Discovery and Characterization of Synthesized and FDA-Approved Inhibitors of Clostridial and Bacillary Collagenases

Abstract

In view of the worldwide antimicrobial resistance (AMR) threat, new bacterial targets and anti-infective agents are needed. Since important roles in bacterial pathogenesis have been demonstrated for the collagenase H and G (ColH and ColG) from Clostridium histolyticum, collagenase Q1 and A (ColQ1 and ColA) from Bacillus cereus represent attractive antivirulence targets. Furthermore, repurposing FDA-approved drugs may assist to tackle the AMR crisis and was addressed in this work. Here, we report on the discovery of two potent and chemically stable bacterial collagenase inhibitors: synthesized and FDA-approved diphosphonates and hydroxamates. Both classes showed high in vitro activity against the clostridial and bacillary collagenases. The potent diphosphonates reduced B. cereus-mediated detachment and death of cells and Galleria mellonella larvae. The hydroxamates were also tested in a similar manner; they did not have an effect in infection models. This might be due to their fast binding kinetics to bacterial collagenases.

Figure 1

Examples of known bacterial collagenase H and Q1 (ColH and ColQ1) inhibitors. (a) Structures of bacterial ColH inhibitors.⁻ (b) Structures of the recently identified inhibitors of ColH and ColQ1.⁻ Zinc-binding groups are highlighted by dashed rectangles. n.d.: not determined.

Figure 2

Chemical structures of compounds 13 and 27 and their activities against the collagenase unit (CU) of ColQ1 and peptidase domain (PD) of ColH.

Scheme 1. Synthesis of Compounds 7–12

Reagents and conditions: (a) oxalic acid, 4 N HCl, reflux, 6 h; (b) POCl₃, DMF, 50 °C, 6 h; (c) triethyl phosphite, sealed tube, 150 °C, 18 h; and (d) bromotrimethylsilane, dry DCM, stirring, room temperature, 18 h.

Scheme 2. Synthesis of Compounds 13 and 16

Reagents and conditions: (a) oxalic acid, 4 N HCl, reflux, 6 h; (b) POCl₃, DMF, 50 °C, 6 h; (c) triethyl phosphite, sealed tube, 150 °C, 18 h; (d) bromotrimethylsilane, dry DCM, stirring, room temperature, 18 h; (e) dioxane/H₂O (1:1), LiOH, 55 °C, 24 h; and (f) POCl₃, DMF, 0 °C to room temperature, 5 min.

Scheme 3. Synthesis of Compound 14

Reagents and conditions: (a) oxalic acid, 4 N HCl, reflux, 6 h; (b) POCl₃, DMF, 50 °C, 6 h; (c) 3,4-dichloroaniline, EDC.HCl, DCM, 18 h; (d) triethyl phosphite, sealed tube, 150 °C, 18 h; and (e) bromotrimethylsilane, dry DCM, stirring, room temperature, 18 h.

Scheme 4. Synthesis of Compound 15

Reagents and conditions: (a) (4-chlorophenyl)boronic acid, dioxane/H₂O (4:1), Na₂CO₃ (2 M), Pd(PPh₃)₄, microwave, 20 min; (b) oxalic acid, 4 N HCl, reflux, 6 h; (c) POCl₃, DMF, 50 °C, 6 h; (d) triethyl phosphite, sealed tube, 150 °C, 18 h; and (e) bromotrimethylsilane, dry DCM, stirring, room temperature, 18 h.

Scheme 5. Synthesis of Hydroxamic Acid Compounds

Reagents and conditions: (a) NaOH, EtOH/H₂O (4:1), rt., 18 h; (b) tert-butyl N-(2-aminoethyl)carbamate, EDC.HCl, HOBt, DIPEA, CH₂Cl₂, rt., 18 h; (c) 4 N HCl, EtOH, 0 °C to rt., 18 h; (d) azide-N-diazoimidazole-1-sulfonamide hydrogen sulfate, K₂CO₃, ZnCl₂, DIPEA, MeOH, rt., 18 h; (e) aq. hydroxylamine (50% in water w/w), KCN (cat.), MeOH, rt., 18 h; and (f) alkyne 34a–34d, prop-2-ynoxybenzene or prop-2-ynylsulfanylbenzene, CuSO₄ (5H₂O), NaAsc, N,N-dimethylformamide/H₂O (1.2:1), rt., 18 h.

Figure 3

Crystal structure of ColG-PD in complex with 13 solved at a resolution of 1.95 Å. Close-up view of the active site in ball-and-stick representation. The inhibitor (cyan) is shown in sticks with a polder map contoured at 2.5 σ above the background. The catalytic zinc ion (dark gray) and the calcium ion (green) are shown as spheres (PDB code: 7ZBV).

Figure 4

Crystal structure of ColG-PD in complex with 27 solved at 1.80 Å resolution. Close-up view of the active site in ball-and-stick representation. The inhibitor (cyan) is shown in sticks with the maximum likelihood weighted 2Fo–Fc electron density map contoured at 1σ. The catalytic zinc ion (dark gray) and the calcium ion (green) and water molecules (red) are shown as spheres (PDB code: 7Z5U).

Figure 5

Activity of ColQ1 inhibitors against the collagenolytic activity of the full-length (FL) ColQ1. Inhibitors prevented the cleavage of 1 mg/mL of Col I chains (i.e., β, α-1, and α-2). The Bacillus cereus ColQ1-FL (50 ng) was incubated with 1 mg/mL Col I for 3 h, and the degradation was then visualized by 12% SDS-PAGE. Col I: 1 mg/mL Col I without any protease. M (kDa): molecular weight standards, Col I: type I collagen, ColQ1: collagenase Q1.

Figure 6

Activities of FDA-approved tiludronate disodium and compound 14 on the fibroblast (NHDF) cells infected with Bacillus cereus. (a) The antigelatinolytic activities of compounds tiludronate disodium and 14 against B. cereus collagenases. The DMEM medium of the infected NHDF cells was applied to the zymograms. Clear regions against blue background indicate that gelatin in the gel has been cleaved. (b) The amount of fibrillar collagens maintained by tiludronate disodium and 14 in the infected NHDF cells (highlighted in the yellow background). (c) The cytotoxicity of B. cereus infection (highlighted in the yellow background) in NHDF cells treated with and without tiludronate disodium and 14. Ctrl represents the noninfected cells (gray column) and the infected cells and nontreated with inhibitors (red column). Statistical analysis was performed with one-way ANOVA, and statistical significance was analyzed by the Tukey test. Significance was calculated by comparing nontreated vs treated cells with compounds (mean ± SD, ****p < 0.0001, **p < 0.01, *p ≤ 0.05, ns: nonsignificant). Ctrl: control. M (kDa): molecular weight marker.

Figure 7

Change in the transepithelial electrical resistance (TEER) of the Madin–Darby canine kidney II (MDCK II) cells challenged with Bacillus cereus bacteria or 50% (v/v) culture supernatant and treated with or without collagenase inhibitors. (a) 14 and 15 preserve the TEER value of the MDCK II infected with B. cereus compared with the nontreated conditions with inhibitor. (b) Compounds 11, 13, 15, and tiludronate disodium maintained the TEER of MDCK II cells challenged with B. cereus supernatant. Each curve represents the average ± standard deviation of at least three independent experiments.

Figure 8

Survival analysis of Galleria mellonella larvae treated with Bacillus cereus AH187 with and without 14 and tiludronate disodium. Each curve represents results of three independent experiments; the statistical difference between groups treated with 100, 50, and 25 μM of compound 14 and B. cereus AH187 and with the group treated only with B. cereus AH187 is p < 0.0001, p = 0.0039, and p = 0.0173, respectively. The statistical difference between groups treated with 100 μM tiludronate disodium and with B. cereus AH187 is p = 0.0032 (log-rank test). The survival rate for the larvae treated with compound 14 and tiludronate disodium in PBS was 100%.