Discovery of Novel Chemical Series of OXA-48 β-Lactamase Inhibitors by High-Throughput Screening

Abstract

The major cause of bacterial resistance to β-lactams is the production of hydrolytic β-lactamase enzymes. Nowadays, the combination of β-lactam antibiotics with β-lactamase inhibitors (BLIs) is the main strategy for overcoming such issues. Nevertheless, particularly challenging β-lactamases, such as OXA-48, pose the need for novel and effective treatments. Herein, we describe the screening of a proprietary compound collection against Klebsiella pneumoniae OXA-48, leading to the identification of several chemotypes, like the 4-ideneamino-4H-1,2,4-triazole (SC_2) and pyrazolo[3,4-b]pyridine (SC_7) cores as potential inhibitors. Importantly, the most potent representative of the latter series (ID2, AC₅₀ = 0.99 μM) inhibited OXA-48 via a reversible and competitive mechanism of action, as demonstrated by biochemical and X-ray studies; furthermore, it slightly improved imipenem's activity in Escherichia coli ATCC BAA-2523 β-lactam resistant strain. Also, ID2 showed good solubility and no sign of toxicity up to the highest tested concentration, resulting in a promising starting point for further optimization programs toward novel and effective non-β-lactam BLIs.

Keywords: HTS; OXA-48; bacterial resistance; β-lactamase inhibitor.

Figure 1

Structure of clinically available BLIs: clavulanic acid (I), sulbactam (II), tazobactam (III), avibactam (IV), vaborbactam (V), relebactam (VI).

Figure 2

Cyclic system retrieval (CSR) curves of the subset and full set. Related AUC and F₅₀ values are also reported.

Figure 3

HTS workflow.

Figure 4

Bar chart showing the compound distribution across the activity percent as outcome of the primary screening. A zoom of the distribution of the negative values are also reported. The activity binning was performed considering half-closed intervals in which the largest endpoint was included. The dashed line indicates the activity threshold.

Figure 5

Scatter plot of primary screening versus HC in terms of activity percent. The dots are shaped by outcome of the primary screening: circles and diamonds correspond to actives and inactives, respectively. The dots are colored by outcome of the HC: green, yellow, and grey correspond to active, interfering, and inactive compounds, respectively.

Figure 6

Stacked bar chart showing percentage of true hits (green) and interfering compounds (yellow) within each chemical class (SC_1–SC_31).

Figure 7

Distribution of activity percent within the scaffolds. In the box plot, a white line within the boxes marks the median value. Dots below the whiskers indicate outliers. For each scaffold, the numbers of hits, median, minimum, and maximum activity percent values are reported in the table.

Figure 8

Donut chart showing the number of compounds in i-iv ranges of activity percent: (i) −100 to −80%, (ii) −80 to −50%, (iii) −50 to −30%, (iv) −30 to −12.60% (HC) or −30 to −20% (HE). Each range was considered as half-closed interval of activity percent in which the largest endpoint was included. The innermost and external rings refer to the HC and HE outcomes, respectively.

Figure 9

Stacked bar chart showing the hit distribution per scaffold within i–iv ranges of activity percent: (i) −100% to −80%, (ii) −80% to −50%, (iii) −50% to −30%, (iv) −30% to −12.60% (HC) or −30% to −20% (HE), as obtained from the HC and HE stages. Each range was considered as half-closed interval of activity percent in which the largest endpoint was included. On the top of the bars, the total hits and the total tested compounds in brackets are reported. For the sake of simplicity, scaffolds without active representatives in the HE are not reported in the plot.

Figure 10

Scatter plot of pAC₅₀ versus HAC of the confirmed OXA-48 inhibitors colored by chemical classes. The size of the dots is related to the LE value series of compounds. Diagonal lines represent areas of LE values defined by the equation LE = (1.37/HAC) × pAC₅₀.

Figure 11

Time dependent inhibition with ID1, ID2, and tazobactam.

Figure 12

Lineweaver–Burk plots showing competitive inhibition of OXA-48 with respect to nitrocefin using ID1 and ID2. Inhibitor concentrations were 0 (blue), 0.126 (orange), 0.632 (grey), and 3.160 µM (yellow).

Figure 13

X-ray cocrystal structure of ID3 in complex with OXA-48 enzyme (PDB code: 7AW5), reported as cyan and yellow representations, respectively. (a) OXA-48 catalytic pocket occupied by ID3 (cyan surface). Carboxylated Lys73 (KCX73), catalytic Ser70 and Tyr211 (oxyanion hole), and the catalytic water molecule (red circle) are reported. (b) ID3 and the most relevant residues of the binding pocket. Hydrogen bonds, electrostatic and π–π interactions are represented as yellow, purple, and grey dashed lines. For the sake of clarity, some portions of the protein have been omitted.

Scheme 1

(a) H₄, MeSO₃H, EtOH, 80 °C, 100 min, 72%; (b) ethyl pyruvate, aryl aldehyde, HCl, EtOH, MW 150 °C, 10–30 min, 8–29%; (c) LiOH, MeOH-THF-water (1:1:1), 40–45 °C, 20–72 h, 27–94%.

Scheme 2

(a) 4-ethylbenzaldehyde, LDA, THF, −78 °C, 15 min, 23–95%; (b) TBDMS-Cl, DIMAP, imidazole, DCM, rt, 16 h, 91%; (c) Dess-Martin periodinane, DCM, rt, 15–30 min, 80–99%; (d) N₂H₄, DIPEA, EtOH-THF (4:1), 80 °C, 1.5–16 h, 43–83%; (e) (4-(tert-butoxycarbonyl)phenyl)boronic acid, K₂CO₃, Pd(dppf)Cl₂.CH₂Cl₂, dioxane-water (4:1), MW 120–140 °C, 1–3h, 49–54%; (f) TBAF, THF, rt, 16 h, 52%; (g) TFA, DCM, rt, 16 h, 34–43% (for 10 and 11); (h) 4M HCl in dioxane, rt, 40 h, 65% (for 12).

Scheme 3

(a) MnO2, DCM, 95 °C, 16 h, 74%; (b) MePPh₃Br, KOtBu, Et₂O, rt, 16 h, 78%; (c) H₂, Pd/C, AcOH, EtOH, 5 bar, rt, 5 h, 91%; (d) 4M HCl in dioxane, rt, 40 h, 31%.

Scheme 4

(a) 2oxopropanoic acid, methyl-4-formylbenzoate, HCl, EtOH, MW 150 °C, 15 min, 18%; (b) HATU, NH₄Cl, DIPEA, DMF, rt, 64 h, 74%; (c) LiOH, MeOH-THF-water (1:1:1), rt, 4 h, 23%.

Figure 14

X-ray cocrystal structure of ID2 in complex with OXA-48 enzyme (PDB code 7AUX). The ligand and the most relevant residues of the binding pocket are reported in green and yellow, respectively. Hydrogen bonds, electrostatic and π–π interactions are represented as yellow, purple, and grey dashed lines. For the sake of clarity, some portions of the protein have been omitted.