Inhibition of Aminoglycoside 6'- N-acetyltransferase Type Ib (AAC(6')-Ib): Structure-Activity Relationship of Substituted Pyrrolidine Pentamine Derivatives as Inhibitors

Abstract

The aminoglycoside 6'-N-acetyltransferase type Ib (AAC(6')-Ib) is a common cause of resistance to amikacin and other aminoglycosides in Gram-negatives. Utilization of mixture-based combinatorial libraries and application of the positional scanning strategy identified an inhibitor of AAC(6')-Ib. This inhibitor's chemical structure consists of a pyrrolidine pentamine scaffold substituted at four locations (R1, R3, R4, and R5). The substituents are two S-phenyl groups (R1 and R4), an S-hydroxymethyl group (R3), and a 3-phenylbutyl group (R5). Another location, R2, does not have a substitution, but it is named because its stereochemistry was modified in some compounds utilized in this study. Structure-activity relationship (SAR) analysis using derivatives with different functionalities, modified stereochemistry, and truncations was carried out by assessing the effect of the addition of each compound at 8 µM to 16 µg/mL amikacin-containing media and performing checkerboard assays varying the concentrations of the inhibitor analogs and the antibiotic. The results show that: (1) the aromatic functionalities at R1 and R4 are essential, but the stereochemistry is essential only at R4; (2) the stereochemical conformation at R2 is critical; (3) the hydroxyl moiety at R3 as well as stereoconformation are required for full inhibitory activity; (4) the phenyl functionality at R5 is not essential and can be replaced by aliphatic groups; (5) the location of the phenyl group on the butyl carbon chain at R5 is not essential; (6) the length of the aliphatic chain at R5 is not critical; and (7) all truncations of the scaffold resulted in inactive compounds. Molecular docking revealed that all compounds preferentially bind to the kanamycin C binding cavity, and binding affinity correlates with the experimental data for most of the compounds evaluated. The SAR results in this study will serve as the basis for the design of new analogs in an effort to improve their ability to induce phenotypic conversion to susceptibility in amikacin-resistant pathogens.

Keywords: Acinetobacter; acetyltransferase; aminoglycoside resistance; aminoglycoside-modifying enzymes; structure–activity relationship.

Figure 1

(A) The complex of 2637.001 and AAC(6′)-Ib obtained from molecular docking. The bound acetyl CoA is also shown. (B) Interaction map of the ligand in its binding site of the AAC(6′)-Ib receptor. The map shows the hydroxyl functionality interaction with Gln91 and the primary amine of 2637.001 hydrogen bonding with Asp179. The pictures show the top-ranked, lowest-energy conformation.

Figure 2

(A) The complex of 2637.004 and AAC(6′)-Ib obtained from molecular docking. The bound acetyl CoA is also shown. (B) Interaction map of the ligand in its binding site of the AAC(6′)-Ib receptor. The primary amine of 2637.004 maintains a hydrogen bond interaction with Asp179. The pictures show the top-ranked, lowest-energy conformation.

Figure 3

(A) The complex of 2637.005 and AAC(6′)-Ib obtained from molecular docking. The bound acetyl CoA is also shown. (B) Interaction map of the ligand in its binding site of the AAC(6′)-Ib receptor. Compound 2637.005 adopts a different orientation in the pocket and now the primary amine interacts with the phenol group of Tyr65 and the hydroxyl group is no longer close enough to hydrogen bond with Gln64. The pictures show the top-ranked, lowest-energy conformation.

Figure 4

(A) The complex of 2637.019 and AAC(6′)-Ib obtained from molecular docking. The bound actyl CoA is also shown. The figure shows a potential for intramolecular pi stacking between the R1 and R5 phenyl groups. (B) Interaction map of the ligand in its binding site of the AAC(6′)-Ib receptor. Compound 2637.019 still maintains a hydrogen bond interaction with Asp115. The pictures show the top-ranked, lowest-energy conformation.