Piperidinols That Show Anti-Tubercular Activity as Inhibitors of Arylamine N-Acetyltransferase: An Essential Enzyme for Mycobacterial Survival Inside Macrophages

Abstract

Latent M. tuberculosis infection presents one of the major obstacles in the global eradication of tuberculosis (TB). Cholesterol plays a critical role in the persistence of M. tuberculosis within the macrophage during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the cholesterol sterol-ring degradation and is essential for intracellular survival. The ability of the NAT from M. tuberculosis (TBNAT) to utilise propionyl-CoA links it to the cholesterol-catabolism pathway. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. TBNAT has been investigated as a potential target for TB therapies. From a previous high-throughput screen, 3-benzoyl-4-phenyl-1-methylpiperidinol was identified as a selective inhibitor of prokaryotic NAT that exhibited antimycobacterial activity. The compound resulted in time-dependent irreversible inhibition of the NAT activity when tested against NAT from M. marinum (MMNAT). To further evaluate the antimycobacterial activity and the NAT inhibition of this compound, four piperidinol analogues were tested. All five compounds exert potent antimycobacterial activity against M. tuberculosis with MIC values of 2.3–16.9 µM. Treatment of the MMNAT enzyme with this set of inhibitors resulted in an irreversible time-dependent inhibition of NAT activity. Here we investigate the mechanism of NAT inhibition by studying protein-ligand interactions using mass spectrometry in combination with enzyme analysis and structure determination. We propose a covalent mechanism of NAT inhibition that involves the formation of a reactive intermediate and selective cysteine residue modification. These piperidinols present a unique class of antimycobacterial compounds that have a novel mode of action different from known anti-tubercular drugs.

New Approaches to Target the Mycolic Acid Biosynthesis Pathway for the Development of Tuberculosis Therapeutics

“…Finally, the screen revealed a novel mechanism-based irreversible class of piperidinol aNAT inhibitors 78 - 82 with good potency against M. tb and M. marinum aNAT enzymes (Table 3) [113, 115]. Compound 78 has IC 50 values of 7.7 ± 0.9 and 1.3 ± 0.0 μM against M. tb aNAT (TBaNAT) and M. marinum aNAT (MMaNAT), respectively.…”

“…The addition of a second basic functionality with compound 81 increased activity significantly, yielding the most potent anti-TB compound both enzymatically and against whole M. tb cells. All compounds had MIC values of 2.7-16.9 μM and 14.8-42.3 μM against M. bovis BCG and M. tb , respectively [115]. Figure 14 shows the plausible mechanism for suicide inhibition of the piperidinols [115].…”

“…All compounds had MIC values of 2.7-16.9 μM and 14.8-42.3 μM against M. bovis BCG and M. tb , respectively [115]. Figure 14 shows the plausible mechanism for suicide inhibition of the piperidinols [115]. The ring opening of the piperidine ring allows for a reverse aldol condensation yielding a Michael acceptor which in turn alkylates and inactivates aNAT.…”

The role of lysine100 in the binding of acetylcoenzyme A to human arylamine N-acetyltransferase 1: Implications for other acetyltransferases

“…Moreover, recent studies have shown a relationship between NAT1 and cancer cell proliferation and survival suggesting that this protein is a potential drug target [7,8]. There have also been a number of reports on the development of small molecule inhibitors for human and non-human NATs [9][10][11][12].…”

<em>In Silico</em> Screening and Analysis of Potential Inhibitors of Arylamine N-Acetyltransferases (NATs) from the Traditional Chinese Medicine: A Study Using Free Available Tools

“…In another therapeutic perspective, studies showed that NAT gene of Mycobacterium tuberculosis (M. tuberculosis) is part of a cluster essential for the survival of the mycobacteria in the environment inside macrophage [10,16,17]. This points to NAT from M. tuberculosis (TBNAT) as a potential drug target for antimycobacterial therapy [18,19].…”