally annotate a secretory mycobacterial asparaginase Rv1538c (MtA) and describe its biochemical properties. MtA primarily existed as dimer along with a minor population of multimers. Circular dichroism and fluorescence spectroscopy demonstrated a compact structure in Tris HCl buffer at pH 8.0. Under these conditions it also displayed optimum activity. It retained ∼40% activity at pH 5.5, supporting its physiological relevance in acidic phagosomal environment. MtA contravened classical Michaelis-Menten kinetics and exhibited product inhibition profile, yielding a Kcat of 869.4 s-1 and an apparent Km of 8.36 mM. We report the presence of several antigenic epitopes and a C-terminal YXXXD/E motif in MtA, hinting towards its potential to interact or influence host immune system. This was supported by our observation of morphological changes in MtA-treated human B lymphoblasts. We propose that MtA is a dual purpose enzyme used by Mtb to survive inside its host by; 1) ammonia-mediated neutralization of the phagosomal acidic pH and 2) inducing stress to primary immune cells and compromising the host immune response. Overall, this study contributes to our understanding of the biological role of mycobacterial asparaginase opening avenues for developing effective TB therapeutics.