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Discovery of ML 267 as a Novel Inhibitor of Pathogenic Sfp phosphopantetheinyl transferase (PPTase)

, , , , , , , and .

Author Information

,a ,a ,a ,a ,b ,a ,a and a.*

a NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD, 20850.
b Department of Chemistry & Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093.
* To whom correspondence should be addressed: Email: vog.hin.liam@dyenolam

Received: ; Last Update: September 3, 2013.

Since the dawn of the antibiotic era 70 years ago, the evolution of drug resistance has been a persistently evolving threat. This can only be combated by the continued development of new therapies that address resistance mechanisms or engage novel cellular targets. Toward this end, phosphopantetheinyl transferase (PPTase) has been highlighted as a potential target for antibacterial development due to its role in the activation of fatty acid synthase, as well as a myriad of virulence factor-producing machinery. The necessity of this locus to bacterial homeostasis has been confirmed by genetic knockout, and it is intriguing to consider that inhibition of this enzyme may both thwart proliferation and render the bacterium avirulent. However, no chemical matter is currently known that exhibits potent inhibitory activity with this enzyme. To further evaluate the therapeutic potential of PPTase as target for the development of a new class of antibacterial agents, we conducted a quantitative high throughput screening campaign and subsequent medicinal chemistry optimization in pursuit of small molecules that inhibit surfactin PPTase (Sfp). Herein, we detail the discovery of ML267, a probe molecule that has been optimized for nanomolar antagonistic activity with this target. ML267 possesses antibiotic activity against representative Gram Positive bacteria, including Community-Acquired Methicillin Resistant Staphylococcus aureus. Moreover, ML267 was profiled for cytoxtoxity (HepG2), acute toxicity (in vivo), and promiscuity against other thiol-sensitive assays (human GST A1-1), and was found to be devoid of activity in these studies. Finally, ML267 possesses a promising in vitro ADME and in vivo PK profile, suggesting its suitability for further testing in animal models of bacterial infection and virulence.

Assigned Assay Grant #: MH083266

Screening Center Name & PI: NIH Chemical Genomics Center, Christopher P. Austin

Chemistry Center Name & PI: NIH Chemical Genomics Center, Christopher P. Austin

Assay Submitter & Institution: Michael D. Burkart, Department of Chemistry & Biochemistry, University of California, San Diego

PubChem Summary Bioassay Identifier (AID): 1819

Probe Structure & Characteristics

Image ml267fu1
CID/ML#Target NameIC50/EC50 (nM) [SID, AID]Anti-target Name[1]IC50/EC50 (μM) [SID, AID]Fold SelectiveSecondary Assay[1] Name: IC50/EC50 (μM) [SID, AID]
CID 53257126/ML267Sfp-PPTaseqHTS: 290 nM IC50 [SID 124398570, AID 602370]Cytotoxicity (HepG2 Cells)>57 μM IC50 [SID 124398570, AID 602373]>196 foldGel assay: 2.14 μM IC50 [SID 124398570, AID 602362]

1. Recommendations for Scientific Use of the Probe

This compound can be used to assess the ability of Sfp-PPTase inhibitors to enhance the yield of synthase enzymes from bacterial protein extracts through a site-specific phosphopantetheinylation approach. Additionally, this compound enables the evaluation of this enzyme as a target for therapeutic development through in vitro and in vivo studies. This compound may also prove useful when used in combination with known anti-infective compounds that target bacterial fatty acid production and potentiate their effects.

2. Materials and Methods

General Methods for Chemistry. All air or moisture sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Anhydrous solvents such as dichloromethane, N,N-dimethylforamide (DMF), acetonitrile, methanol and triethylamine were purchased from Sigma-Aldrich. Preparative purification was performed on a Waters semi-preparative HPLC system. The column used was a Phenomenex Luna C18 (5 micron, 30 × 75 mm) at a flow rate of 45 mL/min. The mobile phase consisted of acetonitrile and water (each containing 0.1% trifluoroacetic acid). A gradient of 10% to 50% acetonitrile over 8 minutes was used during the purification. Fraction collection was triggered by UV detection (220 nm). Analytical analysis was performed on an Agilent LC/MS (Agilent Technologies, Santa Clara, CA). Method 1: A 7 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8 minute run time at a flow rate of 1 mL/min. A Phenomenex Luna C18 column (3 micron, 3 × 75 mm) was used at a temperature of 50° C. Method 2: A 3 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with a 4.5 minute run time at a flow rate of 1 mL/min. A Phenomenex Gemini Phenyl column (3 micron, 3 × 100 mm) was used at a temperature of 50° C. Purity determination was performed using an Agilent Diode Array Detector for both Method 1 and Method 2. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode. 1H NMR spectra were recorded on Varian 400 MHz spectrometers. Chemical shifts are reported in ppm with undeuterated solvent (DMSO-d6 at 2.49 ppm) as internal standard for DMSO-d6 solutions. All of the analogs tested in the biological assays have purity greater than 95%, based on both analytical methods. High resolution mass spectrometry was recorded on Agilent 6210 Time-of-Flight LC/MS system. Confirmation of molecular formula was accomplished using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02).

2.1. Assays

2.1.1. Sfp- and AcpS-PPTase qHTS assays

The assay was performed in 50 mM HEPES-Na pH 7.6, 10 mM MgCl2, 0.01% Nonidet P-40, and 0.01% BSA. Three μL of reagents, consisting of buffer (in columns 3 and 4 as negative control) and Sfp- or AcpS-PPTase (in columns 1, 2, 5 – 48, final concentration of 15 nM or 100 nM, respectively) were dispensed into a 1,536-well Greiner black solid bottom plate (see Table 1 for protocol steps). Compounds (23 nL) were transferred via Kalypsys pintool equipped with a 1,536-pin array. The plate was incubated for 15 min at room temperature, followed by the addition of 1 μL substrate (final concentrations for rhodamine-CoA and BHQ-2-YbbR 5 μM and 12.5 μM, respectively) to start the reaction. The plate was then centrifuged at 1,000 rpm for 15 seconds, and the fluorescence intensity was recorded on a ViewLux High-throughput CCD imager (Perkin-Elmer) using standard BODIPY optics (525 nm excitation and 598 nm emission). The plate was then incubated for 30 or 60 minutes (Sfp or AcpS, respectively), and a second read on the ViewLux was performed. The fluorescence intensity difference over the 30- or 60-minute period (Sfp or AcpS, respectively) was used to calculate the respective reaction rate for each well. All screening operations were performed on a fully integrated robotic system (Kalypsys Inc., San Diego, CA) as described elsewhere. Plates containing DMSO only (instead of compound solutions) were included approximately every 50 plates throughout the screen to monitor any systematic trend in the assay signal associated with reagent dispenser variation or decrease in enzyme specific activity. PubChem AID 602370, AID 602360.

Table 1. Sfp-PPTase qHTS assay protocol.

Table 1

Sfp-PPTase qHTS assay protocol.

2.1.2. PPTase gel assay

A DMSO solution of confirmed hits (0.5 μL) was added to a 1.33X enzyme solution (15 μL, containing 26.6 nM Sfp, 66 mM HEPES•Na, 13.3 mM MgCl2, 0.0133% NP-40, 0.133% BSA, pH 7.6). After a 10 minute incubation, the enzymatic reaction was initiated by the addition of 4 X substrate solution (4 μL, containing 50 μM Rhodamine CoA and 50 μM apo-Actinorhodin-ACP). The reactions were terminated after a 30 minute incubation at room temperature by the addition of 2X quench solution (20 μL, containing 4 M Urea, 25 mM EDTA, 0.004% phenol red, pH 8.0).

Samples were separated under native conditions on a 20% polyacrylamide gel using standard Laemmli conditions. Following the run, gels were imaged with a Chemi-Doc Plus imager (Bio-Rad, Hercules, CA) and band intensity quantified using the ImageJ software package. Pixel density values were normalized to control wells and fit with the 4-parameter Hill equation using in-house tools. PubChem AID: 602362.

2.1.3. Label-free gel assay for phosphopantetheinylation

Test compound (0.5 μL) dissolved in DMSO was added to 1.33X enzyme solution (15 μL) containing 66 nM Sfp, 66 mM HEPES•Na pH 7.6, 13.3 mM MgCl2, 0.0133% NP40, and 1.33 mg/mL BSA. These solutions were incubated at room temperature for 10 minutes, at which point the phosphopantetheinylation reaction was initiated by the addition of 5X substrate solution (4 μL) containing 50 μM apo-ACP and 50 μM coenzyme A in 10 mM HEPES•Na pH 7.6. After incubation at room temperature for 30 minutes, quench/load solution (5 μL) containing 50 mM EDTA pH 8.0, 50% glycerol and 0.005% phenol red was added. The samples were electrophoretically separated on a discontinuous polyacrylamide gel using the Laemmli buffers sans SDS, with urea (2 M final) included in the resolving gel (15% total acrylamide/bisacrylamide concentration).

After separation, the gels were fixed (50% MeOH, 7% AcOH, 30 min), washed thrice with deionized water (200 mL, 5 min per wash), and stained with Sypro Ruby® according to the manufacturer’s recommendations. Imaging was accomplished with a Bio-Rad ChemiDocTM XRS Gel Imager using standard ethidium bromide settings. Protein bands were quantified via densitometry using the ImageJ software package.

2.1.4. Human GST A1-1 counterscreen assay

Reaction buffer consisted of 50 mM HEPES pH 7.5 with 0.01% Tween 20. 3 μL of solution containing 6.67 nM human glutathione-S-transferase (hGST) A1-1 (columns 1 – 2, 5 to 48, final assay concentration 5 nM) or buffer (columns 3 and 4, positive control) with 133 μM glutathione (GSH, final assay concentration 100 μM) were dispensed into a 1,536-well Greiner black solid bottom plate. Test compounds (46 nL) and control compound (Reactive Blue 2) were transferred via Kalypsys pintool and incubated for 15 minutes. Next, 1 μL of substrate PBI 3773 at 40 μM (final assay concentration 10 μM) was delivered to all wells, centrifuged at 1,000 rpm for 15 seconds, and incubated for 40 minutes, followed by the addition of 4 μL of Luciferase Detection Reagent (Promega). After an additional 15 minute incubation, the plate was read in Luminescence mode on a ViewLux High-throughput CCD imager. [1]

2.1.5. Hepg2 cytotoxicity counterscreen

Test compounds’ toxicity was assessed by measuring cellular ATP content using a luciferase-coupled ATP quantitation assay (CellTiter-Glo; Promega, Madison, WI). In this assay, luminescent signal is proportional to amount of ATP, and thus to the number of metabolically competent cells. Briefly, Hepg2 cells were dispensed at 2,000 cells/5 μL/well in tissue-culture treated 1,536-well white/solid bottom assay plates (Greiner Bio-One North America, Monroe, NC) using a Flying Reagent Dispenser (Aurora Discovery, Carlsbad, CA). Cells were incubated at 37° C for 6 hr to allow for cell attachment, followed by addition of compounds via pin tool (Kalypsys, San Diego, CA). After compound addition, plates were incubated for 48 hr at 37° C. At the end of the incubation period, 5 μL of CellTiter-Glo reagent was added, plates were incubated at room temperature in the dark for 30 min, and the luminescence intensity of each well was determined using a ViewLux plate reader (PerkinElmer, Shelton, CT). The positive control was 92 μM and 41 μM of tetra-N-Octylammonium bromide, and the negative control was DMSO.

2.1.6. Microbial susceptibility testing

Innoculum preparation. Bacillus subtilis strains were maintained on lysogeny broth (LB) solidified by the addition of 1.5% w/v agar. Single colonies were used to inoculate cation-adjusted mueller hinton II broth (2 mL) and shaken overnight at 30 ºC. In the morning, this culture (100 μL) was used to seed a fresh LB medium (10 mL) and was shaken at 30 ºC until the culture OD600 reached 0.5. This culture was diluted 1:100 in fresh cation-adjusted mueller hinton II broth to provide the inoculum below.

Bacterial susceptibility. Cation-adjusted mueller hinton II broth (2 μL) was dispensed into wells of a sterile white 1536-well plate. Test compounds (23 nL) prepared as serial dilutions in DMSO were added to the plate by pintool transfer. Innoculum (2 μL) was added; the plates were covered with a vented Kalypsys assay lid and incubated at 30 ºC. After 5 h, Bac-Titer Glo (4 μL; Promega Corp, Madison, WI) was added to the plates. They were incubated 10 minutes at room temperature, and then the luminescence was detected in a ViewLux multimodal plate reader.

2.1.7. Reversibility Experiment

A DMSO solution of test compounds dissolved in DMSO at a concentration 500 times their respective IC50 (1 μL) were added to a 1.66 μM solution of Sfp (49 μL, dissolved in assay buffer (66 mM HEPES-Na, 13 mM MgCl2, 0.13% BSA, 0.013% NP40) and equilibrated at room temperature for 30 minutes. This solution was then serial diluted 10-fold twice in assay buffer containing 2% DMSO. The resulting solution (37.5 μL, 100-fold total dilution) was added to individual wells of a 384-well plate. Assay reactions were immediately initiated by the addition of a 4-X substrate solution (12.5 μL, containing 50 μM FiTC-YbbR peptide and 100 μM Rhodamine CoA dissolved in 10 mM HEPES-Na, pH 7.5), and the reaction time course was monitored in an Envision multilabel plate reader using the standard fluorescein settings.

2.1.8. Membrane damage assessment assay

Assessments of membrane activity were performed in B. subtilis HM489 similarly to the protocol of Singh [2], using the BacLight nucleic acid staining system (Introgen Corp, Calsbad, CA, USA). The organism was maintained on LB medium solidified by the addition of bactoagar to 1.5% w/v, and all liquid culturing was performed in 250 mL baffled glass fernbach flasks containing 25 mL of LB medium. Bacterial cultures were grown from single colonies in LB medium at 30º C with shaking at 300 rpm. When the culture reached an OD of 0.5, the flask was chilled on wet ice for 10 min, and then the cells collected by centrifugation at 10,000 × g for 5 min. The cells were resuspended in sterile 0.85% NaCl (4 mL) and split into two portions (2 mL each); one received ethanol to 70% v/v and was set aside with intermittent shaking for 1 h, while the other was concentrated by centrifugation at 10,000 × g for 5 min. The cells were resuspended gently in 0.85% NaCl and adjusted to an OD value of 0.20. Cell suspension (50 μL) was added to wells of a 96-well plate, followed by DMSO solution of test compounds (1 μL), followed by another portion of cell suspension (49 μL). Then the plate was covered and incubated 30 minutes at room temperature.

During the incubation, cells killed in 70% EtOH were collected by centrifugation (5 min at 10,000 × g), washed twice with 0.85% NaCl, and diluted with 0.85% NaCl to an OD of 0.20. This preparation was then used, in combination with live cell solution, to prepare solutions varying in the ratio of live/dead cells to construct a standard curve.

After the test compound incubation was complete, 100 μL of a 1 X BacLight solution (18 μL each of the Syto9 and propidium iodide solutions per 10 mL deionized H2O) was added to the bacterial suspensions, and the cells were allowed to stain 15 minutes at room temperature. The plate was then read directly in an envision plate reader with 485 nm excitation filter, FITC dichotic mirror module, and emission at both 535 and 615 nm.

2.1.9. HPLC Assay

Enzyme Reaction. Enzyme reactions were conducted in a 25 μL total assay volume in 384-well Greiner polypropylene plates. DMSO solution of test compound (0.5 μL) was added to a 1.25X solution of Sfp (20 μL, 37.5 nM) prepared in 1.25X HPLC assay buffer (62.5 mM MES-Na, 12.5 mM MgCl2, 0.0125% NP40, pH 6.0). After a 10 minute incubation at room temperature, reaction was initiated by the addition of 5.5X substrate solution (containing variable concentrations of CoA and ActACP in 10 mM HEPES-Na, pH 7.5). Following a 30 minute incubation, reactions were quenched by the addition of 50 mM EDTA, pH 8.0 (25 μL), and the plate was heat sealed.

HPLC Separation. Portions of the quenched enzyme reactions (25 μL) were analyzed with an Agilent 1200 instrument fitted with a multiwell plate-compatible injector and diode array detector using a Jupiter C4 column (part number 00B-416-60, 50 × 2 mm length containing 5 μm particles, 300 Å pores, Phenomonex, Torrance, CA, USA), using a constant flow rate of 0.4 mL/min. Mobile phases (Buffer A: H2O with 0.1% v/v TFA; Buffer B: 95% acetonitrile with 0.1% v/v TFA) were prepared from HPLC grade solvents. The separation was accomplished as follows: after equilibration with 5% Buffer B, the samples were injected, and the mobile phase was ramped up to 50% Buffer B over 0.5 minutes, followed by an isocratic flow at this composition for 1.5 minutes. Separation of the apo- and holo- forms of ActACP was then achieved with a linear gradient from 50% to 70% Buffer B over 2 minutes. The mobile phase was then ramped to 95% Buffer B in 0.5 minutes, the column was washed by a 1 minute isocratic flow, and then adjusted to starting conditions with a 0.5 minute linear gradient to 5% Buffer B. Effective binding of the next sample required the inclusion of an end-run equilibration under this condition for 2 minutes. With this program, the two forms of the protein exhibited retention times of 4.7 and 5.3 minutes for the holo- and apo- forms of ActACP, respectively, with a typical baseline resolution between the peaks of 0.3 minutes. The absorbance trace recorded at 210 nm was baseline corrected, integrated, and the peak areas for holo-ActACP were normalized to positive and negative controls.

2.1.10. Minimum Inhibitory Concentration Determination

Methods for MIC determination were made in accordance with standards put forth by the National Clinical Laboratory Standards Institute detailed in documents M07-A8 [3] and M27-A2 [4], for bacterial and fungal species, respectively. Briefly, organisms were maintained on solid medium. Innoculum was prepared from overnight liquid cultures of bacteria or by suspending 2-day old colonies in RPMI 1640 medium. Test articles dissolved in DMSO (1 μL) were added to sterile medium [50 μL, cation-adjusted mueller hinton II broth for bacteria (BD BBL, Franklin Lakes, NJ) or RMPI 1640 for fungi (Invitrogen Corporation, Carlsbad, CA)] followed by innoculum prepared in the same medium (50 μL, containing ~ 1 × 103 cfu) and incubated at 30–37º C for 16–20 h (48 h for fungi). Plates were visually inspected for microbial growth, and the first well containing no visible microbial growth was scored as the MIC. Strains evaluated in this manner included Bacillus subtilis 168, Bacillus subtilis HM489 [5], Escherichia coli K12, Pseudomonas aeruginosa ATCC 9028, Staphylococcus aureus ATCC 6538 (methicillin sensitive), Staphylococcus aureus ATCC BAA-1717 (community-acquired methicillin resistant strain USA300-HOU-MR), Candida albicans ATCC 90028 (fluconazole sensitive), and Candida albicans ATCC 96901 (fluconazole resistant).

2.2. Probe Chemical Characterization

Probe Characterization of ML267.

Probe Characterization of ML267

4-(3-chloro-5-(trifluoromethyl)pyridin-2-yl)-N-(4-methoxypyridin-2-yl)piperazine-1-carbothioamide: LC-MS Retention Time: t1 (Method 1) = 4.883 min and t2 (Method 2) = 3.036 min; 1H NMR (400 MHz, DMSO-d6) δ 3.58 – 3.67 (m, 4 H), 3.88 (s, 3 H), 4.05 – 4.13 (m, 4 H), 6.83 (s, 1 H), 7.15 (s, 1 H), 8.16 – 8.26 (m, 2 H) and 8.56 – 8.61 (m, 1 H); HRMS (ESI) m/z (M+H)+ calcd. for C17H18ClF3N5OS, 432.0867; found 432.0856.

Internal IDMLS IDSIDCIDML #TypeSource
NCGC00244182MLS00387405912439857053257126ML267ProbeNCGC
NCGC00242296MLS00337060410422479249853301AnalogNCGC
NCGC00242279MLS00337060310422477349852967AnalogNCGC
NCGC00242267MLS00337060510422475949853368AnalogNCGC
NCGC00238589MLS00337060710422446049852823AnalogNCGC
NCGC00241077MLS00337060810422456849852959AnalogNCGC
Figure 1. List and structures of probe and related analogs that have been submitted to the MLSMR.

Figure 1List and structures of probe and related analogs that have been submitted to the MLSMR

Figure 2. Stability of ML267 in DPBS (pH 7.

Figure 2

Stability of ML267 in DPBS (pH 7.4) buffer (Panel A) and PPTase buffer at room temperature over 48 h (Panel B). Stability of ML267 at pH 2 (Panel C) and pH 9 at room temperature over 4 h (Panel D)

2.3. Probe Preparation

Preparation of 4-(3-chloro-5-(trifluoromethyl)pyridin-2-yl)-N-(4-methoxypyridin-2-yl)piperazine-1-carbothioamide (ML267) as depicted in scheme 1

Scheme 1. Synthetic route to ML267.

Scheme 1

Synthetic route to ML267.

A mixture of 4-methoxypyridin-2-amine (0.124 g, 1.0 mmol) and 1,1′-thiocarbonyldiimidazole (0.187 g, 1.05 mmol) in dichloromethane (2 mL) was stirred for 15 min at room temperature. 1-(3-chloro-5-(trifluoromethyl)pyridin-2-yl)piperazine (0.292 g, 1.1 mmol) was added to the clear yellow solution, and the reaction mixture was stirred at 40° C for 1 h. The solvent was evaporated and the crude product was taken up in 2 mL DMSO and purified via reverse phase chromatography to give 4-(3-chloro-5-(trifluoromethyl)pyridin-2-yl)-N-(4-methoxypyridin-2-yl)piperazine-1-carbothioamide, TFA (ML267) as a white solid.

General procedures for the preparation of aryl piperazines as depicted in scheme 2

Scheme 2. General procedures for the preparation of aryl piperazines.

Scheme 2

General procedures for the preparation of aryl piperazines.

Procedure A: A mixture of arylboronic acid (3.2 mmol, 2 eq), copper (II) acetate (0.029 g, 0.16 mmol, 10 mol%) and 4 Å molecular sieves (0.2 g) in dichloromethane (4 mL) was stirred for 5 minutes at room temperature. t-Butyl piperazine-1-carboxylate (0.3 g, 1.61 mmol, 1 eq) was then added, followed by bubbling with oxygen for 5 minutes. The vial was then sealed and filled with oxygen. The mixture was stirred overnight at 45° C. After completion of the reaction, the reaction mixture was filtered through a metal scavenger cartridge (2 times to remove the copper completely). The crude products were then deprotected with TFA/dichloromethane, and the products were either purified by HPLC or used directly in the next step.

Procedure B: A mixture of Aryl halide (1 mmol, 1 eq), t-Butyl piperazine-1-carboxylate (1.2–1.5 eq), a base (Cs2CO3 or sodium tert-butoxide, 1.5 eq), a ligand (XantPhos or BINAP or JohnPhos or XPhos, 5 mol%) and Pd source [Pd(OAc)2 or Pd2(dba)3, 2.5 – 5 mol%] in toluene (2 mL) was bubbled with argon for 5 minutes. The vial was capped and stirred at 80 – 110° C for 2–12 h. After completion of the reaction, the solvent was evaporated. The crude solid was dissolved in methanol/methylene chloride and stirred with a metal scavenger and filtered through celite. The crude products were purified on via biotage® flash chromatography or reversed-phase HPLC and then deprotected with TFA.

Procedure C: A mixture of Aryl iodide (1 mmol, 1 eq), t-Butyl piperazine-1-carboxylate (1.2–1.5 eq), Cs2CO3 or potassium phosphate (2 mmol, 2 eq), a ligand (2-isobutyrylcyclohexanone or racemic BINOL, 20 mol%) and copper [1] iodide (10–20 mol%) in DMF (2 mL) was bubbled with argon for 5 minutes. The vial was capped and stirred at room temperature or at 70 ºC for 2–12 h. After completion of the reaction, the product was extracted with ethyl acetate. The organic layer was washed with 10% HCl, water and brine. The crude solid was then dissolved in methanol/methylene chloride and stirred with a metal scavenger, filtered through celite and purified via biotage® flash chromatography or reversed-phase HPLC, and then deprotected with TFA.

3. Results

3.1. Dose Response Curves for Probe

Figure 3. Dose response of Sfp-PPTase activity (●) and the HepG2 cytotoxicity counterscreen [1].

Figure 3Dose response of Sfp-PPTase activity (●) and the HepG2 cytotoxicity counterscreen [1]

3.2. Cellular Activity

Figure 4. In vitro cellular activity of 1 (CID 4566836) and ML267 against Bacillus subtilis HM489(●), and HepG2 human hepatocarcinoma [1] cell lines after 48 hr incubation.

Figure 4In vitro cellular activity of 1 (CID 4566836) and ML267 against Bacillus subtilis HM489(●), and HepG2 human hepatocarcinoma [1] cell lines after 48 hr incubation

3.3. Profiling Assays

Figure 5. Inhibitory activity of ML267 (●) and 1, CID: 4566836 [1] against the human glutathions S-transferase isoform A1-1 (hGST A1-1).

Figure 5Inhibitory activity of ML267 (●) and 1, CID: 4566836 [1] against the human glutathions S-transferase isoform A1-1 (hGST A1-1)

Table 2ADME profile for ML267 (27)

All experiments were conducted at Pharmaron Inc.

CompoundPBS buffer (pH 7.4) Solubility (μM)Mouse Liver Microsome Stability (T1/2)aCYP 2D6 % Inhibition @ 3μMbCYP 3A4 % Inhibition @ 3μMcPBS buffer (pH 7.4) Stability % remaining at 48 hoursMouse Plasma Stability % remaining at 2 hours
ML2672049.5 min2520100%100%
a

represents the stability in the presence of NADPH. The probe compound showed no degradation without NADPH present over a 1 hr period.

b

dextromethorphan was used as the substrate.

c

6β-hydroxytestosterone was used as the substrate.

Table 3In vivo PK (mouse) at 30 mpk IP and 3 mpk IV

All experiments were conducted at Pharmaron Inc. using male CD1 mice (6–8 weeks of age). Data was collected in triplicate at 8 time points over a 24 h period.

CompoundRouteaT1/2[1]Cmax (ng/mL)AUCinf (h*ng/mL)Vd (L/kg)MRTb[1]%FCl (mL/min/kg)P/B Ratioc
ML267IV2.4N/A69831.53.4NA7.22.5
ML267IP2.01763368860NANA98.5NA2.1
a

Both formulated as a solution (5% DMSO and 10% Solutol in H2O). IV: Dosed at 3 mpk; IP: Dosed at 30 mpk.

b

Mean residence time (the time for elimination of 63.2% of the IV dose).

c

Plasma to brain ratio [AUClast(plasma)/AUClast(brain)].

4. Discussion

4.1. Comparison to Existing Art and How the New Probe is an Improvement

Comparison of ML267 to Prior Art

Table 4Comparison of ML267 to Wyeth Compound 16, an optimized lead described by Wyeth Research

CompoundBiochemical IC50 (μM)Minimum inhibitory concentration (μg/mL)
SfpAcpSB. subtilis HM489B. subtilis 168S. aureus MSS. aureus MRE. coliP. aeruginosaC. albicans FSC. albicans FR
ML2670.2906.930.851.76.83.4a>100>100>100>100
Wyeth compound 16NDb
>114c
1.4b
>114C
ND64 bND64 b>128 bND>128bND
a

ND: Not Determined, Potency confirmed by independent laboratory: NYU Anti-infectives Screening Core, NYU Lagnone Medical Center, New York, New York.

b

Values reported in [6].

c

Values determined at NCGC.

Very few PPTase inhibitors have been described to date. The primary series arising from the private sector constitutes a collection of anthranilates reported by Wyeth Research, and were developed during a campaign targeting AcpS-PPTase. None of these compounds possess remarkable in vitro antibacterial activity, a result that likely led to termination of the project. The top compound arising from this work is Wyeth Compound 16, and the performance of this compound benchmarks the state of the art to which ML267 may be compared. To assert due diligence, we prepared an independent sample of Wyeth compound 16 to directly qualify its performance in a head-to-head fashion for the biochemical experiments. Chemical characterization of the resynthesized version of the Wyeth compound, including LC-MS assessment of purity, were completed inhouse. Evaluation of this compound in the primary screening assay surprisingly demonstrated null activity with both Sfp and AcpS-PPTase (data not shown). When this compound was evaluated in microbial susceptibility experiments, modest antibacterial activity was observed, with IC50 values of 42 and 49 μM in B. subtilis strains HM489 and 168 (data not shown). The latter strain was utilized to allow a direct comparison of our observations to those reported by Wyeth Research, and confirmed that these microbial susceptibility experiments provide a potency estimation not grossly skewed from the MIC reported in the literature. The inability to reproduce the biochemical activity of the substance raises questions regarding the biological results reported in [6], however further investigation of the subject was not warranted at this time.

Given the benefit of the doubt, benchmark values for potency in biochemical and biological assays to which ML267 should be held are referenced from their disclosure [6]. In this manner, probe ML267 significantly improves the state of the art for the field of PPTase biology, and provides a first in class compound for the Sfp-subtype denotation of this enzyme class. With regard to potency at the directed target, we find that ML267 shows a 5-fold increase over Wyeth Compound 16, displaying an IC50 in the primary screen of 290 nM, with additional coverage of AcpS-PPTase at a potency of 6.93 μM. Furthermore, even greater improvements were seen in MIC experiments, where ML267 a remarkable 38-fold improvement in potency in the common laboratory strain B. subtilis 168, a result that was further enhanced in the genetically modified test strain B. subtilis HM489, whose viability is solely dependent upon an active Sfp gene product.

With respect to MIC activity in human pathogens, we observed noteworthy inhibition of Gram positive representative strains of S. aureus, with an 18-fold increase over Wyeth compound 16 in a methicillin resistant strain, with an observed MIC of 3.4 μg/mL. In an act of further attentiveness, this activity was confirmed by an external contract laboratory specializing in anti-infective screening, a result we found particularly motivating. Testing in both Gram negative and fungal pathogens showed that our spectrum of activity was not enveloping with regard to microbial life, a result that is considered acceptable at this stage of an anti-infective discovery campaign [7]. While these organisms are expected to also depend on PPTase activity for viability, this limited scope of activity may be the result of genetic variability between PPTase loci, the reduced penetrability of the Gram-negative outer membrane (a formidable barrier) [8], or increased efflux capacities in fungal pathogens [9].

In summary, the in vitro antimicrobial profile of ML267, provides the research community with a unique tool that may be leveraged to 1.) evaluate the role of Sfp-PPTase in bacterial cell viability, 2.) determine the potential of Sfp-PPTase as a drug target in the case of Gram positive bacterial infections, and 3.) assess the role that polyketide, nonribosomal peptide, and fatty acid synthases play in virulence biology. The favorable potency observed against CA-MRSA in in vitro antimicrobial studies, coupled to the favorable in vitro and in vivo ADME and PK properties provide a glimmer of hope that a new class of antibiotic compounds acting through novel mechanism of action may soon be within reach.

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Footnotes

*

Purity >95% as determined by LC/MS and 1H NMR analyses.

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