Inactivation of tetracycline antibiotics by tetracycline destructases (TDases) remains a clinical and agricultural threat. TDases can be classified as type 1 Tet(X)-like TDases and type 2 soil-derived TDases. Type 1 TDases are widely identified in clinical pathogens. A combination therapy of tetracycline and a TDase inhibitor is much needed to rescue the clinical efficacy of tetracyclines. Anhydrotetracycline is a pan-TDase inhibitor that inhibits both type 1 and type 2 TDases. Here, we present structural, biochemical, and phenotypic evidence that anhydrotetracycline binds in a substrate-like orientation and competitively inhibits the type 1 TDase Tet(X6) to rescue tetracycline antibiotic activity as a sacrificial substrate. Anhydrotetracycline interacting residues of Tet(X6) are conserved within type 1 TDases, indicating a conserved binding mode and mechanism of inhibition. This mode of binding and inhibition is distinct from anhydrotetracycline’s inhibition of type 2 TDases. This study forms the framework for development of next-generation therapies to counteract enzymatic tetracycline resistance.
Tetracyclines (TCs) are an important class of antibiotics
threatened
by an emerging new resistance mechanismenzymatic inactivation.
These TC-inactivating enzymes, also known as tetracycline destructases
(TDases), inactivate all known TC antibiotics, including drugs of
last resort. Combination therapies consisting of a TDase inhibitor
and a TC antibiotic represent an attractive strategy for overcoming
this type of antibiotic resistance. Here, we report the structure-based
design, synthesis, and evaluation of bifunctional TDase inhibitors
derived from anhydrotetracycline (aTC). By appending a nicotinamide
isostere to the C9 position of the aTC D-ring, we generated bisubstrate
TDase inhibitors. The bisubstrate inhibitors have extended interactions
with TDases by spanning both the TC and presumed NADPH binding pockets.
This simultaneously blocks TC binding and the reduction of FAD by
NADPH while “locking” TDases in an unproductive FAD
“out” conformation.
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