Propargyl-Linked Antifolates Are Potent Inhibitors of Drug-Sensitive and Drug-Resistant <i>Mycobacterium tuberculosis</i>

<div><p><i>Mycobacterium tuberculosis</i> continues to cause widespread, life-threatening disease. In the last decade, this threat has grown dramatically as multi- and extensively-drug resistant (MDR and XDR) bacteria have spread globally and the number of agents that effectively treat these infections is significantly reduced. We have been developing the propargyl-linked antifolates (PLAs) as potent inhibitors of the essential enzyme dihydrofolate reductase (DHFR) from bacteria and recently found that charged PLAs with partial zwitterionic character showed improved mycobacterial cell permeability. Building on a hypothesis that these PLAs may penetrate the outer membrane of <i>M</i>. <i>tuberculosis</i> and inhibit the essential cytoplasmic DHFR, we screened a group of PLAs for antitubercular activity. In this work, we identified several PLAs as potent inhibitors of the growth of <i>M</i>. <i>tuberculosis</i> with several of the compounds exhibiting minimum inhibition concentrations equal to or less than 1 μg/mL. Furthermore, two of the compounds were very potent inhibitors of MDR and XDR strains. A high resolution crystal structure of one PLA bound to DHFR from <i>M</i>. <i>tuberculosis</i> reveals the interactions of the ligands with the target enzyme.</p></div

Examples of propargyl-linked antifolates evaluated for antibacterial activity.

<p>Examples of propargyl-linked antifolates evaluated for antibacterial activity.</p

Propargyl-Linked Antifolates Are Potent Inhibitors of Drug-Sensitive and Drug-Resistant <i>Mycobacterium tuberculosis</i> - Fig 3

<p>a) Crystal structure of MtbDHFR (blue) bound to NADPH (yellow) and compound UCP1106 (green). Active site residues are labeled. b) Superposition of MtbDHFR: NADPH: UCP1106 (same colors as a) with a structure of human DHFR bound to NADPH and UCP1015 (magenta)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161740#pone.0161740.ref032" target="_blank">32</a>]. Residue Phe 31 (human structure) has two conformations.</p

Evaluation of enzyme inhibition and antibacterial activity of propargyl-linked antifolates.

<p>Evaluation of enzyme inhibition and antibacterial activity of propargyl-linked antifolates.</p

Physicochemical properties of PLAs.

<p>Physicochemical properties of PLAs.</p

Data collection and Refinement Statistics.

<p>Data collection and Refinement Statistics.</p

Oxa, Thia, Heterocycle, and Carborane Analogues of SQ109: Bacterial and Protozoal Cell Growth Inhibitors

We synthesized a library of 48 analogues of the Mycobacterium tuberculosis cell growth inhibitor SQ109 in which the ethylenediamine linker was replaced by oxa, thia, or heterocyclic species, and in some cases, the adamantyl group was replaced by a 1,2-carborane or the <i>N</i>-geranyl group by another hydrophobic species. Compounds were tested against <i>M. tuberculosis</i> (H37Rv and/or Erdman), Mycobacterium smegmatis, Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, Trypanosoma brucei, and two human cell lines (human embryonic kidney, HEK293T, and the hepatocellular carcinoma, HepG2). The most potent activity was found against <i>T. brucei</i>, the causative agent of human African trypanosomiasis, and involved targeting of the mitochondrial membrane potential with 15 SQ109 analogues being more active than was SQ109 in cell growth inhibition, having IC₅₀ values as low as 12 nM (5.5 ng/mL) and a selectivity index of ∼300