Fragment-based drug design of a bacterial kinase inhibitor capable of increasing the antibiotic sensitivity of clinical isolates

Abstract

According to the World Health Organization (WHO), antimicrobial resistance is a serious global health issue. Overcoming antibiotic resistance involves several strategies, including the inhibition of resistance mechanisms. Among the various resistance mechanisms, aminoglycoside phosphotransferases (APHs) catalyze the transfer of the γ-phosphate from a nucleotide donor to various aminoglycosides, leading to their inactivation. In this work, using a fragment-based drug design (FBDD) approach, we have identified and characterized a promising APH inhibitor capable of increasing the sensitivity of Pseudomonas aeruginosa and Staphylococcus aureus resistant to aminoglycosides. It is therefore a good candidate for the future development of APH inhibitors to be prescribed in combination with aminoglycosides. This molecule is a competitive inhibitor of adenosine 5'-triphosphate (ATP), the phosphate donor of APHs. Further studies are required to optimize this molecule to improve its specificity for APHs and its bioavailability in bacteria.

Fig. 1. Crystal structures of APH(2”)-IVa in complex with 4 fragments selected during primary screen.

Crystal structures and corresponding omit maps contoured at a sigma level ± 1 of APH(2”)-IVa in complex with (a) F69 determined at 2.38 Å (PDB 9QNQ), (b) F245 at 2.00 Å (PDB 9QNS), (c) F274 at 1.78 Å (PDB 9QMR) and (d) F382 at 2.65 Å (PDB 9QNN). Inhibitors are represented in gray sticks and residues involved in interactions are shown as yellow lines. Interactions are shown as dashed lines: van der Waals interactions in gray, hydrogen bonds in blue and ionic bonds in orange. e Minimal scaffolds identified for optimal interaction with the hinge of APH(2”)-IVa, where X represents a halogen atom.

Fig. 2. Characterization of halogen-containing analogues of identified scaffolds interacting with the hinge of APH and potential vectors for hit optimization.

a, b APH(2”)-IVa enzymatic activity inhibition by 500 µM of fragments and structure of the analogues tested. c–g Crystal structures and corresponding omit maps contoured at a sigma level of ± 1 of APH(2”)-IVa in complex with 1 at 2.47 Å (PDB 9QNW), 2 at 1.88 Å (PDB 9QOK), 3 at 1.86 Å (PDB 9QPB), 4 at 2.13 Å (PDB 9QOL) and 5 at 2.13 Å (PDB 9QOM). Inhibitors are represented in gray sticks and residues involved in interactions are shown as yellow lines. Interactions are shown as dashed lines: van der Waals interactions in gray, hydrogen bonds in blue and ionic bonds in orange. h Potential vectors, represented as red arrows, for optimization of the most promising hits, F69 and 2.

Fig. 3. Structure of the selected derivatives of F69.

Crystal structures of APH(2”)-IVa in complex with (a) 11 determined at 2.41 Å (PDB 9QNY) or (b) 36 determined at 2.30 Å (PDB 9QOE), and corresponding omit maps contoured at a sigma level of ± 1. Inhibitors are represented in gray sticks, residues involved in interactions are shown as yellow lines and the water molecule is represented as a red sphere. Interactions are shown as dashed lines: van der Waals interactions in gray, hydrogen bonds in blue and ionic bonds in orange.

Fig. 4. Affinity of APH(2”)-IVa for 36 and its ATP-competitive inhibition.

a Differential heat released (top panel) and ITC binding curves (bottom panel) of 36 binding to APH(2”)-IVa. b Hyperbolic fitting of raw data (top panel) and Lineweaver-Burk representation (bottom panel) of the ATP competitive inhibition of APH(2”)-IVa by 36. Final concentrations were 0.1 µM APH(2”)-IVa, 0-2 mM MgATP, 100 µM kanamycin A, 2 mM PEP, 140 µM NAD and 0-100 µM 36. The red circles, yellow triangles, blue squares and green stars correspond respectively to 0, 20, 50 and 100 µM of 36.

Fig. 5. Characterization of derivatives of 36.

a Structure of the derivatives of 36 tested. (b) APH(2”)-IVa inhibition constants of 36 derivatives determined as in Fig. 4b. c–e Crystal structures and corresponding omit maps contoured at a sigma level of ± 1 of APH(2”)-IVa in complex with 36-3 determined at 2.11 Å (PDB 9QNX), 36-5 at 2.30 Å (PDB 9QP9) and 36-9 at 2.04 Å (PDB 9QPL). Inhibitors are represented in gray 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 gray, hydrogen bonds in blue and ionic bonds in orange.

Fig. 6. Characterization of azaindole derivatives at vector 2.

a Structure of the more potent analogues of 2 at vector 2. b APH(2”)-IVa inhibition constants of the analogues of 2 determined as in Fig. 4b.

Fig. 7. Characterization of the inhibitory effect and binding mode of 5-chloro-7-azaindole derivatives at vector 3.

a Structure of the derivatives of 2-aminopyridine and 5-chloro-7-azaindole tested. b Crystal structures of APH(2”)-IVa in complex with 68 determined at 1.92 Å (PDB 9QOI) and corresponding omit map contoured at a sigma level of ± 1. (c, d) APH(2”)-IVa inhibition constants of analogues of (c) F68 or (d) 2 determined as in Fig. 4b, except for 83-86 where their concentrations were decreased to 0-10 µM. e, f Crystal structures of APH(2”)-IVa in complex with 83 determined at 2.29 Å (PDB 9QOD) and 85 at 1.99 Å (PDB 9QOC), and corresponding omit maps. Inhibitors are represented in gray 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 gray, hydrogen bonds in blue and ionic bonds in orange.

Fig. 8. Characterization of the effect of 83 on the activity of several APHs.

a Inhibition of the activity of APH(2”)-IVa, APH(3’)-Ib, APH(3’)-Ic, APH(3’)-IIa and APH(3’)-IIb by 100 µM of 83. b IC₅₀ of 83 with APH(2”)-IVa from E. casseliflavus or APH(3’)-IIb from P. aeruginosa determined by HPLC. c Crystal structure of APH(3’)-IIb in complex with 83 determined at 1.97 Å (PDB 9QOS) and corresponding omit map contoured at a sigma level of ± 1. Inhibitor is represented in gray sticks, residues involved in interactions as beige lines and water molecules are represented as red spheres. Interactions are shown as dashed lines: van der Waals interactions in gray, hydrogen bonds in blue and ionic bonds in orange. d–f ITC curves and thermodynamic profiles of binding of 83 to APH(2”)-IVa, APH(3’)-IIa and APH(3’)-IIb, respectively.

Fig. 9. Characterization of the effect of 83 on bacterial growth kinetics.

Measurement of the 24-h growth kinetics of (a) P. aeruginosa C0307 or (b) S. aureus C0032 in the presence of increasing concentrations of kanamycin A and different concentrations of 83. The characteristics of these strains are shown in the Supplementary Table 6. The symbols circles, squares, triangles and diamonds correspond respectively to 0, 125, 250 and 500 µM of 83. The colors black, yellow, green, cyan, mauve and red correspond respectively to 0, 8, 16, 32, 64 and 128 µg mL⁻¹ of Kanamycin A in (a) and 0, 256, 512, 1024, 2048 and 4096 µg mL⁻¹ of Kanamycin A in (b).

Fig. 10. Comparison of the structures of APH-competitive nucleotide inhibitor complexes.

a, b Structures of 83 in complex with (a) APH(2”)-IVa (PDB 9QOD) and (b) APH(3’)-IIb (PDB 9QOS). c, d Structures of CKI-7 in complex with (c) APH(3’)-IIIa (PDB 3Q2J) and (d) APH(9)-Ia (PDB 3Q2M). e Structure of quercetin in complex with APH(2”)-IVa (PDB 4DFU). f, g Structures of (f) 1-NA-PP1 (PDB 4GKH) and (g) 1-NM-PP (PDB 4GKI) in complex with APH(3’)-Ia. Inhibitors are represented as sticks, residues involved in interactions are shown as lines: yellow for APH(2”)-Iva (a, e), beige for APH(3’)-IIb (b), brown for APH(3’)-IIIa (c), mauve for APH(9)-Ia (d) and red for APH(3’)-Ia (f, g). Water molecules are represented as red spheres. Interactions are shown as dashed lines: van der Waals interactions in gray, hydrogen bonds in blue and ionic bonds in orange.