Alternative Strategies for Circumventing Antibiotic Resistances
In addition to the investigation of the inhibition of APHs using ePK inhibitors that target ATP binding, the discovery of the structural similarities between aminoglycoside and protein kinases also led to the examination of the ability of APHs to phosphorylate protein kinase substrates (Daigle et al. 1999b). APH(30)-IIIa and APH(2")-Ia (of the bifunctional enzyme) were shown to be capable of phosphorylating peptide substrates, but at a much slower rate than the phosphorylation of aminoglycosides. The binding modes of peptide substrates were examined by modelling studies using the structure of APH(30)-IIa. The crystal structure of APH(30)-IIa in complex with kanamycin A is the newest addition to the array of structural information on aminoglycoside-modifying enzymes (Nurizzo et al. 2003). The cAMP kinase inhibitor PKI in the conformation observed in the crystal structure of cAMP-dependent protein kinase (Zheng et al. 1993) was modelled into the active site of APH(30)-IIa (Smith and Baker 2002). The peptide can be readily accommodated in the binding site of APH(30)-IIa with very few steric hindrances detected between the inhibitor and the resistance enzyme.
The possibility of using cationic peptides as starting molecules for the development of broad-spectrum inhibitors of resistance enzyme activities was therefore examined (Boehr et al. 2003). Due to the large number of negatively charged residues in the binding pockets of aminoglycoside-modifying enzymes, positively charged peptides are the preferred substrates. Both APH(3')-IIIa and APH(2")-Ia were inhibited by protegrin, indolicidin and its analogue, CP10A, in a non-competitive manner with both ATP and kana-mycin. The inhibition patterns demonstrate that the peptides bind to both the free enzyme and to enzyme-substrate complexes. Together, these results suggest that the peptide inhibitors have multiple binding modes and may span both the ATP and aminoglycoside binding sites. Furthermore, the modelling study of cAMP kinase PKI and APH(3')-IIa corroborated the inhibition kinetics experiments that showed that PKI fully occupied the amino-
glycoside binding site as well as a large portion of the putative ATP binding site (Smith and Baker 2002). Although some antimicrobial peptides are able to penetrate the cytoplasm of the bacterium, many are known to act on the cytoplasmic membrane (Wu et al. 1999). Unfortunately, none of the peptides that inhibited resistance enzymes in vitro displayed synergistic antimicrobial properties with aminoglycosides in organisms harbouring resistance genes (Boehr et al. 2003).
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