Substrate‐Inspired Fragment Merging and Growing Affords Efficacious LasB Inhibitors

Abstract Extracellular virulence factors have emerged as attractive targets in the current antimicrobial resistance crisis. The Gram‐negative pathogen Pseudomonas aeruginosa secretes the virulence factor elastase B (LasB), which plays an important role in the infection process. Here, we report a sub‐micromolar, non‐peptidic, fragment‐like inhibitor of LasB discovered by careful visual inspection of structural data. Inspired by the natural LasB substrate, the original fragment was successfully merged and grown. The optimized inhibitor is accessible via simple chemistry and retained selectivity with a substantial improvement in activity, which can be rationalized by the crystal structure of LasB in complex with the inhibitor. We also demonstrate an improved in vivo efficacy of the optimized hit in Galleria mellonella larvae, highlighting the significance of this class of compounds as promising drug candidates.

on the applied treatment. Two negative control groups supplemented with no injection to control the quality of larvae and a buffer control group injected with sterile PBS were included.
A positive control group was also included, and the larvae were administered with 65% (v/v) PA14 supernatant. To test the anti-virulence effect of LasB inhibitors, a mixture of 65% (v/v) PA14 supernatant, LasB inhibitor and 300 μM TCEP were incubated at 37 °C for 30 min and injected into the larvae. All groups were incubated at 37 °C and inspected once per day for 4 days post-treatment and to record mortality. The larvae were considered dead if they are black and do not move when stimulated by contact with the forceps. The survival analysis was performed using GraphPad Prism v8, data were plotted using the Kaplan-Meier method, and statistical significance between groups was calculated with log-rank test.
General Chemistry. All reagents were used from commercial suppliers without further purification. Procedures were not optimized regarding yield. NMR spectra were recorded on a Bruker AV 500 (500 MHz) spectrometer at room temperature. Chemical shifts are given in parts per million (ppm) and referenced against the residual proton, 1 H, or carbon, 13 C, resonances of the >99% deuterated solvents as internal reference. Coupling constants (J) are given in Hertz (Hz). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet of doublets, dt =doublet of triplets, m = multiplet, br = broad and combinations of these) coupling constants and integration. Liquid chromatography-mass spectrometry (LC-MS) was performed on an LC-MS system, consisting of a DionexUltiMate 3000 pump, autosampler, column compartment, and detector (Thermo Fisher Scientific, Dreieich, Germany) and ESI quadrupole MS (MSQ Plus or ISQ EC, Thermo Fisher Scientific, Dreieich, Germany). High-resolution mass spectra were determined by LC-MS/MS using Thermo Scientific Q Exactive Focus Orbitrap LC-MS/MS system. Purity of the final S4 compounds was determined by LC-MS using the area percentage method on the UV trace recorded at a wavelength of 254 nm and found to be >95%. Figure S1. Comparison of compound 7g and compound 4 binding to LasB. Superposition of the LasB-Compound 4 (aqua; PDB 6f8b) and LasB-7g (slate) structures are shown. The movement of loop leading to closure of the binding pocket upon binding to 7g (light pink sticks) is highlighted by dotted arrows. For simplicity, only the major conformation of 7g observed in the crystal structure in shown. The color scheme used: 4 (magenta), 7g (light pink), Zn 2+ (gray) and Ca +2 (green).    Figure S4. Analysis of putative tunnel identified by CAVER in the structure of LasB in complex with compound 7g. A) LasB (slate) and the computed tunnel (grey) are shown as surface representation. B) Surface diagram showing the position of 7g in the tunnel. Total volume of the tunnel and 7g was calculated to be approximately 360 Å 3 and 86 Å 3 . Table S1. Data collection and refinement statistics.

Synthesis of intermediate and final compounds
General procedure i: Synthesis of chloro acid derivatives 4a-4c from the corresponding amino acid Amino acid (1.0 eq) was dissolved in 6 N HCl (2 mL/mmol or until mostly dissolved) under nitrogen atmosphere and cooled to -5 °C. NaNO2 (1.5-2.5 eq) was dissolved in water (0.3 mL/mmol amino acid) and added dropwise slowly. The mixture was stirred overnight while warming to r.t. The reaction mixture was extracted with EtOAc/THF (3:1). The combined organic extracts were washed with saturated aq. NaCl solution and dried over anh.
Na2SO4 and filtered. The solvent was removed under reduced pressure to afford the product.
The crude was used in the next step without further purification.
General procedure ii: Synthesis of derivatives 5a, 5d-5g The acid (1.0 eq), SOCl2 (2.0 eq) and a few drops of DMF were heated to 70 °C for 1 h. The cooled mixture was added dropwise to a solution of the corresponding aniline (1.1 eq) in DMF (1 mL/mmol) a cooled to 0 °C. The mixture was stirred at r.t overnight. The reaction was quenched with water and extracted with EtOAc (3x). The combined organic extracts were washed with saturated aq. NaCl solution, dried over anh. Na2SO4 and filtered. The solvent was removed under reduced pressure to afford the crude product. The purification was done by column chromatography or flash chromatography.

General procedure iii: Synthesis of derivatives 5b and 5c
2-Chloro-3-cyclohexylpropanoic acid or 2-chloro-3-cyclopropylpropanoic acid (1.2 eq) and EDC . HCl (1.2 eq) were added to a solution of the corresponding aniline (1.0 eq) in DCM. The resultant mixture was stirred at r.t. for 3-4 h. The reaction was monitored with TLC or LC-MS.
The solution was washed with 1 M HCl followed by saturated aqueous NaCl solution (1x) then dried over anh. Na2SO4. The organic phase was filtered and concentrated under reduced pressure to afford the crude product. The crude was used in the next step without further purification.

General procedure iv: Synthesis of thioacetate derivatives 6a-6g
The amide (1.0 eq) was dissolved in acetone under argon atmosphere. To this solution, CH3COSK (1.5-2.0 eq) was added, and the reaction was stirred at r.t. for 2-6 h. It was monitored by TLC or LC-MS. The reaction was quenched with water and extracted with EtOAc S13 (3x). The combined organic extracts were washed with saturated aq. NaCl solution (1x), dried over anh. Na2SO4 and filtered. The solvent was removed under reduced pressure to afford the crude product. The purification was done by flash chromatography.

General procedure v: Hydrolysis of thioacetate for derivatives 7a-7g
The thioacetate (1.0 eq) was dissolved in methanol (5 mL/mmol) under argon atmosphere and 2 M aqueous NaOH solution (2.0 eq) or solid NaOH (3.0 eq) was added. The reaction was stirred at r.t. for 1-3 h before quenching with 1 M or 2 M HCl. The reaction was extracted with EtOAc and washed with 0.5 M HCl. The combined organic extracts were washed with saturated aqueous NaCl solution (1x) and then dried over anh. Na2SO4. The solvent was removed under reduced pressure to afford the crude product. The purification was done by column chromatography or preparative HPLC (H2O+0.05%FA/ACN+0.05%FA 95:5 → 5:95).

S15
Compound 5e was prepared according to general procedure ii, using compound 4a (259 mg,