Design and Synthesis of Fluorescent Acyclic Nucleoside Phosphonates as Potent Inhibitors of Bacterial Adenylate Cyclases

Abstract

Bordetella pertussis adenylate cyclase toxin (ACT) and Bacillus anthracis edema factor (EF) are key virulence factors with adenylate cyclase (AC) activity that substantially contribute to the pathogenesis of whooping cough and anthrax, respectively. There is an urgent need to develop potent and selective inhibitors of bacterial ACs with prospects for the development of potential antibacterial therapeutics and to study their molecular interactions with the target enzymes. Novel fluorescent 5-chloroanthraniloyl-substituted acyclic nucleoside phosphonates (Cl-ANT-ANPs) were designed and synthesized in the form of their diphosphates (Cl-ANT-ANPpp) as competitive ACT and EF inhibitors with sub-micromolar potency (IC₅₀ values: 11-622 nm). Fluorescence experiments indicated that Cl-ANT-ANPpp analogues bind to the ACT active site, and docking studies suggested that the Cl-ANT group interacts with Phe306 and Leu60. Interestingly, the increase in direct fluorescence with Cl-ANT-ANPpp having an ester linker was strictly calmodulin (CaM)-dependent, whereas Cl-ANT-ANPpp analogues with an amide linker, upon binding to ACT, increased the fluorescence even in the absence of CaM. Such a dependence of binding on structural modification could be exploited in the future design of potent inhibitors of bacterial ACs. Furthermore, one Cl-ANT-ANP in the form of a bisamidate prodrug was able to inhibit B. pertussis ACT activity in macrophage cells with IC₅₀ =12 μm.

Keywords: adenylate cyclase; anthrax; antibacterial agents; fluorescence; whooping cough.

Figure 1

Structures of adefovir diphosphate (PMEApp), (M)ANT-nucleotides and target Cl-ANT-ANPpp analogues.

Figure 2

Emission spectra representing binding of 15, 27 and 30 to the catalytic site of ACT and the effect of calmodulin (CaM) on this process. Tested compounds were added to the assay buffer at final concentrations of 100 nM (27, 30) or 300 nM (15) and the emission was scanned following the excitation at 280 nm (A) and 295 nm (B) – dashed lines. ACT (dotted lines) or ACT and CaM (solid lines) were added successively to yield final concentration of 300 nM. Data are expressed as a percent of maximal fluorescence. Superimposed recordings of representative experiments are shown.

Figure 3

Saturation of ACT active site with 15, 27 and 30. Final concentrations of ACT and CaM were 300 nM of each, nucleotides were used at final concentrations from 10 nM to 500 nM. The fluorescence increase at 430 nm was calculated by subtracting the fluorescence at 430 nm after the addition of ACT to Cl-ANT-ANPpp analogues from the maximal fluorescence at 430 nm following the final addition of CaM to the mixture. Similar data were obtained in three independent experiments. Half-saturation concentration EC₅₀ was calculated using GraphPad Prism 5.0 software.

Figure 4

Direct fluorescence of 15, 27 and 30. 100 nM of compound, 2.4 µM ACT, and 2.4 µM CaM were added to well in sequence. The excitation wavelength was 350 nm, and emission was immediately scanned from 380 to 550 nm. Superimposed recordings of a representative experiment are shown. Similar data were obtained in three independent experiments.

Figure 5

Time-resolved activation of ACT by CaM and stepwise abolishment of FRET by PMEApp. Excitation wavelength was 280 nm and emission was detected at 430 nm over time. 100 nM of 27 or 30, 300 nM ACT, 300 nM CaM, and PMEApp in the final concentrations of 0 nM (peak 1), 5 nM (peak 2), 25 nM (peak 3), 50 nM (peak 4), 100 nM (peak 5), 200 nM (peak 6), 300 nM (peak 7), 500 nM (peak 8) and 1000 nM (peak 9), were added in sequence. A record of a representative experiment is shown. Similar data were obtained in three independent experiments.

Figure 6

Left: Comparison of docking pose of compound 27 with PMEApp in the crystal structure (PDB 1ZOT).^[17] Right: Detail of binding of compound 27 in the ACT lipophilic pocket.

Scheme 1

Synthesis of the Cl-ANT acyclic nucleosides. Reagents and conditions: a) DMTrCl, Py, RT, overnight; b) i) NaH, DMF, 5-chloroisatoic anhydride, 14 h; ii) 80% AcOH, RT, 2h; c) i) MsCl, Py, RT, 2 h; ii) NaN₃, DMF, HMPA, 100 °C, 14 h; iii) H₂/Pd/C, MeOH, RT, 24 h; d) i) 5-chloroisatoic anhydride, DMF, THF; ii) 80% AcOH, RT, 2h.

Scheme 2

Synthesis of Cl-ANT-ANPs. Reagents and conditions: a) NH₃/EtOH, MW, 100 °C, 30 min; b) 5-chloroisatoic anhydride, NaH, THF, RT, 24 h; c) TMSBr, Py, RT, 12 h, then L-phenylalanine isopropyl ester hydrochloride, TEA, 2,2’-dipyridyldisufide, PPh₃, Py, 70 °C, 72 h; d) i) MsCl, Py, RT, 2 h; ii) NaN₃, DMF, HMPA, RT, 5 days; iii) H₂/Pd/C, MeOH, RT, 12 h; e) 5-chloroisatoic anhydride, DMF, THF, DMAP, RT, overnight; f) i) TMSBr, Py, RT, 14 h; ii) morpholine, t-BuOH, DCC, H₂O; III) tributylammonium pyrophosphate, DMSO, RT, 48 h.

Scheme 3

Synthesis of Cl-ANT acyclic nucleosides with extended linker. Reagents and conditions: a) i) N⁶-benzoyladenine, NaH, DMF, 60 °C, 14 h; ii) 80% AcOH, 60 °C, 30 min; b) TBSCl, imidazole, DMF, RT, 14 h; c) 1M NaOMe/MeOH, MeOH, RT, 16 h; d) i) 5-chloroisatoic anhydride, NaH, DMF, 80 °C, 3 h; ii) 80% AcOH, 50 °C, 8 h; e) i) MsCl, Py, RT, 2 h; ii) NaN₃, DMF, RT, 5 days; iii) H₂/Pd/C, MeOH, RT, 14 h; f) i) 5-chloroisatoic anhydride, DMF, THF, DMAP, RT, 16 h; ii) 80% AcOH, 50 °C, 2 h.

Scheme 4

Synthesis of Cl-ANT-ANPs derivatives with extended linker. Reagents and conditions: a) pTsOCH₂P(O)(OiPr)₂, Mg(tBuO)₂, DMF, 60°C, 72 h; b) TBAF, THF, RT, 16 h; c) 5-chloroisatoic anhydride, NaH, DMF, 50 °C, 48 h; d) i) MsCl, Py, RT, 2 h; ii) NaN₃, DMF, RT, 5 days; iii) H₂/Pd/C, MeOH, RT, 14 h; iv) 5-chloroisatoic anhydride, DMF, THF, DMAP, RT, 16 h; e) TMSBr, Py, RT, 12 h, then L-phenylalanine isopropyl ester hydrochloride, TEA, 2,2’-dipyridyldisufide, PPh₃, Py, 70 °C, 72 h; f) i) TMSBr, Py, RT, 14 h; ii) morpholine, t-BuOH, DCC, H₂O; III) tributylammonium pyrophosphate, DMSO, RT, 48 h.