Semisynthetic Analogues of Anhydrotetracycline as Inhibitors of Tetracycline Destructase Enzymes. Markley Jana L,Fang Luting,Gasparrini Andrew J,Symister Chanez T,Kumar Hirdesh,Tolia Niraj H,Dantas Gautam,Wencewicz Timothy A ACS infectious diseases The synthesis and biological evaluation of semisynthetic anhydrotetracycline analogues as small molecule inhibitors of tetracycline-inactivating enzymes are reported. Inhibitor potency was found to vary as a function of enzyme (major) and substrate-inhibitor pair (minor), and anhydrotetracycline analogue stability to enzymatic and nonenzymatic degradation in solution contributes to their ability to rescue tetracycline activity in whole cell Escherichia coli expressing tetracycline destructase enzymes. Taken collectively, these results provide the framework for the rational design of next-generation inhibitor libraries en route to a viable and proactive adjuvant approach to combat the enzymatic degradation of tetracycline antibiotics. 10.1021/acsinfecdis.8b00349