Development of a one-pot assay for screening and identification of Mur pathway inhibitors in Mycobacterium tuberculosis

Abstract

The cell wall of Mycobacterium tuberculosis (Mtb) consists of peptidoglycan, arabinogalactan and mycolic acids. The cytoplasmic steps in the peptidoglycan biosynthetic pathway, catalyzed by the Mur (A-F) enzymes, involve the synthesis of UDP-n-acetylmuramyl pentapeptide, a key precursor molecule required for the formation of the peptidoglycan monomeric building blocks. Mur enzymes are indispensable for cell integrity and their lack of counterparts in eukaryotes suggests them to be promising Mtb drug targets. However, the caveat is that most of the current assays utilize a single Mur enzyme, thereby identifying inhibitors against only one of the enzymes. Here, we report development of a one-pot assay that reconstructs the entire Mtb Mur pathway in vitro and has the advantage of eliminating the requirement for nucleotide intermediates in the pathway as substrates. The MurA-MurF enzymes were purified and a one-pot assay was developed through optimization of successive coupled enzyme assays using UDP-n-acetylglucosamine as the initial sugar substrate. The assay is biochemically characterized and optimized for high-throughput screening of molecules that could disrupt multiple targets within the pathway. Furthermore, we have validated the assay by performing it to identify D-Cycloserine and furan-based benzene-derived compounds with known Mur ligase inhibition as inhibitors of Mtb MurE and MurF.

Molecular Targets Related Drug Resistance Mechanisms in MDR-, XDR-, and TDR-Mycobacterium tuberculosis Strains

“…The cytoplasmic phases in peptidoglycan biosynthetic pathway are catalyzed by the Mur (A-F) enzymes. The imperative role of Mur enzymes in cell integrity and the minimum availability of their complements in eukaryotes highlighted them as promising anti-TB drug targets (Eniyan et al, 2016 ). Besides these, several other potential targets have also been proposed, such as, MbtA—involved in the iron metabolism of M. tuberculosis ; cytochrome b subunit (QcrB) and type II NADH dehydrogenase—involved in energy generation; fatty acid synthases (FASs) and polyketide synthases (PKSs) involved in cell wall biosynthesis (Fernandes et al, 2015 ).…”

Cell wall peptidoglycan inMycobacterium tuberculosis: An Achilles’ heel for the TB-causing pathogen

“…2013; Eniyan et al . 2016). The mycobacterial Mur ligases catalyse amide bond formation via a similar reaction mechanism to that seen in the E. coli enzymes, i.e.…”

Reaching toward underexplored targets in antibacterial drug design

“…The lack of antibacterial activity of MurB inhibitors is a persistent problem; nevertheless, this case demonstrates the successful use of available structural data in design of Mur enzyme inhibitors. The recent crystal structure of MurB from Mycobacterium tuberculosis (MtbMurB; Eniyan, Dharavath, Vijayan, Bajpai, & Gourinath, ) and a one‐pot assay that reconstructs the entire Mtb Mur pathway in vitro (Eniyan, Kumar, Rayasam, Perdih, & Bajpai, ) will be useful in the development of novel inhibitors of peptidoglycan biosynthesis in M. tuberculosis (Moraes et al, ).…”

“…The recent crystal structure of MurB from Mycobacterium FIGURE 1 MurA to MurF intracellular cascade toward a key peptidoglycan building block UDP-N-acetylmuramyl (UDP-MurNAc) pentapeptide from uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Individual enzymes together with their substrates are presented above in respective arrows in addition to the chemical structure of MurA inhibitor fosfomycin tuberculosis (MtbMurB; Eniyan, Dharavath, Vijayan, Bajpai, & Gourinath, 2018) and a one-pot assay that reconstructs the entire Mtb Mur pathway in vitro (Eniyan, Kumar, Rayasam, Perdih, & Bajpai, 2016) will be useful in the development of novel inhibitors of peptidoglycan biosynthesis in M. tuberculosis (Moraes et al, 2015).…”

“…Multiple inhibition by a single agent would have an advantage by being less prone to high-level, target-based resistance by single genetic modifications in the bacteria (Chopra, 2013;Silver, 2011). A novel recently developed one-pot assay which reassembles the entire Mur pathway in vitro (Eniyan et al, 2016) provides great assistance for high-throughput screening of compounds that could disrupt multiple Mur enzymes. Upto-date, great number of MurC to MurF inhibitors has been reported and described in details in several reviews starting from substrate and transition-state analogs, potential ATP-competitive inhibitors, diphosphate mimetics to inhibitors obtained by high-throughput screening and structure-based drug design (Kotnik, Anderluh, & Prezelj, 2007;Barreteau et al, 2008;Hrast et al, 2014;Kouidmi et al, 2014;Silver, 2003Silver, , 2006Silver, , 2011.…”