Identification of the anti-mycobacterial functional properties of piperidinol derivatives

Abstract

Background and purpose: Tuberculosis (TB) remains a major global health threat and is now the leading cause of death from a single infectious agent worldwide. The current TB drug regimen is inadequate, and new anti-tubercular agents are urgently required to be able to successfully combat the increasing prevalence of drug-resistant TB. The purpose of this study was to investigate a piperidinol compound derivative that is highly active against the Mycobacterium tuberculosis bacillus.

Experimental approach: The antibacterial properties of the piperidinol compound and its corresponding bis-Mannich base analogue were evaluated against M. smegmatis and Gram-negative organisms. Cytotoxicity studies were undertaken in order to determine the selectivity index for these compounds. Spontaneous resistant mutants of M. smegmatis were generated against the piperidinol and corresponding bis-Mannich base lead derivatives and whole genome sequencing employed to determine the genetic modifications that lead to selection pressure in the presence of these compounds.

Key results: The piperidinol and the bis-Mannich base analogue were found to be selective for mycobacteria and rapidly kill this organism with a cytotoxicity selectivity index for mycobacteria of >30-fold. Whole genome sequencing of M. smegmatis strains resistant to the lead compounds led to the identification of a number of single nucleotide polymorphisms indicating multiple targets.

Conclusion and implications: Our results indicate that the piperidinol moiety represents an attractive compound class in the pursuit of novel anti-tubercular agents.

Linked articles: This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc.

Figure 1

Compounds identified as inhibitors of the NAT enzyme. Structures of piperidinol compound 1 and the bis‐Mannich base compound 2.

Figure 2

Time kill curve of compounds 1 and 2. Exponentially growing cultures of M. smegmatis were treated with 2x MBC of compound 1, 2 and rifampicin. CFUs were counted at the time points indicated. The averages of five independent experiments are shown and the data are presented as mean ± SEM.

Figure 3

Cytotoxicity testing of compounds 1 and 2 against human A549 human lung epithelium cell line and ovine blood. (A) Effect on the cell viability of the human A549 cell line in the presence of compound 1 or 2. A549 cells were exposed to compound 1 or 2 at the final concentrations indicated for 24 h, and the cell viability was determined after this time by the addition of resazurin. Percentage cell viability is compared with an A549 cell‐only control. (B) Haemolysis of ovine red blood cells in the presence of compound 1 or 2. Ovine red blood cells were exposed to compound 1 or 2 at the final concentrations indicated for 1 h after which the percentage haemolysis was determined by measuring the absorbance at 450 nm. Percentage lysis is compared with the 100% lysis after addition of lysis buffer (10 mM Tris, pH 7.8, 0.32 M sucrose, 5 mM MgCl₂, 10% Triton X‐100). In both cases, the averages of five independent experiments are shown, and the data are presented as mean ± SEM.