Targeting bacterial kinases as a strategy to counteract antibiotic resistance

Abstract

Antibiotic resistance is rapidly emerging as one of the most critical health threats, with resistant microorganisms progressively diminishing the effectiveness of established antibiotics. As a result, the development of therapeutic approaches that effectively target resistant pathogens is of utmost importance. In this study, we developed inhibitors for APH(2")-IVa, a bacterial kinase conveying resistance to aminoglycoside antibiotics. Starting from a hit of a fragment-based screening, we explored the inhibitory motif by structure-based design, ultimately leading to a series of triazole analogues. Advanced analogues displayed promising ADME properties, emerging selectivity vs a panel of human kinases, permeability in both Gram-positive and Gram-negative bacteria, and a moderate antibiotic efficacy for clinical strains of P. aeruginosa. Taken together, our results suggest inhibition of bacterial kinases could be a promising option to reinstall the efficacy of aminoglycoside antibiotics.

Fig. 1. Crystal structures of APH(2”)-IVa in complex with the two identified hit scaffolds, 1 and 2.

Crystal structures of APH(2”)-IVa in complex with 1 (A, PDB 9QOD) and 2 (B, PDB 9QNQ) and their corresponding omit maps contoured at a sigma level of ± 1. Inhibitors are represented in grey sticks, water molecule as red sphere and residues involved in interactions are shown as yellow lines. Interactions are shown as dashed lines: Van der Waals interactions in grey, hydrogen bonds in blue and ionic bonds in orange.

Fig. 2. Synthesis of analogs of 1 and 2.

A Synthesis of 7-azaindole derivatives 10 and 14a:a) a) m-CPBA, DME:n-Hexane (2:1, v/v), rt; b) tetramethylammonium bromide, methanesulfonic anhydride, DMF, -10 °C to rt; c) c) benzenesulfonyl chloride; NaH, DMF, 0 °C to rt; d) Pd(dppf)Cl₂, TMEDA/DMF (1:1) 90 °C; e) DMP, DCM, 0 °C to rt; f) NH₂NH₂ • H₂O, H₂SO₄, NaOH, EtOH, reflux to rt; g) Pd(PPh₃)₄, TMEDA/DMF (1:1) 80 °C; h) K₂CO₃, MeOH, rt; i) NaN₃, sodium ascorbate, CuSO₄(H₂O)₅, H₂O/t-BuOH (1:1), rt. B Synthesis of the pyridine-2-amine derivatives 19 and 22. a) NCS, DMF, -20 °C to rt; b) Pd(dppf)Cl₂, XPhos, K₂CO₃, dioxane/H₂O (6:1), 80 °C, 4 h; c) 3 M HCl, MeOH; d) CuI, PPh₃, Pd(OAc)₂, ACN/Et₃N, 85 °C, 20 h; e) Jones reagent, acetone, 0 °C to rt; f) NH₂NH₂•H₂O, EtOH.

Fig. 3. Crystal structure of APH(2”)-IVa in complex with 14a.

Crystal structure of APH(2”)-IVa in complex with 14a (PDB 9QOT) and its corresponding omit map contoured at a sigma level of ± 1. Inhibitor is represented in grey sticks, residues involved in interactions are shown as yellow lines and water molecule is represented as red sphere. Interactions are shown as dashed lines: van der Waals interactions in grey, hydrogen bonds in blue and ionic bonds in orange.

Fig. 4. Synthesis of the triazole analogs 14b-n, 14b1-14b4, 14c1-14c6.

A a) Azides, sodium ascorbate, CuSO₄(H₂O)₅, H₂O/t-BuOH (1:1), rt. B a) di(1H-imidazol-1-yl)methanone, NH₃, DMF, rt; b) Benzylamine or p-methoxybenzylamine, DIPEA, HATU, DMF, 25 °C. C Commercially available azides used for derivatization. D Custom-made azides used for derivatization.

Fig. 5. Crystal structures of APH(2”)-IVa in complex with the triazole analogs 14a, 14b, 23, 24, 14f and 14k.

Crystal structures of APH(2”)-IVa in complex with 14a (A, PDB 9QOT),14b (B, PDB 9QOX), 23 (C, PBD 9RL1), 24 (D, PBD 9QOW), 14 f (E, PDB 9QOZ) and 14k (F, PDB 9QOU) and their corresponding omit maps contoured at a sigma level of ± 1. Inhibitors are represented in grey sticks, residues involved in interactions are shown as yellow lines. Interactions are shown as dashed lines: van der Waals interactions in grey, hydrogen bonds in blue and ionic bonds in orange.

Fig. 6. Crystal structures of APH(2”)-IVa in complex with the triazole analogs 14b1, 14c1, 14c3, 14c4, 14c5, 14c6, 14m and 14n.

Crystal structures of APH(2”)-IVa in complex with 14b1 (A, PDB 9QP6), 14c1 (B, PDB 9QP0), 14c3 (C, PDB 9QP1), 14c4 (D, PDB 9QP2), 14c5 (E, PDB 9QP3), 14c6 (F, PDB 9QP5) 14m (G, PDB 9QP7), 14n (H, PDB 9QPA) and their corresponding omit maps contoured at a sigma level of ± 1. Inhibitors are represented in grey sticks, residues involved in interactions are shown as yellow lines and water molecules are represented as red spheres. Interactions are shown as dashed lines: van der Waals interactions in grey, hydrogen bonds in blue and ionic bonds in orange.

Fig. 7. Uptake studies for 14a, 10, 14b, 14c, 14c2, 14c3 and 14c4 in Gram-negative and Gram-positive bacterial pathogens.

A Uptake assay for 14a, 10, 14b and 14c in E. coli. B Uptake assay for 14a, 10, 14b and 14c in E. coli ΔtolC strain. C) Uptake assay for 14a, 10, 14b and 14c in K. pneumoniae. D) Uptake assay for 14a, 10, 14b and 14c in P. aeruginosa. E) Uptake assay for 14a, 10, 14b, 14c, 14c2, 14c3 and 14c4 in E. faecalis. F) Uptake assay for 14a, 10, 14b, 14c, 14c2, 14c3 and 14c4 in methicillin-resistant S. aureus. Assays were conducted in biological triplicate. The results are displayed as mean and standard deviation. Concentrations were assessed in membrane, periplasm, cytoplasm and cell lysate (from left to right). Blue bars 14a, red bars 10, green bars 14b, purple bars 14c, orange bars 14c2, black bars 14c3, brown bars 14c4.

Fig. 8. Checkerboard assays.

MIC determinations with P. aeruginosa C0214 in the presence of increasing concentrations of kanamycin A (from left to right) and of 10 A, 14n B or 14c C (from top to bottom). Cells of the table are stained with a linear color gradient according to OD_600nm, from white at 0 to dark blue at the maximum value in the plate, indicated next to the color scale. The first column shows the effect of inhibitors in the absence of kanamycin A. The last column represents the negative control (medium with inhibitors but without bacteria). Growth curves are shown in Supplementary Fig. 4.