Protein expression and purification
The gene coding for the E. coli DnaK substrate-binding (β) domain (393–507) was amplified by PCR and subcloned into pET21a using the NdeI and BamHI cloning sites. The resulting protein contains 17 extra amino acid residues (MGSSHHHHHHGLVPRGS) at the N-terminus. The protein was expressed in the E. coli strain BL21(DE3) pLysS and purified using Ni2 + affinity chromatography. Uniformly, 15N-labeled DnaK was produced by growing the bacteria in M9 minimal media containing 15NH4Cl as the sole nitrogen source. The NMR samples were dissolved in 20 mM sodium phosphate buffer (pH 7.5) containing 90%/10% (H2O/D2O) or 99.5% D2O. DnaK, human Hsp70, DnaJ, and GrpE were purchased from Stressgen (Ann Arbor, MI, USA).
Apidaecin1a (GKPRPYSPRPTSHPRPIRV), pyrrhocoricin (VDKGSYLPRP-TPPRPIYNRN), drosocin (GNNRPVYIPGPRPPHPRI), and the model substrate NRLLLTG were synthesized by the Medical College of Wisconsin (Milwaukee, WI, USA).
Spectra were acquired on a 600-MHz Bruker Avance spectrometer (Bruker BioSpin Corp., Billerica, MA, USA) equipped with a TCI cryoprobe. Ligand binding was monitored by comparing the aliphatic region of 1D 13C-filtered 1H NMR spectra of 20 μM protein solutions (containing 90%/10% H2O/D2O or 99.5% D2O buffered with 20 mM sodium phosphate, pH 7.5; T = 300 K) in the presence or absence of 80 μM mixtures of 10 compounds, and then individual compounds from mixtures that caused significant perturbations in the spectra were identified.
The binding affinity of each ligand was initially estimated with a single titration point according to the following equation:
are total concentration of target protein and ligand, respectively. The parameter p
represents the fractional population of bound versus free species at equilibrium, which for fast exchanging ligands is measured as:
is the observed protein chemical shift during the titration, and δfree
are the chemical shifts for the protein in the fully unbound and fully bound (saturated) states, respectively.
Isothermal titration calorimetry
Titrations were performed using a VP-ITC instrument from MicroCal (Northampton, MA, USA). Full-length DnaK or β-domain were used at 10–100 μM in 20 mM sodium phosphate buffer (pH 7.4) and 5% dimethyl sulphoxide (DMSO). Compounds were used at 15-fold excess in the same buffer. Data were analyzed using MicroCal Origin software provided by the ITC manufacturer (MicroCal, Northampton, MA, USA).
Minimum inhibitory concentration measurements
cultures were seeded in 96-well plates by mixing 90 μ
L aliquots of an OD600
= 0.001 culture with 10 μ
L of compound solutions at various concentrations and grown overnight. Optical density (OD) at 530 nm was measured using a Perkin Elmer Victor 2 Multilabel plate reader (PerkinElmer, Shelton, CT, USA). Testing was carried out in 50% Mueller-Hinton II broth (BBL) in H2
O. Wild-type Yersinia pseudotuberculosis
) was grown overnight at 25 °C and approximately 106
bacteria were inoculated into each 1 mL of broth containing serial dilutions of the compound to be tested. After overnight incubation at 40 °C, the OD was read at 600 nm and compared with the growth without inhibitor. The minimal inhibitory concentration (MIC) was read as the concentration producing greater than fourfold reduction in final OD600
To a stirred solution of free amine (1.0 equiv.) and Et3N (2.0 equiv.) in 5 mL dichloromethane (DCM) was added a solution of thiophene-2-carbonyl chloride (1.1 equiv.) in 5 mL DCM at −30 °C (). The resulting solution was stirred for 1 h and then allowed to warm to room temperature. After removal of the solvent, the residue was purified by flash column chromatography in hexane-ethyl acetate or DCM-methanol to provide the correspondent product (yield 75–95%). Synthesis scale was 100 mg, for which about 0.5 mmol of starting material was required.
Preparation of the thiophene-2-carbonyl amide derivatives.
N-(naphthalen-1-ylmethyl)thiophene-2-carboxamide 1H NMR (CDCl3, 600 MHz): δ 8.17 (s, 1H), 7.87 (s, 1H), 7.73 (s, 1H), 7.46 (m, 6H), 6.99 (s, 1H), 6.39 (s, 1H), 5.01 (s, 2H); HRESI-TOF-MS: calcd for C16H13NOS 268.0791 [M + H]+, found 268.0796.
N-(1H-indol-5-yl)thiophene-2-carboxamide 1H NMR (CDCl3, 600 MHz): δ 8.19 (s, 1H), 7.92 (s, 1H), 7.71 (s, 1H), 7.62 (s, 1H), 7.52 (s, 1H), 7.33 (m, 2H), 7.23 (s, 1H), 7.12 (s, 1H), 6.54 (s, 1H); HRESI-TOF-MS: calcd for C13H10N2OS 243.0587 [M + H]+, found 243.0592.
N-(quinazolin-2-yl)thiophene-2-carboxamide 1H NMR (CDCl3, 600 MHz): δ 9.35 (s, 1H), 7.90 (m, 3H), 7.61 (m, 4H), 6.89 (s, 1H); HRESI-TOF-MS: calcd for C13H9N3OS 256.0539 [M + H]+, found 256.0545.
N-(benzo[b]thiophen-7-ylmethyl)thiophene-2-carboxamide 1H NMR (CDCl3, 600 MHz): δ 8.35 (s, 1H), 7.82 (m, 5H), 7.47 (s, 1H), 6.97 (s, 1H), 5.30 (s, 2H); HRESI-TOF-MS: calcd for C14H11NOS2 274.0355 [M + H]+, found 274.0361.
N-(3,4-dichlorobenzyl)thiophene-2-carboxamide 1H NMR (DMSO-d6, 600 MHz): δ 9.11 (s, 1H), 7.79 (m, 2H), 7.60 (d, J = 7.2 Hz, 1H), 7.56 (s, 1H), 7.30 (d, J = 7.2 Hz, 1H), 7.16 (s, 1H), 4.43 (s, 2H); HRESI-TOF-MS: calcd for C12H9Cl2NOS 285.9855 [M + H]+, found 285.9861.
N-(2,6-dichlorobenzyl)thiophene-2-carboxamide 1H NMR (DMSO-d6, 600 MHz): δ 9.11 (s, 1H), 7.79 (m, 2H), 7.51 (s, 1H), 7.35 (m, 2H), 7.17 (s, 1H), 4.44 (s, 2H); HRESI-TOF-MS: calcd for C12H9Cl2NOS 285.9855 [M + H]+, found 285.9860.
N-(2-hydroxybenzyl)thiophene-2-carboxamide 1H NMR (DMSO-d6, 600 MHz): δ 8.96 (s, 1H), 8.09 (s, 1H), 8.03 (s, 1H), 7.78 (s, 1H), 7.39 (m, 2H), 7.28 (m, 2H), 7.14 (s, 1H), 4.42 (s, 2H); HRESI-TOF-MS: calcd for C12H11NO2S 232.0438 [M − H]−, found 232.0440.
N-(3,5-dichlorobenzyl)thiophene-2-carboxamide 1H NMR (DMSO-d6, 600 MHz): δ 8.67 (s, 1H), 7.79 (s, 1H), 7.75 (s, 1H), 7.51 (m, 2H), 7.38 (s, 1H), 7.11 (s, 1H), 4.66 (s, 2H); HRESI-TOF-MS: calcd for C12H9Cl2NOS 285.9855 [M + H]+, found 285.9862.
(4-(2-hydroxyethyl)piperazin-1-yl)(thiophen-2-yl)methanone 1H NMR (DMSO-d6, 600 MHz): δ 7.75 (s, 1H), 7.38 (s, 1H), 7.11 (s, 1H), 3.61 (m, 4H), 3.51 (m, 2H), 2.42 (m, 6H); HRESI-TOF-MS: calcd for C11H16N2O2S 241.1005 [M + H]+, found 241.1012. Commercially available BI-88B12 analogs were purchased from Chembridge Corp., (San Diego, CA, USA). Structures for purchased and synthesized compounds are shown in .
SBD or FL following values in the ITC column indicates to which protein the compound was titrated