Evaluation of Expanded 2-Aminobenzothiazole Library for Inhibition of Pseudomonas aeruginosa Virulence Phenotypes

Abstract

Bacterial resistance to antibiotics is a rapidly increasing threat to human health. New strategies to combat resistant organisms are desperately needed. One potential avenue is targeting two-component systems, which are the main bacterial signal transduction pathways used to regulate development, metabolism, virulence, and antibiotic resistance. These systems consist of a homodimeric membrane-bound sensor histidine kinase, and a cognate effector, the response regulator. The high sequence conservation in the catalytic and adenosine triphosphate-binding (CA) domain of histidine kinases and their essential role in bacterial signal transduction could enable broad-spectrum antibacterial activity. Through this signal transduction, histidine kinases regulate multiple virulence mechanisms including toxin production, immune evasion, and antibiotic resistance. Targeting virulence, as opposed to development of bactericidal compounds, could reduce evolutionary pressure for acquired resistance. Additionally, compounds targeting the CA domain have the potential to impair multiple two-component systems that regulate virulence in one or more pathogens. We conducted structure-activity relationship studies of 2-aminobenzothiazole-based inhibitors designed to target the CA domain of histidine kinases. We found these compounds have anti-virulence activities in Pseudomonas aeruginosa, reducing motility phenotypes and toxin production associated with the pathogenic functions of this bacterium.

Figure 1.. Histidine kinase structure and roles in P. aeruginosa virulence.

A) Generic structure and domain organization of a TCS. HKs contain a periplasmic sensory domain (maroon) with a transmembrane domain (navy blue) to connect the cytosolic kinase domains comprised of the dimerization and histidine phosphorylation (DHp) domain with the catalytic histidine residue (orange) and the catalytic ATP-binding (CA) domain (teal). HKs generally have a cognate response regulator (purple) containing a conserved aspartate residue where phosphorylation occurs. B) CA domain, homology boxes that represent highly conserved sequence motifs. N-box (yellow), F-box (purple), G1-Box (orange), G2-box (green), G3-box (pink). C) Examples of HKs implicated in pyocyanin production and swarming motility in P. aeruginosa. CheA (yellow), a non-traditional cytosolic HK, receives signals through methyl-accepting chemotaxis proteins (MCPs).

Figure 2.. Predicted binding motifs of 2-aminobenzothiazole molecules.

A) Numbering of 2-aminobenzothiazole inhibitors and hydrogen bond donor-acceptor-donor motif that is crucial for binding to key residues within the CA-domain binding pocket. Docked binding poses of Rilu-1 (B) and Rilu-2 (C) with HK853 (PDB: 3DGE). Both interact with highly-conserved Asp411 and have π-π stacking interactions with conserved Tyr384. The larger group off of the six-position in Rilu-2 occupies more space within the pocket. Red indicates oxygen, blue indicates nitrogen, yellow indicates sulfur, and gray indicates carbon for defined atoms.

Figure 3.. Data obtained for binding by ligand- and protein-detection NMR.

A. Binding evaluated by ligand detection using ¹H and ¹⁹F DLB, T₂-CPMG and STD. 1D ¹H and 2D HSQC were used to evaluate binding by protein detection. The symbol (√) is used when binding is observed by the selected method. N/A indicates that the experiment was not performed. B. Binding of Rilu-1 to HK853 (residues 232–489). Differential line broadening and shifting (DLBS) and T₂-CPMG experiments show the aromatic region of 1D ¹H NMR spectra of 300 μM of Rilu-1 in its free state (blue trace) and in presence of 15 μM of HK (red trace). STD indicates the ON and OFF resonance spectra of 300 μM of Rilu-1 in presence of 15 μM of HK (blue and red traces, respectively), the differential spectra (STD diff) are represented in green. ¹⁹F Differential line broadening experiment shows ¹⁹F NMR spectra of 300 μM of Rilu-1 in its free state (blue trace) and in presence of 15 μM of HK (red trace).

Figure 4.. Competition studies with ADP for binding to HK by ¹⁹F NMR.

Titration experiments performed by 1D ¹⁹F NMR with Rilu-1 (A) and C-14 (B) for binding to HK (232–489) in the absence and in presence of ADP. For each titration, a 1D ¹⁹F spectrum of the compound was acquired free in buffer (blue), in presence of HK protein (red), and with increasing molar equivalent ratios of ADP (green to purple to light blue). The free and bound states of both compounds are identified on the spectra.

Figure 5.. Swarming motifs of PA14 strain on modified fastidious anaerobe broth (FAB) agar.

Comparison of DMSO vehicle-control to plates contain 100 μM of Rilu-2, 6 or 7. Rilu-2 resulted in a substantial decrease in the swarming capabilities of the bacteria, but did not prevent growth. Plates were imaged using a Typhoon FLA 9500 scanner (GE healthcare) on DY-520XL filter setting. Images were redshifted to improve contrast of bacteria tendrils from agar plate and analyzed using ImageJ (NIH).

Scheme 1:. Synthesis of selected analogue set.

A. i. NBS, CH₂Cl₂ ii. Benzoyl isothiocyanate, acetone iii. NaOH, H₂O, CH₃OH iv. NaH, DMF B. i. NaIO₄, RuCl₃, H₃CCN, CCl₄, H₂O 20 °C ii. CF₃CO₂H, CH₂Cl₂, 20 °C C. i. KSCN, Br₂, AcOH D. i. Pd₂(dpa)₃, Xantphos, Cs₂CO₃, dioxane ii. Fe, NH₄Cl, CH₃CH₂OH, H₂O, 80 °C iii. KSCN, AcOH, Br₂ iv. Boc₂O, Et₃N, CH₂Cl₂ v. NaIO₄, RuCl₃, H₃CCN, CCl₄, H₂O 25 °C vi. CF₃CO₂H, CH₂Cl₂ vii. CH₃I, NaH, DMF. viii. Oxone, THF/H₂O, 0–25 °C ix. H₂, Pd/C, NH₃^·H₂O, CH₃OH, 25 °C E. i. NBS, DMF ii. NaH, SEMCl, DMF iii. Tert-butyl-N-(4-sulfanylphenyl)carbamate, Pd₂(dpa)₃, Xantphos, Cs₂CO₃, dioxane iv. Lutidine, TMSOTf, CH₂Cl₂ v. KSCN, Br₂, AcOH vi. Raney Ni, H₂, CH₃OH vii. oxone, H₃CCN, H₂O viii. CF₃CO₂H, CH₂Cl₂ F. i. NaH, SEMCl, THF ii. 4-Nitrobenzenethiol, Pd₂(dpa)₃, Xantphos, Cs₂CO₃, dioxane iii. Fe, NH₄Cl, CH₃CH₂OH, H₂O, 80 °C iv. KSCN, Br₂, AcOH v. Boc₂O, Et₃N, CH₂Cl₂ vi. NaIO₄, RuCl₃, H₃CCN, CCl₄, H₂O vii. CF₃CO₂H CH₂Cl₂ G. i. 4-Nitrobenzenethiol, CuI, Cs₂CO₃, DMF, 110 °C ii. Fe, NH₄Cl, CH₃CH₂OH, H₂O, 80 °C iii. KSCN, Br₂, AcOH iv. Oxone, THF/H₂O H. i. 4-Nitrobenzenethiol, K₂CO₃, DMF, 80 °C ii. Fe, NH₄Cl, CH₃CH₂OH, H₂O, 80 °C iii. KSCN, Br₂, AcOH iv. Boc₂O, N(CH₂CH₃)₃, C₄H₈O v. NaIO₄, RuCl₃, H₃CCN, CCl₄, H₂O, 25 °C vi. CF₃CO₂H CH₂Cl₂, 20 °C.