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.

Structure-activity relationship of pyrrolidine pentamine derivatives as inhibitors of the aminoglycoside 6′-N-acetyltransferase type Ib

“…S1, Supplementary material). The purity and identity of compounds were verified as before [24] using a Shimadzu 2020 Liquid chromatography-mass spectrometry (LCMS) system. Chromatographic separations were carried out on a Phenomenex Luna C18 analytical column (5 μm, 150 mm × 4.6 mm i.d.)…”

“…The mobile phases A and B for LCMS analysis were LCMS-grade water and LCMS-grade acetonitrile, respectively (Sigma-Aldrich and Fisher Scientific, both with 0.1% formic acid for a pH of 2.7). The procedure for analyzing 5 μL aliquots was identical to that described in our previous study [24]. Figures S2 and S3 (Supplementary material) show the relevant information on the compounds’ characterization and degree of purification.…”

“…An initial test to determine the levels of inhibition produced by the compounds was done by measuring OD 600 after 20 h of growth in Mueller-Hinton broth supplemented with 16 μg/ml amikacin and 8 μM of the potential inhibitor. These concentrations were selected based on previous studies using compounds with the same scaffold [23, 24]. Each compound was tested in four separate experiments by duplicate.…”

“…Checkerboard assays were performed in Mueller-Hinton broth as described previously [23, 24]. Each compound was tested on at least three independent checkerboard experiments, in which each dose combination was tested by duplicate.…”

“…This finding highlights the necessity for an inhibitor with enough potency to effectively combat a range of pathogenic bacteria, even in environments with high enzyme concentrations. In addressing this challenge, we conducted further structure-activity relationship (SAR) studies to investigate how changes in the compounds' stereochemistry and substitutions of functionalities impact their inhibitory effectiveness [24]. Expanding upon our previous SAR analyses, this work aims to continue exploring the connection between molecular modifications and their inhibitory effects.…”