Title:

Pyrrolamide DNA Gyrase Inhibitors: Fragment-based NMR Screening to Antibacterial Agents

Authors:

Ann E. Eakin, et al.

Supplemental Data Section

Production of recombinant DNA gyrase proteins

Construction of Expression Plasmids

Full length E. coligyrA (Accession Number P0AES4.1) and gyrB (Accession Number P0AES7.2) genes were cloned downstream of the tac promoter in pTTQ18 (7) vector to generate pZEN1477 (GyrA) and pZEN1840 (GyrB). E. coligyrB 24kD N-terminal domain (Accession number P0AES7.2, aa 1-220) was cloned downstream of the tac promoter in pTTQ18 to generate pZEN1697. S. aureusgyrB24kD N-terminal domain with additional flexible loop deletion (Accession number P0A0K8.2, aa14-104, 128-233) was cloned downstream of the T7 promoter in pZT7#3.3 (Pioli, D., Hockney, R. C., Kara, B. V., and Bundell, K. R. 4 February, 1999, international patent application WO1999005297) to generate pBA1037. DNA sequences of the cloned genes were confirmed by sequencing on an ABI PRISM 3100 DNA sequencer (Applied Biosystems, Carlsbad CA) using Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Carlsbad CA). Computer analyses of DNA sequences were performed with Sequencher (Gene Codes Corporation, Ann Arbor MI).

Recombinant protein overexpression

E. coli full length GyrA: pZEN1477 was transformed into E. coli JM109 (New England Biolabs, Ipswich, MA) and plated on LB agar containing 50 g/ml ampicillin. After overnight growth at 37oC, several transformants were inoculated into 3 liters of LB broth containing 50 g/ml ampicillin and grown at 37°C with aeration to mid-logarithmic phase (OD600 = 0.5) after which IPTG was added to a final concentration of 1 mM. After 3 hours at 37°C, the cells were harvested by centrifugation at 5,000 x g for 15 min at 25°C.

E. coli full length GyrB: pZEN1840 was transformed into E. coli BL21(DE3) (Novagen, Madison WI) and plated on LB agar containing 50 g/ml ampicillin. After overnight growth at 37oC, several transformants were inoculated into 3 liters of LB broth containing 50 g/ml ampicillin and grown at 37°C with aeration to mid-logarithmic phase (OD600 = 0.5) after which IPTG was added to a final concentration of 1 mM. After 2 hours at 30°C, the cells were harvested by centrifugation at 5,000 x g for 15 min at 25°C.

E. coli15N-labeled GyrB 24kDaN-terminal domain: pZEN1697 was transformed into E. coli BL21 (Novagen, Madison WI) and plated on LB agar containing 50 g/ml ampicillin and grown overnight at 37°C.Uniformly 15N-labeled protein was prepared by innoculating transformants from the above-described plate into M9 minimal media containing 50 g/ml ampicillin and supplemented with 1 g/L [15N] NH4Cl and 10 g/L glucose as the sole nitrogen and carbon sources. After growing at 37°C with aeration to mid-logarithmic phase (OD600 = 0.5), IPTG was added to a final concentration of 1 mM to induce expression. After 22 hours at 30°C, the cells were harvested by centrifugation at 5,000 x g for 15 min at 25°C.

S. aureusGyrB 24kDa N-terminal domain: pBA1037 was transformed into E. coli BL21(DE3) (Novagen, Madison WI) and plated on LB agar containing 10 g/ml tetracycline. After overnight growth at 37°C, several transformants were inoculated into 3 liters of LB broth containing 50 g/ml tetracycline and grown at 37°C with aeration to mid-logarithmic phase (OD600 = 0.5) after which IPTG was added to a final concentration of 1 mM. After 2 hours at 30°C, the cells were harvested by centrifugation at 5,000 x g for 15 min at 25°C.

Cell pastes were stored at –20°C and protein expression and solubility checked by SDS-PAGE.

Purification of full length of E. coli Gyrase Subunits (GyrA and GyrB)

Recombinant GyrA and GyrB proteins from E. coli were purified by similar methods, as described in the following. Frozen cell paste (from 6 L cell culture) was resuspended in 100 ml of Lysis Buffer [50 mM Tris/HCl, pH 7.5, 1 mM EDTA, 5 mM DTT, 10% glycerol, 25 mM NaCl, 1 mM PMSF, 2 Protease inhibitor cocktail tablets (Roche Diagnostics, Indianapolis, IN)]. The cells were disrupted by French press at 18,000 psi twice, and the crude extract was centrifuged at 25,000 rpm (45Ti rotor, Bechman) for 30 min at 4°C. The supernatant was loaded at a flow rate of 1.5 ml/min onto a 20 ml of Q-Sepharose HP (HR16/10) column (GE Healthcare Life Sciences) pre-equilibrated with Buffer A (50 mM Tris/HCl, pH 7.5, 1 mM EDTA, 5 mM DTT, 10% (v/v) glycerol, 25 mM NaCl). The column was then washed with Buffer A, and the protein was eluted by a linear gradient from 25 mM to 1 M NaCl in Buffer A. Fractions containing GyrA or GyrB were pooled, and dialyzed against 1 L of Buffer A overnight at 4°C. The dialyzed sample was loaded at a flow rate of 1.5 ml/min onto a 20 ml of Heparin Sepharose CL-6B (HR16/10) column (GE Healthcare Life Sciences) pre-equilibrated with Buffer A. After the column was washed with 100 ml of Buffer A, the protein was eluted by a linear gradient from 25 mM to 1 M NaCl in Buffer A. The fractions containing GyrA or GyrB were pooled and solid (NH4)2SO4 (0.4 g/ml) was added and mixed on ice for an hour. The sample was centrifuged at 11,000 rpm for 30 min at 4°C (JA12 rotor, Beckman), the pellet was then dissolved in 5 ml of Buffer A. The 5 ml sample was applied at a flow rate of 1.5 ml/min to a 320 ml Sephacryl S-200 (HR 26/60) (GE Healthcare Life Sciences) pre-equalibrated with Buffer B (50 mM Tris/HCl, pH 7.5, 1 mM EDTA, 5 mM DTT, 10% (v/v) glycerol, 150 mM NaCl). The fractions containing GyrA or GyrB were pooled and dialyzed against 1 L Storage buffer (50 mM Tris/HCl, pH 7.5, 1 mM EDTA, 100 mM KCl, 2 mM DTT, 20% (v/v) glycerol) overnight at 4ºC. The protein was characterized by SDS-PAGE analysis and judged to be at 95% purity. The protein was stored at –80°C.

Purification of GyrB 24kDa N-terminal domains from E. coli and S. aureus

Both N-terminal domain fragments were purified by similar methods described in the following. The frozen cell paste was suspended in 50 ml of Lysis Buffer [50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 mM DTT, 10% (v/v) glycerol, 1 mM PMSF, 1 Protease inhibitor cocktail tablet (Roche Diagnostics, Indianapolis, IN)]. Cells were disrupted by passing them twice through a French press operated at 18,000 psi, and the crude extract was centrifuged at 25,000 rpm (45Ti rotor, Beckman-Coulter, Brea, CA) for 30 min at 4ºC. The supernatant was loaded at a flow rate of 2.0 ml/min onto a 20 ml Q-Sepharose HP (HR16/10) column (GE Healthcare Life Sciences, Piscataway, NJ) pre-equilibrated with Buffer A [50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 mM DTT, 10% (v/v) glycerol]. The column was then washed with Buffer A, and the protein was eluted by a linear gradient from 0 to 1 M NaCl in Buffer A. Fractions containing GyrB24K were pooled, and 3 M (NH4)2SO4 in 50 mM Tris/HCl, pH 7.5, 1 mM EDTA, 2 mM DTT, 10% (v/v) glycerol was added to a final concentration of 1 M (NH4)2SO4. The sample was applied at a flow rate of 2.0 ml/min to a 20 ml Phenyl Sepharose HP (HR16/10) column (GE Healthcare Life Sciences, Piscataway, NJ) pre-equilibrated with Buffer B [50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 mM DTT, 10% (v/v) Glycerol, 1M (NH4)2SO4]. The column was washed with Buffer B, and the protein was eluted by a linear gradient from 1 to 0 M (NH4)2SO4 in Buffer A. Fractions containing GyrB24K were pooled, and solid (NH4)2SO4 (0.4 g/ml) was added to precipitate the proteins and mixed on ice for 1 hour. The sample was centrifuged at 25,000 rpm for 30 min at 4°C (45Ti rotor, Beckman-Coulter, Brea, CA), and the pellet was then dissolved in 5 ml of Buffer A. The 5 ml sample was applied at a flow rate of 1.5 ml/min to a 320 ml Sephacryl S-100 (HR 26/60) (GE Healthcare Life Sciences, Piscataway, NJ) pre-equalibrated with Buffer C [50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 mM DTT, 10% (v/v) glycerol, 150 mM NaCl]. The fractions containing GyrB24K were pooled and dialyzed against 1 L Storage Buffer [50 mM Tris/HCl, pH 7.5, 1 mM EDTA, 100 mM KCl, 2 mM DTT, 20% (v/v) glycerol]. The protein was characterized by SDS-PAGE analysis and analytical LC-MS. The extent of isotope labeling was checked for the 15N-labeled E. coli GyrB 24kDa N-terminal domain by comparing the observed mass (by mass spectroscopy) with that expected from the sequence. By this method, the protein was determined to be 98% labeled. Both proteins were stored at –80°C.

Gyrase ATPase assay

Unless otherwise specified, reagents and buffers were sourced from Sigma Aldrich Inc. (St. Louis, MO) and were of ACS reagent grade, or higher purity. All buffer solutions were prepared from high purity water (≥18 M•cm) obtained from a Milli-Q purifier (Millipore, Billerica, MA), to avoid adventitious metal cations and phosphate.

Gyrase was reconstituted from purified E. coli GyrA and GyrB stocks to a concentration of 1M each subunit in a solution containing 2 mg/mL salmon sperm DNA (Invitrogen, Carlsbad, CA), 50 mM HEPES/NaOH pH 7.5, 75 mM ammonium acetate and allowed to incubate at room temperature approximately 5 minutes. Enzyme was then diluted into enzyme working buffer containing all assay components (except the initiator, magnesium chloride) at double the final assay concentrations. The composition of enzyme working buffer contained 100 mM HEPES pH 7.5, 150 mM ammonium acetate, 500M ATP, 1.0 mM EDTA, 5% (w/v) glycerol, 400 nM BSA 0.01% Brij-35, 2 mM dithiothreitol, and reconstituted enzyme solution sufficient to yield 0.8 nM each GyrA and GyrB subunits. The initiator solution contained 16 mM magnesium chloride in aqueous 5% (w/v) glycerol.

Two-fold dilutions of compounds were made in neat DMSO at 50x the final assay concentration. Assays were conducted in 384-well polystyrene, untreated, Thermo/Matrix flat-bottomed plates (Fisher Scientific, Pittsburgh, PA). Assay wells received 0.6 L compound, followed by 15 L of enzyme solution in 2x final assay buffer, and reactions were initiated by the addition of 15 L of the magnesium initiator solution. Reactions were allowed to progress 18-24 hours at room temperature (approx. 22-24 °C) prior to quenching with 30 L of malachite green reagent to measure phosphate resulting from ATP hydrolysis. Five minutes after the quench reagent was added, absorbance at 650 nm (A650) was recorded.

Malachite green phosphate detection reagent was prepared as follows. Malachite green hydrochloride (1.35 g, Spectrum Chemical, Gardena, CA) was added to 3L of water, and stirred covered for 30 minutes. Separately, 42 g of ammonium molybdate tetrahydrate was added to 1L of 4 N HCl, stirred 30 minutes, until all solids dissolved. The ammonium molybdate solution was added to the malachite green solution with stirring over ~2 minutes, covered and let stir for an additional 30 minutes and then filtered (0.2 m PES filter). The final reagent remains stable for >2 months if stored in the dark at room temperature.

To calculate IC50 values, raw A650 nm was converted to percent inhibition calculated from the means of 32 wells each per assay plate of 0% inhibition (DMSO control) and 100% inhibition (2 M novobiocin control) wells. Curve fitting to determine IC50 values was performed using the equation %I = 100 / {1 + (IC50 / [I])n} as implemented by the XLfit software package (ID Business Solutions, Guildford, UK).

GyrB crystallography and structure refinement

Crystals of the S. aureus GyrB 24 kDa N-terminal loop-deletion domain with pyrrolamide 1 were grown using vapor diffusion methodologies. 10 nL of pyrrolamide 1 (100 mM in DMSO) was added to 0.5 mL protein (15 mg/ml in 20 mM Tris pH 8.0, 100 mM KCl, 2 mM DTT) prior to addition of 0.5 mL of well solution (19-30% Peg3350, 0.1M Hepes pH 7.5, 0.2M MgCl2). Crystals appeared within 1-2 days and grew to full size (~200 M) within 1 week after incubation at 20°C. Crystals were cryoprotected in a stepwise fashion with 5 minute incubations through solutions containing mother liquor first with 5% glycerol and then 10% glycerol prior to freezing in liquid nitrogen. Pyrrolamide 2 was soaked into crystals previously grown with pyrrolamide 1by adding 4 mM pyrrolamide 2 to the cryogen and incubating overnight in the final solution prior to harvesting. Data were collected on a FRe+ anode (Rigaku) using a Saturn 944 detector (Rigaku). Indexing and data processing were performed using the d*Trek program (6) or XDS (3) and Scala (2). Molecular replacement solutions were found using an unpublished structure of Sau GyrB N-terminal domain (data not shown) with the program AMoRe (5). After manual ligand placement, iterative cycles of refinement and model building were performed using Refmac5 (4) and COOT (1). A summary of data collection and refinement statistics are listed in Table S1.

Sequencing of DNA from Variant Strains Isolated in Spontaneous Resistance Experiments

Eight isolates from the spontaneous resistance studies which displayed elevated MIC values for pyrrolamide 4 were selected for DNA sequence analysis and comparison to DNA from the S. aureus parent strain. Genomic DNA was prepared using a standard genomic DNA preparation kit (Promega, Madison WI).For each of the isolates, the gyrB gene was amplified using a high-fidelity PCR mix (Roche, Nutley, NJ) and the SagB-Up and SagB-Down primers (MWG Biotech, Table S2). The polymerase chain reaction (PCR) product was purified using a QIAquick PCR Purification kit (Qiagen, Valencia, CA) and sequenced in an Applied Biosystems 3100 Genetic Analyzer (ABI, Foster City, CA) using the SagA through SagI primers (Table S2). If changes were observed, a second independent PCR product was amplified and sequenced to confirm the variation was genuine and not the result of an incorporation error during PCR amplification. Sequence data was analysed using Sequencher v.4.7 (GeneCodes, Ann Arbor, MI) and differences to the gyrB sequence from theS. aureus ARC516 parent strain recorded.The nucleotide and translated amino acid sequences from the region of the gyrB gene surrounding the observed changes between the parent strain and resistance variant strains are shown in Figure S1. The gyrA, parC and parE genes were sequenced using the same methodology (sequences not shown, since all were identical to sequences found in parent strain).

References for Supplemental Section

  1. Emsley, P., B. Lohkamp, W. G. Scott and K. Cowtan. 2010. Features and development of Coot. Acta Cryst. D66:486-501.
  2. Evans, P.R. 2005. Scaling and assessmentof data quality. Acta Cryst. D62:72-82.
  3. Kabsch, W. 2010. XDS. Acta Cryst. D66:125-132.
  4. Murshudov, G.N., A. A. Vagin and E. J. Dodson. 1997. Refinement of macromolecular structures by the maximum-likelihood method. Acta Cryst. D53:240-255.
  5. Navaza, J. 1994. AMoRe: an automated package for molecular replacement. Acta Cryst.A50:157-163.
  6. Pflugrath, J.W. 1999. The finer things in X-ray diffraction data collection. Acta Cryst.D55:1718-1725.
  7. Stark, M. J. R. 1987. Multicopy expression vectors carrying the fuc repressor gene for regulated high-level expression of genes in Escherichia coli. Gene51:255-267.

Figure Legend

Figure S1. Sequence Comparison of gyrB genes from parent and variant strains of S. aureus isolated in sponstaneous resistance studies with pyrrolamide 4. The nucleotide sequencesfor two regions of the gyrB gene are shown(lower case font) above the translated amino acid sequences (abbreviations in capitalized font) from the parent strain and two types of pyrrolamide 4-resistant isolates of S. aureus. Of the 8 resistant isolates sequenced, five displayed a single nucleotide change predicted to result in the Arg144Ile change and 3 displayed a different single nucleotide change predicted to result in the Thr173Ala change. The codons involved in the observed sequence differences between parent and resistant strains are indicated by boxes, with the specific nucleotide differences observed and predicted amino acid changes highlighted in bold, italic font.

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