Gram-negative pathogens resistant to amikacin and other aminoglycosides of clinical relevance usually harbor the 6’- N -acetyltransferase type Ib [AAC(6')-Ib], an enzyme that catalyzes inactivation of the antibiotic by acetylation using acetyl-CoA as donor substrate. Inhibition of the acetylating reaction could be a way to induce phenotypic conversion to susceptibility in these bacteria. We have previously observed that Zn 2+ acts as an inhibitor of the enzymatic acetylation of aminoglycosides by AAC(6')-Ib, and in complex with ionophores it effectively reduced the levels of resistance in cellulo . We compared the activity of 8-hydroxyquinoline, three halogenated derivatives, and 5-[N-Methyl-N-Propargylaminomethyl]-8-Hydroxyquinoline in complex with Zn 2+ to inhibit growth of amikacin-resistant Acinetobacter baumannii in the presence of the antibiotic. Two of the compounds, clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) and 5,7-diiodo-8-hydroxyquinoline, showed robust inhibition of growth of the two A . baumannii clinical isolates that produce AAC(6')-Ib. However, none of the combinations had any activity on another amikacin-resistant A . baumannii strain that possesses a different, still unknown mechanism of resistance. Time-kill assays showed that the combination of clioquinol or 5,7-diiodo-8-hydroxyquinoline with Zn 2+ and amikacin was bactericidal. Addition of 8-hydroxyquinoline, clioquinol, or 5,7-diiodo-8-hydroxyquinoline, alone or in combination with Zn 2+ , and amikacin to HEK293 cells did not result in significant toxicity. These results indicate that ionophores in complex with Zn 2+ could be developed into potent adjuvants to be used in combination with aminoglycosides to treat Gram-negative pathogens in which resistance is mediated by AAC(6')-Ib and most probably other related aminoglycoside modifying enzymes.

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Clinical resistance to amikacin and other aminoglycosides is usually due to the enzymatic acetylation of the antimicrobial molecule. A ubiquitous resistance enzyme among Gram-negatives is the aminoglycoside 6′-N-acetyltransferase type Ib [AAC(6′)-Ib], which catalyzes acetylation using acetyl-CoA as a donor substrate. Therapies that combine the antibiotic and an inhibitor of the inactivation reaction could be an alternative to treat infections caused by resistant bacteria. We previously observed that metal ions such as Zn2+ or Cu2+ in complex with ionophores interfere with the AAC(6′)-Ib-mediated inactivation of aminoglycosides and reduced resistance to susceptibility levels. Ag1+ recently attracted attention as a potentiator of aminoglycosides′ action by mechanisms still in discussion. We found that silver acetate is also a robust inhibitor of the enzymatic acetylation mediated by AAC(6′)-Ib in vitro. This action seems to be independent of other mechanisms, like increased production of reactive oxygen species and enhanced membrane permeability, proposed to explain the potentiation of the antibiotic effect by silver ions. The addition of this compound to aac(6′)-Ib harboring Acinetobacter baumannii and Escherichia coli cultures resulted in a dramatic reduction of the resistance levels. Time-kill assays showed that the combination of silver acetate and amikacin was bactericidal and exhibited low cytotoxicity to HEK293 cells.

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.

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Resistance to amikacin in Gram-negatives is usually mediated by the 6'-N-acetyltransferase type Ib [AAC(6')-Ib], which catalyzes the transfer of an acetyl group from acetyl CoA to the 6' position of the antibiotic molecule. A path to continue the effective use of amikacin against resistant infections is to combine it with inhibitors of the inactivating reaction. We have recently observed that addition of Zn2+ to in-vitro enzymatic reactions, obliterates acetylation of the acceptor antibiotic. Furthermore, when added to amikacin-containing culture medium in complex to ionophores such as pyrithione (ZnPT), it prevents the growth of resistant strains. An undesired property of ZnPT is its poor water-solubility, a problem that currently affects a large percentage of newly designed drugs. Water-solubility helps drugs to dissolve in body fluids and be transported to the target location. We tested a pyrithione derivative described previously (Magda et al. Cancer Res 68:5318–5325, 2008) that contains the amphoteric group di(ethyleneglycol)-methyl ether at position 5 (compound 5002), a modification that enhances the solubility. Compound 5002 in complex with zinc (Zn5002) was tested to assess growth inhibition of amikacin-resistant Acinetobacter baumannii and Klebsiella pneumoniae strains in the presence of the antibiotic. Zn5002 complexes in combination with amikacin at different concentrations completely inhibited growth of the tested strains. However, the concentrations needed to achieve growth inhibition were higher than those required to achieve the same results using ZnPT. Time-kill assays showed that the effect of the combination amikacin/Zn5002 was bactericidal. These results indicate that derivatives of pyrithione with enhanced water-solubility, a property that would make them drugs with better bioavailability and absorption, are a viable option for designing inhibitors of the resistance to amikacin mediated by AAC(6')-Ib, an enzyme commonly found in the clinics.

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16Gram-negative pathogens resistant to amikacin and other aminoglycosides of clinical 17 relevance usually harbor the 6'-N-acetyltransferase type Ib [AAC(6')-Ib], an enzyme that 18 catalyzes inactivation of the antibiotic by acetylation using acetyl-CoA as donor 19 substrate. Inhibition of the acetylating reaction could be a way to induce phenotypic 20 conversion to susceptibility in these bacteria. We have previously observed that Zn +2 21 acts as an inhibitor of the enzymatic acetylation of aminoglycosides by AAC(6')-Ib, and 22Addition of 8-hydroxyquinoline, clioquinol, or 5,7-diiodo-8-hydroxyquinoline, alone or 33 in combination with Zn +2 , and amikacin to HEK293 cells did not result in significant 34 toxicity. These results indicate that ionophores in complex with Zn +2 could be developed 35 into potent adjuvants to be used in combination with aminoglycosides to treat Gram-36 negative pathogens in which resistance is mediated by AAC(6')-Ib and most probably 37 other related aminoglycoside modifying enzymes. 38