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.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is one amongst the top 10 causes of death worldwide. The growing rise in antibiotic resistance compounded with slow and expensive drug discovery has further aggravated the situation. ‘Drug repurposing’ is a promising approach where known drugs are examined for a new indication. In the present study, we have attempted to identify drugs that could target MurB and MurE enzymes involved in the muramic acid synthesis pathway (Mur Pathway) in Mtb. FDA-approved drugs from two repositories i.e. Drug Bank (1932 drugs) and e-LEA3D (1852 drugs) were screened against these proteins. Several criteria were applied to study the protein-drug interactions and the consensus drugs were further studied by molecular dynamics (MD) simulation. Our study found Sulfadoxine (–7.3 kcal/mol) and Pyrimethamine (–7.8 kcal/mol) to show stable interaction with MurB while Lifitegrast (–10.5 kcal/mol) and Sildenafil (–9.1 kcal/mol) showed most reliable interaction with MurE. Furthermore, binding free energy (ΔG_bind), RMSD and RMSF data and the number of hydrogen bonds corroborated the stability of interactions and hence these drugs for repurposing should be explored further. Communicated by Ramaswamy H. Sarma

Antimicrobials are essential for combating infectious diseases. However, an increase in resistance to them is a major cause of concern. The empirical use of drugs in managing COVID-19 and the associated secondary infections have further exacerbated the problem of antimicrobial resistance. Hence, the situation mandates exploring and developing efficient alternatives for the treatment of bacterial and fungal infections in patients suffering from COVID-19 or other viral infections. In this review, we have described the alternatives to conventional antimicrobials that have shown promising results and are at various stages of development. An acceleration of efforts to investigate their potential as therapeutics can provide more treatment options for clinical management of drug-resistant secondary bacterial and fungal infections in the current pandemic and similar potential outbreaks in the future. The alternatives include bacteriophages and their lytic enzymes, anti-fungal enzymes, antimicrobial peptides, nanoparticles and small molecule inhibitors among others. What is required at this stage is to critically examine the challenges in developing the listed compounds and biomolecules as therapeutics and to establish guidelines for their safe and effective application within a suitable time frame. In this review, we have attempted to highlight the importance of rational use of antimicrobials in patients suffering from COVID-19 and boost the deployment of alternative therapeutics.