Using the increased evolution of aminoglycoside (AG)-resistant bacterial strains the need to develop AGs with (i) enhanced antimicrobial activity (ii) the ability to evade resistance mechanisms and (iii) the capability of targeting the ribosome with higher efficiency is more and more pressing. shed light on the mechanism by which they act and revealed three with clearly enhanced ribosome-targeting activity. (C) and (L). Compounds bearing aromatic substituents generally exhibited promising antibacterial activity (MIC 9.4 μg/mL) against (A) (B) (E) (I) NorA (J) (K) (N) (H) and (S). Several of these including compounds 3m-n p x-z aa cc-dd ff gg and ii were even capable of inhibiting the growth of various bacterial strains at Ibuprofen (Advil) an MIC 2.4 μg/mL. Furthermore additional substitution at the TolC (O) while compound 3z (MIC 18.8 μg/mL) was 8 times more active than TOB (MIC 150 μg/mL) against (G). Since these compounds demonstrated comparable or better potency against strains we further investigated their mechanisms of action. We Rabbit Polyclonal to MBD3. compared their abilities to inhibit growth of Δ7 prrn containing wild-type (WT) A1408G or G1491U ribosomes. Strain Δ7 prrn lacks all seven chromosomal rRNA operons and instead contains a single plasmid-borne rRNA operon.[15] Hence each of these strains contains a homogeneous population of WT or mutant ribosomes. Mutations A1408G and G1491U target the primary AG binding site of helix h44 of the 30S subunit and each mutation confers resistance to a number of AGs.[16] TOB inhibited Δ7 prrn WT (MIC 18.8 μg/mL) and failed to inhibit either Δ7 prrn A1408G or G1491U (MIC >150 μg/mL) (Table S2) indicating that TOB inhibits growth by binding its h44 site consistent with previous results.[16a 16 Many of the TOB variants showed a similar activity profile against these strains inhibiting growth in an A1408- and G1491-dependent manner. Several of Ibuprofen (Advil) these (3m-n p x-z aa-dd ff-hh) all carrying an aryl ring substituent are more potent than TOB and retain target specificity. Two compounds (3f and 3g) were found to have Ibuprofen (Advil) indistinguishably strong antibacterial activity against Δ7 prrn WT Ibuprofen (Advil) A1408G and G1491U showing that these compounds act through a distinctmechanism independent of h44. These compounds substituted with linear alkyl chains (C14 and C16 respectively) were previously found to have cellulolytic activity [13] which may be their primary mechanism of action. Compounds 3d and 3e with slightly shorter alkyl chains (C10 and C12 respectively) have lower activity with little-to-no h44-dependence. Finally a number of compounds were found to be virtually inactive against all the tested strains. These include compounds 3b c h and i; substituted respectively with C6 C8 C18 or C22 linear alkyl chains; compound 3s modified with 4-ribosomes with various concentrations of TOB variants. Table 1 Translocation inhibition (IC50 values in μM) activities of TOB variants in WT or mutant A1408G ribosomes. Next the effects of several TOB variants on translocation were analyzed (Fig. 1 Table 1). Many behaved similarly to the parental compound although differences correlating with the structure of the substituent were observed. Four compounds inhibited translocation more strongly than TOB in both WT and A1408G ribosomes. Three of these (3n aa dd) have A1408G) dropped from 44 to 9. Compounds 3u and 3x (with (Fig. 1 Table 1). Compounds 3n aa and dd with strains. In these cases inhibition of ribosomes and remains strictly dependent on A1408 showing that higher potency comes without loss of target specificity. These TOB variants thus bind the primary h44 site with increased affinity. How these 6″-aryl substituents promote TOB binding remains to be determined. Co-crystal structures of ribosomes bound to related AGs provide some clues to the basis of enhanced binding.[5a] Rings I and II of these TOB variants make similar sets of contacts to nucleotides 1407-1409 and 1491-1495 of helix h44. Ring III of gentamicin contacts nucleotides 1405-1407 forming hydrogen bonds with G1405 and C1407. Presumably the analogous ring III of TOB occupies a similar position. The 6″-aryl groups of the TOB variants may form a favourable stacking interaction with a nearby rRNA nucleotide such as C1404 and thereby stabilize binding. For the linear alkyl substituted compounds potency and mechanism of action change as a function of chain length. Compound 3a with the shortest chain (C4) of the series has biological activity indistinguishable from TOB inhibiting cell growth in an A1408- and G1491-dependent manner. When the length of the chain is increased to C6 (3b) antibacterial.