The Rational Design, Synthesis, and Antimicrobial Properties of Thiophene Derivatives That Inhibit Bacterial Histidine Kinases

Abstract

The emergence of multidrug-resistant bacteria emphasizes the urgent need for novel antibacterial compounds targeting unique cellular processes. Two-component signal transduction systems (TCSs) are commonly used by bacteria to couple environmental stimuli to adaptive responses, are absent in mammals, and are embedded in various pathogenic pathways. To attenuate these signaling pathways, we aimed to target the TCS signal transducer histidine kinase (HK) by focusing on their highly conserved adenosine triphosphate-binding domain. We used a structure-based drug design strategy that begins from an inhibitor-bound crystal structure and includes a significant number of structurally simplifiying "intuitive" modifications to arrive at the simple achiral, biaryl target structures. Thus, ligands were designed, leading to a series of thiophene derivatives. These compounds were synthesized and evaluated in vitro against bacterial HKs. We identified eight compounds with significant inhibitory activities against these proteins, two of which exhibited broad-spectrum antimicrobial activity. The compounds were also evaluated as adjuvants for the treatment of resistant bacteria. One compound was found to restore the sensivity of these bacteria to the respective antibiotics.

Figure 1

The two-component system signaling (TCS) cascade. A phosphoryl group is transferred from the Catalytic domain (CA) to a conserved His-residue of the histidine kinase and from there at a conserved sp-residue of response regulator (RR). A typical function for the RR is gene regulation.

Figure 2

A: X-ray crystal structure of the ATP-binding domain of WalK (PDB: 3SL2). B: Binding mode of ATP with ATP-lid loop.

Figure 3

Superposition of Histidine kinase in green with ATP in pink and Gyrase B in blue with its pyrrolamide inhibitor in gold. B: Evaluation of the histidine kinase pyrrolamide complex with DSX. Blue spheres represents favorable interactions where as red spheres represents bad interactions. Blue lines represent favorable distances whereas red lines represent bad distances. C: Zoom of the pyrrolamide ligand inside the Histidine domain of WalK. D: Pyrrolamide inhibitor.

Figure 4

Design Strategy

Figure 5

Chemical library designed.

Figure 6

A: Binding pose of compound 6a into the ATP-binding site of WalK. B: Evaluation of compound 6a docked into ATP-Binding site using PLANTS with DSX. Blue spheres represents favorable interactions whereas red spheres represents bad interactions. Blue lines represent favorable distances whereas red lines represent bad distances.

Figure 7

A screen with cytoplasmic WalK fragments identifies eight compounds with kinase inhibitory activity. Two Bacillus subtilis cytoplasmic histidine kinases constructs, WalK^272-612 and WalK^204-612, were subjected to autophosphorylation in the presence of 1 mMγ-³²P-labeled ATP and in the presence or absence (control) of 1 mM of putative kinase inhibitors. Autophosphorylation of WalK kinase was terminated after 3 minutes by addition of SDS-sample buffer and the reactions were subjected to SDS-PAGE followed by phosphor-imaging. Bands on the gel indicate the extent of the autophosphorylation reaction. Compounds, whose presence caused visual reduction in autophosphorylation activity are indicated by an arrow and were selected for further analysis. (A) Compounds used were 6g, 6h, 6b, 7a, 6c, 6a, 6j, and 6k; top panel WalK^204-612; bottom panel WalK^272-612. (B) Compounds used were 6t, 6s, 6d, 6e, 6f, 6m, 6o, and 6u; top panel WalK^204-612; bottom Panel WalK^272-612. (C) Compounds used were 7c, 6i, 6l, 6n, 7b, 6p, 6q, and 6r; top panel WalK^204-612; bottom panel WalK^272-612.

Figure 8

Compound 6e inhibits autophosphorylation of histidine kinases PhoR, ResE and WalK but not serine/threonine kinase IreK or DNA-gyrase. Autophosphorylation of kinases and DNA gyrase-catalyzed supercoiling were performed as described in materials and methods. In contrast to Figure 1, protein concentrations, ATP concentrations and time points were adjusted for IC₅₀ determination. (A) Autophosphorylation activity of cytoplasmic constructs of PhoR (top panel), ResE (middle panel) and WalK (bottom panel) in the presence of increasing amounts of compound 6e (0; 6.25; 12.5; 25; 50; 100; 200; 400; 800 μM). The autophosphorylation activity was monitored after 60, 8 and 40 min respectively. These time points were chosen imperially to reflect a linear rate of autophosphorylation for each kinase. (B) The serine/threonine kinase IreK from E. faecalis was subjected to autophosphorylation in analogy to the assays performed for histidine kinases with increasing amounts of compound 6e (0; 6.25; 12.5; 25; 50; 100; 200; 400; and 800 μM). The autophosphorylation activity was monitored after 15 min. (C) E. coli DNA gyrase-catalyzed supercoiling in the presence and absence of indicated concentrations of compound 6e was monitored. Ciprofloxacin (200 nM) was used as a positive control for gyrase inhibition. Each reaction contained 1U gyrase and 75ng gyrase substrate according to manufacturer recommendation.

Figure 9

The histidine kinase inhibitors 6d and 6e exhibit antimicrobial activity (A, B). The Kirby-Bauer disc diffusion susceptibility assay was used to determine the antimicrobial activity of the selected compounds (see material and methods). (A) Antimicrobial activity screen of the eight selected compounds against methicillin resistant S. aureus (MRSA). A disk containing 30μg vancomycin was used as control (c). 100μg of compounds 6h (1), 6c (2), 6k (3), 6s (4), 6d (5), 6e (6), 7c (7) and 6i (8) was applied to each disk. (B) Dose-dependent antimicrobial activities of the compounds 6d and 6e against MRSA. 30μg vancomycin was used as control (c). Decreasing amounts of 6d (left) and 6e (right). [1 and 7 = 500 μg; 2 and 8 = 100 μg; 3 and 9 = 20 μg; 4 and 10 = 4 μg; 5 and 11 = 0.8 μg ; 6 and 12 = 0.16 μg] (C) Effect of compounds 6d and 6e on exponentially growing B. subtilis cultures was monitored. To determine whether compounds 6d and 6e exhibit bactericidal or bacteriostatic activity B. subtilis JH642 was grown in LB medium at 37 °C. Vancomycin (Va) and chloramphenicol (Cm) served as controls. Optical density (OD) at a wavelength of 600 nm was routinely measured. At an OD₆₀₀ of 0.5-0.6 (indicated by an arrow) 50μg compounds 6d (squares), 6e (triangles facing up), chloramphenicol (triangles facing down) or vancomycin (diamonds) was added. B. subtilis JH642 was also grown without additives (circles).

Scheme 1

Synthesis of Thiophene derivatives 6a-u^{a a}Reagents and conditions: (a) Pd(PPh₃)₄ or PdCl₂(PPh₃)₂, Na₂CO₃ or K₂CO₃ or Cs₂CO₃, Toluene/EtOH or 1,4-dioxane/H₂O or DMF/H₂O or DMF, 70-90°C.

Scheme 2

Synthesis of acetamide 3^{a a}Reagents and conditions: (a) NH₂OH-HCl, CH₃COONa-3H₂O; (b) TsCl, NaOH.

Scheme 3

Synthesis of Thiophene derivatives 7a-c^{a a}Reagents and conditions: (a) I(CH₂)₂OH, 90°C, N₂.

Scheme 4

Synthesis of amine hydrochloride salts 8a-i^{a a}Reagents and conditions: (a) HCl 12M, THF, 0°C or HCl generated in situ (Addition of concd H₂SO₄ on NaCl)