In silico virtual screening for the identification of novel inhibitors against dihydrodipicolinate reductase (DapB) of Mycobacterium tuberculosis , a key enzyme of diaminopimelate pathway

Abstract

Tuberculosis (TB) caused by Mycobacterium tuberculosis ( M. tb ) is one of the most devastating infectious diseases afflicting a large number of human lives. In spite of the availability of several drugs for treating TB, emergence of drug-resistant strains of the pathogen has made treatment and eradication of TB a challenging task. Hence, there is an imperative need for new intervention strategies to target M. tb . The enzymes of the diaminopimelate pathway, involved in lysine biosynthesis as well as in cell wall biosynthesis, have been considered as important anti-TB drug targets. One such enzyme is dihydrodipicolinate reductase (DapB), catalyzing the reduction of 2,3-dihydrodipicolinic acid to 2,3,4,5-tetrahydro-dipicolinic acid by utilizing NADH/NADPH as its cofactor. Here, we have generated an antisense knockdown mutant strain of dapB and demonstrated a crucial role of dapB in the in vitro and intracellular growth of M. tb . Further, in silico virtual screening was performed by employing a library of ~95,000 compounds for the identification of inhibitory molecules against DapB. The molecules with high docking scores were screened against the enzymatic activity of DapB to determine their IC 50 values. Further, hit molecules that inhibited DapB were screened against the M. tb growth in broth culture and inside macrophages. The molecules exhibiting M. tb inhibition were also evaluated for their cytotoxicity against various mammalian cell lines. In summary, we have identified a lead molecule, B59, with an IC 50 value of 11 µg/mL and MIC 99 value of 20 µg/mL, which can be further optimized to develop potent inhibitory compounds against M. tb DapB. IMPORTANCE Non-compliance to lengthy antituberculosis (TB) treatment regimen, associated side effects, and emergence of drug-resistant strains of Mycobacterium tuberculosis ( M. tb ) emphasize the need to develop more effective anti-TB drugs. Here, we have evaluated the role of M. tb dihydrodipicolinate reductase (DapB), a component of the diaminopimelate pathway, which is involved in the biosynthesis of both lysine and mycobacterial cell wall. We showed that DapB is essential for the in vitro as well as intracellular growth of M. tb . We further utilized M. tb DapB, as a target for identification of inhibitors by employing in silico virtual screening, and conducted various in vitro screening assays to identify inhibitors with potential to inhibit DapB activity and in vitro and intracellular growth of M. tb with no significant cytotoxicity against various mammalian cell lines. Altogether, M. tb DapB serves as an important drug target and a hit molecule, namely, 4-(3-Phenylazoquinoxalin-2-yl) butanoic acid methyl ester has been identified as an antimycobacterial molecule in our study.

Amino Acid Biosynthesis Inhibitors in Tuberculosis Drug Discovery

“…The Lys metabolic branch, also known as the diaminopimelate DAP pathway, is absent in mammals and is crucial for producing other important metabolites, such as dipicolinate and DAP, which are essential for the cross-linking of peptidoglycan polymers in bacterial cell wall synthesis [ 104 , 105 ]. It involves several enzymatic steps ( Figure 20 ), starting from aldol condensation catalyzed by the enzyme dihydrodipicolinate synthase (DapA) to convert homoserine and pyruvate to dihydrodipicolinate.…”

“…The genes of this pathway are essential for Mtb growth, and some of them have already been investigated as targets for potential inhibitors [ 107 , 108 , 109 , 110 ]. The importance of dihydrodipicolinate reductase (DapB), encoded by dapB , has only been recently elucidated [ 104 ]. This enzyme uses NADH/NADPH as a cofactor to reduce dihydrodipicolinate to tetradihydrodipicolinate.…”

“…The hit of this series was quinoxaline derivative B59 ( Figure 21 ), showing good enzymatic activity (32.9 µM), a reasonable MIC (59.8 µM), and negligible cytotoxicity against three different cell lines. This offered a proof of concept that DapB is a potential drug target and that B59 is worth investigating with further hit-to-lead optimization studies to enhance its antimycobacterial activity and improve its cell permeability and drug-like properties [ 104 ].…”