Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), is an infectious disease that causes 1.5 million deaths each year. In Mtb, trehalose, a non-mammalian disaccharide consisting of two glucose molecules joined by a 1,1-a,a-glycosidic bond, is a major component of cell wall-associated glycolipids. Trehalose also plays a significant role in biogenesis and remodeling of the mycobacterial outer membrane. The metabolic pathways associated with trehalose-mediated mycolic acid transfer are conserved in mycobacteria and essential for viability, and thus represent promising targets for drug development. In a recent collaborative study, we discovered that 6-azido-6-deoxy-D-trehalose (6-TreAz) (i) selectively inhibits Mtbbiofilm formation by disruption of TreS-mediated trehalose metabolic remodeling; and (ii) sensitizes drug-tolerant Mtb to the existing anti-TB compound bedaquiline. Thus, 6-TreAz and related trehalose analogues, though difficult to synthesize, can potentially serve as valuable tools for understanding mycobacterial trehalose metabolic remodeling and exploiting it to develop novel adjunctive therapeutics for TB. Here, we report the synthesis of a panel of azido and amino-trehalose analogues and evaluation of these compounds as inhibitors of Mtb growth and biofilm formation. Given the challenges of chemical synthesis of trehalose analogues, we enlisted the glycosyltransferase enzyme TreT to chemoenzymatically synthesize most of the target compounds in high yield and in only 2 steps. From the compound panel, multiple analogues were shown to selectively inhibit Mtb biofilm formation with better potency than 6-TreAz, and mechanism of action was investigated using knock-out mutants and metabolomics. These compounds can serve as tools to probe mycobacterial trehalose metabolism and potentially as lead compounds in antibiotic development efforts.

Azido- and amino-trehalose inhibitors to study and target trehalose metabolic remodeling in mycobacteria

ACS Spring 2022

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