Synthesis and evaluation of Trehalose-Pks13 inhibitor conjugates targeting mycobacteria

Abstract

One obstacle to developing new drugs targeting Mycobacterium tuberculosis (Mtb) is its unique cell wall, which forms a significant permeability barrier to drug transport. Recently, transporters of trehalose and other disaccharides within this structure have been identified. We hypothesized that conjugating small molecules active against Mtb with trehalose could facilitate selective uptake of the trehalose conjugate into the cell. This strategy might enhance penetration of the hydrophobic mycomembrane or enable selective targeting of mycobacteria. To test this hypothesis, we used Cu(I)-catalyzed azide-alkyne Huisgen cycloaddition to conjugate 6-azido trehalose to known polyketide synthase 13 (Pks13) inhibitors, such as 2-aminothiophenes (AT), and benzofurans (BzF) with alkyne moieties, and tested the conjugates' activity against mycobacteria. We found that, in some instances, trehalose served to significantly enhance either the antimycobacterial potency or improve selectivity (by reducing toxicity) of the Pks13 inhibitors. Somewhat surprisingly, in M. smegmatis (Msm), the activity of trehalose-modified AT derivatives was independent of the trehalose transporter LpqY-SugABC, suggesting an alternative mechanism(s) of passage into the cell. Thus, the mechanisms underlying trehalose-enhanced inhibitor activity remains to be elucidated. Future studies applying this Trojan Horse strategy to alternative inhibitor chemotypes will be needed to assess the potential of this approach to overcoming the mycomembrane permeability barrier.

Keywords: Cytotoxicity; Mycobacterium tuberculosis; Mycolic acid; Pks13; Trehalose; Trojan horse.

Fig. 1.

A. Concept of trehalose-drug conjugate uptake in mycobacteria. B. Inhibition of Pks13 (blue subunits: ACP-KS-AT-ACP-TE) via a Pks13 targeting trehalose-drug conjugate which inhibits mycomembrane synthesis. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2.

Conversion of known Mtb Pks13-targeting compounds into trehalose conjugates.

Fig. 3.

Compounds 1, 2, and 10 inhibit Msm in a LpqY-SugABC-independent manner. Msm wild type or mutant was cultured in the presence of the indicated compound or left not treated (NT) as negative control or treated with isoniazid (INH) as positive control. Growth was measured using OD₆₀₀ reading. Error bars represent the standard deviation of three replicates.

Scheme 1.

Synthesis of propargylated Pks13 inhibitors.

Scheme 2.

Synthesis of trehalose-Pks13 inhibitor conjugates by copper-catalyzed azide-alkyne Huisgen cycloaddition.

Scheme 3.

Synthesis of control compounds 14 and 15.