A screen for compounds that inhibit the bacterial siderophore biosynthetic enzyme BasE
Aryl acid adenylating enzymes (AAAEs) are involved in siderophore biosynthesis in Mycobacterium tuberculosis, Yersinia pestis, Escherichia coli, Vibrio cholerae, Bacillus subtilis, and Acinetobacter baumannii. The design and synthesis of a fluorescent probe Fl-Sal-AMS 6 based on the tight-binding inhibitor 5'-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS) enabled the performance of a 384-well more ..
BioActive Compounds: 94
Aryl acid adenylating enzymes (AAAEs) are involved in siderophore biosynthesis in Mycobacterium tuberculosis, Yersinia pestis, Escherichia coli, Vibrio cholerae, Bacillus subtilis, and Acinetobacter baumannii. The design and synthesis of a fluorescent probe Fl-Sal-AMS 6 based on the tight-binding inhibitor 5'-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS) enabled the performance of a 384-well format high-throughput screen against BasE, an AAAE from A. baumannii involved in production of the siderophore acinetobactin. Several small molecule inhibitors with new chemotypes were identified, and compound 23 containing a pyrazolo[5,4-a]pyridine scaffold emerged as the most promising ligand with a KD of 78 nM, which was independently confirmed by isothermal calorimetry. Inhibition was subsequently verified in an ATP-[32P]-pyrophosphate exchange steady-state kinetic assay.
The plasmid pCDD058 was transformed into BL21 STAR (DE3), which was grown in 500 mL of LB containing kanamycin (50 micrograms/mL) at 37 degrees C to an OD600 of 0.6. The culture was induced with 0.4 mM IPTG and grown an additional 4 h at 30 degrees C. Cells were pelleted from the culture media and resuspended in 30 mL lysis buffer (50 mM HEPES, 300 mM NaCl, 10 mM Imidazole pH 8.0) supplemented with 1 mg/mL lysozyme. After 30 min on ice, cells were disrupted using a Branson Sonifier 250 [30% duty for 2 min at each output intensity of 3, 4, and 5] and the lysate was cleared by centrifugation. 50% Ni-NTA (4.0 mL) was added to the cleared lysate and the mixture was incubated at 4 degrees C for 1 h. The Ni-NTA resin was collected in a gravity column and washed with 16 mL wash buffer (50 mM HEPES, 300 mM NaCl, 20 mM Imidazole pH 8.0). The protein was eluted from the resin using 3 mL elution buffer (50 mM HEPES, 300 mM NaCl, 250 mM Imidazole pH 8.0). The protein was then desalted into storage buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 5% glycerol) using a PD-10 column (GE Healthcare) yielding approximately 80 mg/L by Bradford assay using bovine serum albumin as a standard.
A master mix containing all assay components (30 mM Tris-HCl pH 7.5, 1 mM MgCl2, 1 mM TCEP, 0.0025% (w/v) Igepal CA-630, 200 nM BasE, 20 nM Fl-Sal-AMS) was prepared immediately before the assay. The master mix (30 microL) was dispensed to all wells in the first 23 columns of Corning 3575 plates, and the master mix (30 microL) containing 1 microM 2,3-DHB-AMS to all wells in column 24, using a Matrix Wellmate liquid dispenser. To the first 22 columns of the assay plate were transferred 100 nL of DMSO stock solutions of the compound libraries using a pin array transfer robot. The plates were shaken for 30 sec on a Lab-Line plate shaker and incubated for 3 h at 25 degrees C.
Fluorescence polarization was determined in a Perkin-Elmer Envision plate reader with excitation and emission wavelengths of 480 and 535 nm, respectively.
Fluorescence polarization (FP, in mP) was calculated using the following equation: FP = ((FIparallel - GIperpendicular)/(FIparallel + GIperpendicular)) x 1000. Results were normalized by calculating the normalized percent inhibition plate by plate, using the following equation: NPI = ((FPneg - FPexp)/(FPneg - FPpos)) x 100. Wells with NPI > 25% for both replicates were considered tentative positives and further evaluated by checking for excessively high or low fluorescence intensity (FI) when compared with the average FI for all wells in the plate. Tentative positives with FI deviations > 10% of the plate average FI for both replicates were excluded from further consideration (activity outcome = 1 if Normalized FI < 90 or > 110; activity outcome = 2 if Normalized FI >= 90 and <= 110). Activity scores were determined using average NPI. Average NPI <= 0 was scored as 0 for activity; average NPI >= 100 was scored as 100 for activity. Average NPI between 0 and 100 was used to generate activity scores for intermediate values.
Data Table (Concise)