Identification of Small Molecules Blocking the Pseudomonas aeruginosa Type III Secretion System Protein PcrV

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that employs its type III secretion system (T3SS) during the acute phase of infection to translocate cytotoxins into the host cell cytoplasm to evade the immune system. The PcrV protein is located at the tip of the T3SS, facilitates the integration of pore-forming proteins into the eukaryotic cell membrane, and is required for translocation of cytotoxins into the host cell. In this study, we used surface plasmon resonance screening to identify small molecule binders of PcrV. A follow-up structure-activity relationship analysis resulted in PcrV binders that protect macrophages in a P. aeruginosa cell-based infection assay. Treatment of P. aeruginosa infections is challenging due to acquired, intrinsic, and adaptive resistance in addition to a broad arsenal of virulence systems such as the T3SS. Virulence blocking molecules targeting PcrV constitute valuable starting points for development of next generation antibacterials to treat infections caused by P. aeruginosa.


General procedure for Williamson ether synthesis and ester hydrolysis:
To a solution of 4-bromo-2,3-dimethylphenol (584 mg, 2.7 mmol) anhydrous K2CO3 (2 equiv., 753 mg, 5.44 mmol) and KI (1.2 equiv., 542 mg, 3.2 mmol) in acetone (12 ml, 0.25 M of the phenol) was added ethyl 4-bromobutyrate (1.2-2 equiv. until completion according to TLC) the mixture was refluxed (not exceeding 80 °C) for 18-24 h and the reaction was monitored by TLC (EtOAc/Heptane 1:3) until the starting phenol was completely consumed. The solid material was filtered off, washed with acetone and the filtrate was concentrated under vacuum to afford the crude product, which was dissolved in THF/MeOH/H2O (3:1:1, 20 ml) in a microwave vial and LiOH.H2O (5 equiv., 571 mg, 13.6 mmol) was added. The mixture was heated in a microwave reactor for 15-30 min at 65 °C until completion according to TLC (EtOAc/Heptane 1:1, stained with bromocresol green). Volatile solvents were evaporated under vacuum and the remaining aqueous layer was acidified with HCl (6 M) to pH < 3 and the solid precipitate was collected and dried under vacuum to afford the product in 93% yield as white powder.
Method B -Exemplified by compound 9 3-Hydroxybenzotrifluoride (16.2 mg, 0.10 mmol), ethyl 4-bromobutyrate (23.4 mg, 0.12 mmol) and K2CO3 3-5 equiv was mixed in CH3CN (1 ml) and stirred in 80°C oil bath for 24 h and the reaction was monitored with LCMS. The reaction mixture was filtered to remove salt and CH3CN S15 was removed under vacuum. THF (0.4 ml), MeOH (0.4 ml) and aqueous NaOH solution (1 M, 0.4 ml) were added to the reaction tube. The resulting solution was stirred at room temperature for 3 h. The reaction was monitored with LCMS. The reaction mixture was neutralized with 0.4 ml of HCl solution (1 M). After removal of the organic solvent under vacuum, the product was extracted with CH2Cl2 (3 ml x 2) and dried over Na2SO4. The pure product was obtained as white solid after silica gel chromatography (Biotage 10 g, MeOH/CH2Cl2 5%) in 40% yield.

General procedure for amide coupling:
Method C -Exemplified by compound 20 Acid derivative (50 mg, 0.17 mmol) and TBTU (73 mg, 0.22 mmol, 1.3 equiv.) were dissolved in anhydrous DMF (0.85 ml, 0.2 M of the acid) before DIPEA (180 µl, 6 equiv.) was added, and the mixture was stirred at room temperature for 30 min. To the above mixture was added a solution of the HCl salt of amino acid ester (43 mg, 0.22 mmol, 1.3 equiv.) in anhydrous DMF (1.1 ml), and the reaction mixture was continued to stir at room temperature for 18 h. LCMS and TLC (CH2Cl2/MeOH 5%) showed full conversion of the starting material. The mixture was diluted with EtOAc and washed with HCl (1M), H2O and brine. The organic layer was dried over Na2SO4 and concentrated under vacuum. The crude was dissolved in THF/MeOH/H2O (3:1:1, 2 ml) and LiOH . H2O (36 mg, 0.87 mmol, 5 equiv.) was added and the mixture was stirred in microwave reactor at 65 °C for 15 min upon which LCMS showed full conversion to acid. The mixture was acidified with HCl (6 M) and concentrated under vacuum. The crude was dissolved in MeOH/DMSO (1:1) and purified by preparative HPLC using CH3CN/H2O (20→100%, with (A: 0.75% HCOOH in H2O, B: 0.75% HCOOH in CH3CN, 10→100% B over 20 min) to afford the product of interest in 56% yield over two steps.

4-(4-chlorophenoxy)butanoic acid -(3)
Synthesis: To a solution of sodium (429 mg, 18.6 mmol) in EtOH (18.6 ml, 1 M) was added p-chlorophenol (2 g, 15.5 mmol) and the reaction mixture was stirred for 5 min before adding γbutrylactone (1.43 ml, 18.6 mmol). The reaction mixture was stirred for 5 h at 100 °C after which time the solvent was slowly evaporated by continuous heating to 150 °C for additional 12 h. The remaining brownish solid was dissolved in H2O, followed by addition of HCl (1 M) until precipitation occurred. The precipitate was isolated by filtration, washed with water and dried under vacuum to yield the title compound in 74% yield as whitish-brownish solid .

Synthesis:
Step a: Phenyl iodide derivative (850 mg, 3.2 mmol), PdCl2(PPh3)2 (6 mol %) and CuI (3 mol %) was dissolved in anhydrous and degassed THF/TEA (1:3, 21 ml, 0.15 M). The mixture was degassed further by bubbling N2 and propargyl alcohol (3.0 equiv., 409 µl) was added under N2 atmosphere. The mixture was stirred at 40 °C in microwave reactor for 30 min upon which the reaction was completed according to TLC (EtOAc/Heptane 1:3, Rf = 0.1). The reaction mixture was diluted with EtOAc/MeOH 10%, filtered over a pad of Celite and concentrated under vacuum. The crude was loaded to silica gel and purified by chromatography using EtOAc/Heptane 10→33% linear gradient and product was isolated as yellow oil in quantitative yield.