Molecular mutagenesis of ppGpp: turning a RelA activator into an inhibitor

The alarmone nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance and virulence, making (p)ppGpp-mediated signaling a promising target for development of antibacterials. Although ppGpp itself is an activator of the ribosome-associated ppGpp synthetase RelA, several ppGpp mimics have been developed as RelA inhibitors. However promising, the currently available ppGpp mimics are relatively inefficient, with IC50 in the sub-mM range. In an attempt to identify a potent and specific inhibitor of RelA capable of abrogating (p)ppGpp production in live bacterial cells, we have tested a targeted nucleotide library using a biochemical test system comprised of purified Escherichia coli components. While none of the compounds fulfilled this aim, the screen has yielded several potentially useful molecular tools for biochemical and structural work.


Multiple round in vitro transcription assay
The assays were performed as per Bernardo et al. (2006) 4 with minor modifications. Reactions were carried out in T-buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 0.1 mM EDTA and 0.275 mg of BSA per ml) at 30°C. Inhibitors were titrated to final concentrations of 0, 0.025, 0.05, 0.15, 0.4 and 0.6 mM. E. coli s 70 -RNAP holoenzyme (Epicentre, final concentration 5 nM) and template plasmid DNA containing s 70 -rrnB P1 promoter, (pRLG6214, final concentration 0.5 nM) was used for all experiments. Before initiation of transcription, E. coli s 70 -RNAP holoenzyme and T buffer were incubated at 30°C for 5 min. Above mix was added to predispensed mixture of inhibitor and the template plasmid DNA containing s 70 -rrnB P1 promoter (pRLG6214), incubated for further 20 min to initiate open complex formation. 2.5 µl of a mixture of ATP (final concentration 0.5 mM), GTP and CTP (final concentration 0.2 mM each), UTP (final concentration 0.08 mM) and [a-32 P]-UTP (5 µCi at > 3000 Ci/mmol, PerkinElmer) was added to initiate transcription by RNAP. After 7 min, 1 µl heparin (0.125 mg/ml final concentration) was added to the reaction mixture to prevent re-initiation and further incubated for 5 min to allow completion of initiated transcripts. Transcription was terminated by addition of formamide loading buffer and samples were electrophoresed on a 7M urea-5% polyacrylamide gel and quantified by phosphorimaging.
Growth assays with Bacillus subtilis BSB1 wild type strain SAS deletion and ppGpp 0 strain of B. subtilis 5 were grown in S7 liquid medium 6 except for amino acid requirements, which were met as described elsewhere 7 . On 96-well plate, 90 µl of B subtilis starter culture was mixed with 10 µL of compound dilution (in 5 mM Tris pH 7.2) so that final OD600 was 0.025 (l = 1 cm). Following incubation was without shaking at 37°C and growth was monitored with Tecan Infinite M200 at OD600 (raw OD600 values are reported). B. subtilis starter culture was prepared as follows. Late exponential phase cells in S7, with full set of amino acids, were diluted ≈10-fold in S7 lacking either valine or lysine so that, after addition of 8% DMSO, OD600 was 0.25. Next, cell suspensions were aliquoted, frozen in liquid nitrogen, and stored at -80°C. Just before the experiment, cells were thawed on ice and appropriate amount of media was added.

General
Unless stated otherwise, all used solvents were anhydrous. TLC was performed on silica gel pre-coated aluminium plates Silica gel/TLC-cards, UV 254 (Fluka), and compounds were detected by UV light (254 nm), by heating (detection of dimethoxytrityl group; orange color), by spraying with 1% solution of ninhydrine to visualize amines, and by spraying with 1% solution of 4-(4nitrobenzyl)pyridine in ethanol followed by heating and treating with gaseous ammonia (blue color of mono-and diesters of phosphonic acid). Preparative column chromatography was carried out on silica gel (40-60µm; Fluka), and elution was performed at the flow rate of 40 ml/min. The following solvent systems were used for TLC and preparative chromatography: toluene-ethyl acetate 1:1 (T); chloroform-ethanol 9:1 (C1); ethyl acetate-acetone-ethanol- Thio-ppGpp (entry 3) was synthesized enzymatically following the same procedure as used for ppGpp using 6-thio-GDP (Jena) as a substrate.  The monosubstituted intermediate (0.58 g, 0.67 mmol) was dissolved in the mixture of EtOH (25 ml) and water (25 ml), NaHCO3 (0.23 g, 2.72 mmol) and Pd/C (0.2 g) were added. The mixture was then hydrogenated overnight at the pressure of 100 kPa. After filtration over cellite the mixture was concentrated in vacuo and purified ousing preparative HPLC on reversed phase using linear gradient of MeOH in 0.1M aq. TEAB. The final deprotection was performed according to literature procedure 9 (overnight treatment with conc. aq. NH3 followed by treatment with 0.1M aq. HCl). After preparative HPLC on reversed phase using linear gradient of MeOH in 0.1M aq. TEAB and converting to ammonium salt by passing through a column of Dowex 50 in ammonia form desired product was obtained in 43% overall yield (149 mg, 0.29 mmol). 1

Scheme 6
Sodium hydride (270 mg, 6.72 mmol) was added to the solution of 2-N-isobutyryl-2'-O-tetrahydropyranylguanosine (0.37 g, 0.85 mmol) and dibenzyl tosyloxymethanephosphonate (1.86 g, 4.14 mmol) in DMF (10 ml) at rt under argon atmosphere. The reaction mixture was stirring overnight. The reaction mixture was cooled to 0 °C and acetic acid (0.38 ml, 6.72 mmol) was added. The reaction mixture was stirred additional 10 min and concentrated in vacuo. Tetrabenzyl ester intermediate was obtained by column chromatography on silica gel using linear gradient of ethanol in chloroform and without characterization dissolved in EtOH (10 ml). Pd/C (50 mg) was added and the reaction mixture was hydrogenated at 1 atm of H2 overnight. The suspension was filtered, the filtrate concentrated in vacuo and dissolved in conc. aq. NH3. The mixture was left aside overnight, concentrated, and the final product was obtained by preparative HPLC on reversed phase using linear gradient of methanol in 0.1% aq. TEAB in % overall yield (115 mg, 0.21 mmol) after conversion to ammonium salt by passing through small column of Dowex 50 in NH4 + form.

Scheme 12
Relacin was treated with 2M ethanolic methylamine (10 ml/mmol). Progress of the reaction was carefully monitored using HPLC. After complet conversion the desired deprotected relacin by preparative HPLC on reversed phase using linear gradient of MeOH in 0.1M aq. TEAB affording, after converting to ammonium salt by passing through a column of Dowex 50 in NH4 + form, title product in almost quantitative yield. 1

Scheme 13
Mesitylensulfonyl chloride was added to the mixture of dibenzyl-Relacin 10 (0.64 g, 0.77 mmol), triethylamine (0.22 ml, 1.6 mmol) and DMAP (52 mg, 0,43 mmol) in DCM (10 ml). The reaction mixture was stirred at rt under argon atmosphere overnight. The mixture was concentrated under reduced pressure and intermediate was obtained by column chromatography on silica gel using linear gradient of ethanol in chloroform in 30% yield (0.23 g, 0.23 mmol) that was without further characterization used in next step consisting in stirring with NaHS (0.2g, 2,26 mmol) in MeOH (5 ml) at 60 °C for 3h and then 48h at rt. The solvent was removed in vacuo the reaction mixture was purified on silica gel by fast linear gradient of ethanol in chloroform. Obtained intermediate was without further characterization (only LCMS) dissolved in 0.1M K2CO3 in water/MeOH (1:1) and stirred at rt for 1 h. pH of the reaction mixture was adjusted with AcOH to 7, end concentrated under reduced pressure. The final product was obtained by preparative HPLC on reversed phase using linear gradient of MeOH in 0.1M aq. TEAB affording, after converting to ammonium salt by passing through a column of Dowex 50 in NH4 + form, title product in 19% overall yield (26 mg, 0,043 mmol).

Azanucleotides
All azanucleotides were prepared according to general synthetic scheme 1S. In the first step a nucleobase has been attached to appropriately protected iminosugar (nitrogen containing heterocycle). Subsequently a phosphonic function has been attached followed by final deprotection.

Scheme 14
Piperidine phosphonates (entries 19-31) Piperidine phosphonates were prepared by already developed phosphonylation methods 11-16 [ref] from piperidine nucleosides 17 . Herein we present the synthesis of the most relevant piperidine nucleotides in respect to RelA. The whole series of piperidine nucleotides will be published separately elsewhere.

Scheme 15
Diisopropyl phenylphosphonoformiate (0.59 g, 2 mmol) was added to the suspension of 9-(piperidin-4-yl)guanine (0.4 g, 1.71 mmol) in DMF (20 ml). The reaction mixture was stirred at 90 °C overnight and concentrated under reduced pressure. Diisopropyl intermediate was obtained by column chromatography on silica gel using linear gradient of ethanol in chloroform in 55% yield (0.4 g, 0.94 mmol). The intermediate (characterized by means of LCMS only) was coevaporated with MeCN (2x 10 ml), dissolved in MeCN (10 ml) and Me3SiBr (0.5 ml, 3.75 mmol) was added under argon atmosphere. The reaction mixture was stirring under argon atmosphere at rt overnight, concentrated in vacuo, quenched mmol). The intermediate (characterized by means of LCMS only) was coevaporated with MeCN (2x 10 ml), dissolved in MeCN (10 ml) and Me3SiBr (0.29 ml, 2.19 mmol) was added under argon atmosphere. The reaction mixture was stirring under argon atmosphere at rt overnight, concentrated in vacuo, quenched with 2M TEAB (2 ml) and EtOH (10 ml), evaporated, and the final product was obtained by preparative HPLC on reversed phase using linear gradient of MeOH in 0.1M aq. TEAB affording, after converting to ammonium salt by passing through a column of Dowex 50 in Na + form, title product in 53% overall yield (86 mg, 0,23 mmol) in the form of white amorphous solid.