Design, synthesis, and evaluation of substituted nicotinamide adenine dinucleotide (NAD+) synthetase inhibitors as potential antitubercular agents

Abstract

Nicotinamide adenine dinucleotide (NAD+) synthetase catalyzes the last step in NAD+ biosynthesis. Depletion of NAD+ is bactericidal for both active and dormant Mycobacterium tuberculosis (Mtb). By inhibiting NAD+ synthetase (NadE) from Mtb, we expect to eliminate NAD+ production which will result in cell death in both growing and nonreplicating Mtb. NadE inhibitors have been investigated against various pathogens, but few have been tested against Mtb. Here, we report on the expansion of a series of urea-sulfonamides, previously reported by Brouillette et al. Guided by docking studies, substituents on a terminal phenyl ring were varied to understand the structure-activity-relationships of substituents on this position. Compounds were tested as inhibitors of both recombinant Mtb NadE and Mtb whole cells. While the parent compound displayed very weak inhibition against Mtb NadE (IC50 = 1000 μM), we observed up to a 10-fold enhancement in potency after optimization. Replacement of the 3,4-dichloro group on the phenyl ring of the parent compound with 4-nitro yielded 4f, the most potent compound of the series with an IC50 value of 90 μM against Mtb NadE. Our modeling results show that these urea-sulfonamides potentially bind to the intramolecular ammonia tunnel, which transports ammonia from the glutaminase domain to the active site of the enzyme. This hypothesis is supported by data showing that, even when treated with potent inhibitors, NadE catalysis is restored when treated with exogenous ammonia. Most of these compounds also inhibited Mtb cell growth with MIC values of 19-100 μg/mL. These results improve our understanding of the SAR of the urea-sulfonamides, their mechanism of binding to the enzyme, and of Mtb NadE as a potential antitubercular drug target.

Different ways to transport ammonia in human and Mycobacterium tuberculosis NAD+ synthetases

“…The analysis herein described is significant for future efforts in the development of an anti-TB drug. Previously, the crystal structure of tbNadE bound to NaAD + (PDB code 3DLA) was used for docking studies of 118 urea-sulfonamide analogs 43 but this structure has a fully exposed synthetase active site with the P2 loop opened. Based on the structural and sequence differences in the synthetase active site of tbNadE and hsNadE herein shown, a potent tbNadE inhibitor should leverage the differences in the NaAD + -binding residues, such as Lg loop (Trp490 TB ), α20 (Phe631 TB and Phe634 TB ), and α13 (Glu455 TB and Val 452 TB ), (ii) the P2 loop residues (Phe564 TB , Lys560 TB , Ser557 TB , and Glu552 TB ) ( Fig.…”

NAD metabolic dependency in cancer is shaped by gene amplification and enhancer remodelling

“…Sequence alignments between the human and B. Subtilis show limited conservation (23%), but a remarkably high degree of sequence conservation in the NaAD binding and ATP binding sites21( Figure 4C). NADSYNi has been suggested to bind competitively to the NaAD binding site of B. Subtilis 19,22,23 , raising the possibility that NADSYNi could also inhibit the human NADSYN1 enzyme. NADSYN1i inhibited purified human NADSYN1 enzyme activity in a dose-dependent fashion ( Figure 4C) and potently and selectively inhibited PH-amplified cancer cell line growth, lowering NAD levels, while having negligible effect on non-PH amplified cancer or normal cells ( Figure 4C).…”

Network-Based Metabolism-Centered Screening of Potential Drug Targets in Klebsiella pneumoniae at Genome Scale

“…The two genes not predicted as essential in the generic host medium compared to HBF-based medium are nadD and nadE. Indeed, there are many works supporting their potentials as drug targets in diverse bacterial pathogens and investigation of possible inhibitors against them (Sorci et al, 2009;Huang et al, 2010;Rodionova et al, 2014;Wang et al, 2017). This highlighted the power of HBF with 120 metabolites compared to the generic host medium with 192 metabolites to properly simulate the host environment.…”