Covalent Irreversible Inhibitors of Tetracycline Destructases

Next-generation tetracycline antibiotics are threatened by an emerging resistance mechanism ─ enzymatic inactivation. The relevant enzymes ─ tetracycline destructases (TDases) ─ are structural homologues of class A flavin monooxygenase (FMO) that oxidize tetracycline antibiotics, leading to various inactive degradation products. Small molecule inhibitors of antibiotic-inactivating enzymes are critical clinical therapeutics used to manage bacterial resistance with combination therapy. While reversible TDase inhibitors have been reported, we sought to develop covalent inhibitors that are better aligned with clinically effective covalent β-lactamase inhibitors. Here, we report the design, chemical synthesis, and biochemical characterization of the first covalent irreversible inhibitors of TDases based on C9-derivatives of anhydrotetracycline (aTC). The reactive warheads were installed via a one-step Mannich reaction linking either an <i>N</i>-(1-methyl)cyclopropylamine or <i>N</i>-propargylamine group to the C9-position of the aTC D-ring via an amino methylene linkage. We also synthesized two nonspecific FMO inhibitors, <i>N</i>-(1-methyl)cyclopropylbenzylamine (<b>1</b>) and <i>N</i>-methyl-<i>N</i>-benzyl-propargylamine (<b>2</b>) as mechanistic probes to distinguish reactivity with the essential FAD cofactor in TDases via one- or two-electron transfer pathways, respectively. We evaluated the compounds as potential inhibitors of representative TDases from the two major classes─<i>Type 1</i> (TetX6 and TetX7) and <i>Type 2</i> (Tet50). The aTC-based compounds <b>3</b>-<b>5</b> inhibited both <i>Type 1</i> and <i>Type 2</i> TDases with notable differences in potency and inhibition mechanism. The inhibition of <i>Type 1</i> TDases was more potent but reversible with no time dependence. The inhibition of <i>Type 2</i> TDases was time-dependent and irreversible even after exhaustive dialysis, consistent with a covalent mechanism of inhibition. Molecular modeling of the inhibitors supports unique inhibitor binding modes for <i>Type 1</i> and <i>Type 2</i> TDases that are consistent with the observed differences in the inhibition modes. Blue light irradiation of the <i>Type 2</i> TDase enhanced this reactivity. Treatment of Tet50 with probe molecules <b>1</b> and <b>2</b> under blue light exposure enabled the identification of covalent FAD adducts via mass spectrometry that are consistent with the expected one- and two-electron transfer reaction modes of the cyclopropylamine and propargylamine warheads with the FAD cofactor. At concentrations as low as 2 μg/mL, the aTC-based covalent inhibitors <b>3</b>-<b>5</b> recovered tetracycline activity against <i>E. coli</i> overexpressing TDases. Our findings suggest that the inhibition of TDases through covalent trapping of the FAD cofactor is a viable strategy for overcoming TDase-mediated antibiotic resistance.