TETRACYCLINE ANALOGS AFFECTING BINDING TO TN10-ENCODED TET REPRESSOR TRIGGER THE SAME MECHANISM OF INDUCTION

We examined the influence of substituents in tetracycline (tc) analogs modified at positions 2 and 4-9 and anhydrotetracycline (ate) on indu ction of the Tn10-encoded Tet repressor (TetR) by a quantitative in vi tro induction assay. The equilibrium association constants of the modi fied tc to TetR were independently determined to distinguish effects o n binding from those on induction. We found a correlation between the binding affinity and induction of TetR for most re analogs. While a su bstitution at position 5 revealed only minor effects, changes at posit ion 6 increased binding and induction efficiencies up to 20-fold. A ch lorine at position 7 or 8 enhanced binding and induction about 4- and 9-fold, respectively, Substituents at position 9 decreased binding up to 5-fold. Epimerization of the dimethylamino function at position 4 i n 4-epi-tc resulted in about 300-fold-reduced binding and 80-fold-redu ced induction. Substitution of this grouping by hydrogen in 4-de(dimet hylamino)-tc resulted in no binding and no induction. The respective a te analog failed to induce as well, although binding was still observe d, The dimethylamino function may, thus, play a role in triggering the conformational change of TetR necessary for induction, Substitution o f the 2-carboxamido by a nitrilo function did not influence binding an d induction efficiencies. Ate showed about 30-fold increased binding a nd induction, being the most effective inducer tested in this study. T he equilibrium association constants of most TetR-[Mg-tc](+) and TetR- ([Mg-tc](+))(2) analog complexes with tet operator are decreased about 10(2)- and 10(8)-fold, respectively, as compared to those of free Tet R. This suggests that these tc analogs share the same molecular mechan ism of TetR induction.