Structure-guided optimization of protein kinase inhibitors reverses aminoglycoside antibiotic resistance

Abstract

SYNOPSIS Activity of the aminoglycoside phosphotransferase APH(3’)-Ia leads to resistance to aminoglycoside antibiotics in pathogenic Gram-negative bacteria, and contributes to the clinical obsolescence of this class of antibiotics. One strategy to rescue compromised antibiotics such as aminoglycosides is targeting the enzymes that confer resistance with small molecules. Previously we demonstrated that eukaryotic protein kinase (ePK) inhibitors could inhibit APH enzymes, due to the structural similarity between these two enzyme families. However, limited structural information of enzyme-inhibitor complexes hindered interpretation of the results. As well, cross-reactivity of compounds between APHs and ePKs represents an obstacle to their use as aminoglycoside adjuvants to rescue aminoglycoside antibiotic activity. Here, we structurally and functionally characterize inhibition of APH(3’)-Ia by three diverse chemical scaffolds – anthrapyrazolone, 4-anilinoquinazoline and pyrazolopyrimidine (PP) – and reveal distinctions in the binding mode of anthrapyrazolone and PP compounds to APH(3’)-Ia versus ePKs. Using this observation, we identify PP-derivatives that select against ePKs, attenuate APH(3’)-Ia activity and rescue aminoglycoside antibiotic activity against a resistant E. coli strain. The structures presented here and these inhibition studies provide an important opportunity for structure-based design of compounds to target aminoglycoside phosphotransferases for inhibition, potentially overcoming this form of antibiotic resistance.

Antimicrobial interactions: mechanisms and implications for drug discovery and resistance evolution

“…Motivated by the prior success of this approach, considerable efforts to develop new beta-lactamase inhibitors are underway and several such inhibitors are currently in clinical trials [53]. New compounds inhibiting enzymes conferring resistance to carbapenem beta-lactams and aminoglycosides were identified by screening natural products from environmental microorganisms [54 ] and using a structure-guided approach [55], respectively. In both cases, the identified compounds were previously known for entirely different activities on plant leaves and as eukaryotic protein kinase inhibitors, respectively, highlighting the potential for repurposing drugs.…”

Resistance to the Antimicrobial Agent Fosmidomycin and an FR900098 Prodrug through Mutations in the Deoxyxylulose Phosphate Reductoisomerase Gene ( dxr )

“…The vector pKOV-dxrS222T was created by cutting a 2,875-bp BamHI/PstI fragment from the vector pACYC-dxrS222T and ligating it into pKOV, after removal of the 2,036-bp BamHI/PstI fragment. The pKOVdxrS222T vector was used to transform either E. coli MG1655 or E. coli BW25113 ⌬bamB⌬tolC (a kind gift of Gerard Wright, McMaster University, Ontario, Canada) (44). Colonies were grown on LB with either 30 g/ml (for MG1655) or 6 g/ml (for BW25113 ⌬bamB⌬tolC) of chloramphenicol at 30°C for 48 to 72 h. Colonies were picked and grown overnight at 42°C in LB at the appropriate concentration of chloramphenicol.…”

“…Therefore, we replaced the native dxr locus with dxr-S222T in the efflux-defective E. coli strain BW25113 ⌬bamB⌬tolC (44). In this strain, as in MG1655, expression of the dxr-S222T allele confers substantial resistance to FSM (EC 50 s, 0.37 M [95% CI, 0.22 to 0.64 M] versus 5.0 M [95% CI, 2.4 to 11 M] for BW25113 ⌬bamB⌬tolC and BW25113 ⌬bamB⌬tolC-dxr-S222T, respectively; t test, P Ͻ 0.0005) (Fig.…”

Insights into the antibiotic resistance and inhibition mechanism of aminoglycoside phosphotransferase from Bacillus cereus: In silico and in vitro perspective

“…Similarly, molecular docking study of known APH inhibitors, ie, apigenin, quercetin, damnacanthal, genistein, LYS294002, SB203580, and virtually screened inhibitor ZINC71575479 was performed with APH model using AutoDock 4.2 to explore the inhibition mechanism of APH from B. cereus . The virtually screened inhibitor ZINC71575479 was investigated from our earlier study, which showed efficient binding ability toward NTP‐binding site of various aminoglycoside kinases from different multidrug‐resistant strains .…”