Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform

Abstract

Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay.

Structure Of Eukaryotic Type III Glutamine Synthetase Lacking Ring-Ring Quaternary Contacts

“…It has been determined already that quaternary contacts are formed between the least conserved amino acid regions and show significant deviation from class to class 23 . The protein-protein interface analysis of eu-GSIII showed that the interface area is close to the value of ∼1930Å 2 , which is slightly weaker than 2262 Å 2 in pro-GSIII (Fig.5a) but it is still quite notable and higher or equal to previously reported values of ∼1552Å 2 and ∼1890Å 2 for intra-ring interfaces for GSI and GSII respectively 23 .…”

“…Two datasets were collected: 646 image stacks from the 1.2 mg/ml GSIII stock and 445 image stacks from 0.6 mg/ml GSIII stock. Each image stack had 50 frames at a dosage of 2 e/Å 2 /frame and pixel size of 0.6509 Å. All data processing was performed in cryoSPARC v3.3.1 software 31 .…”

“…For example, since nitrogen is a main limiting factor for plant growth, the agricultural industry has widely used phosphinothricin (PPT), an irreversible GS inhibitor 1 , as an herbicide since the 1970s. Similarly, drug screening trials against tuberculosis GS proteins are undergoing [2][3][4] . Human GS is also being considered as a potential drug target to regulate several neurological disorders 5,6 .…”

Mode-of-action profiling reveals glutamine synthetase as a collateral metabolic vulnerability of M. tuberculosis to bedaquiline

“…2D and SI Appendix, Fig. S13 concentrations and ratios associated with the antimycobacterial activity of BDQ independent of substrate concentrations, demonstrating its intrinsic ability to directly sense and specifically respond, in part, to BDQ-mediated changes in AXP concentrations and energy charge in addition to its other well-known ATPdependent regulation by posttranslational modification (26,27). Moreover, isotopic labeling studies using 15 N-labeled glutamate after a nonlethal 3-d preincubation of Mtb with BDQ revealed an almost complete cessation of glutamine biosynthesis (Fig.…”

Glutamine synthetase structure‐catalysis relationship—Recent advances and applications

“…Recent studies aiming at mtGSI selective inhibition are exploring the ATP binding pocket, 48,67,70 the intermonomer interfaces, 40 or targeting the adenylated form of mtGSI. 71 GS inhibition in humans has been studied in animal models for neurodegenerative disorders. A recent review on this matter was published, where the use of MSO as a therapeutic agent is encouraged.…”