Small molecule inhibitor of lipoteichoic acid synthesis is an antibiotic for Gram-positive bacteria

Abstract

The current epidemic of infections caused by antibiotic-resistant gram-positive bacteria requires the discovery of new drug targets and the development of new therapeutics. Lipoteichoic acid (LTA), a cell wall polymer of gram-positive bacteria, consists of 1,3-polyglycerol-phosphate linked to glycolipid. LTA synthase (LtaS) polymerizes polyglycerol-phosphate from phosphatidylglycerol, a reaction that is essential for the growth of gram-positive bacteria. We screened small molecule libraries for compounds inhibiting growth of Staphylococcus aureus but not of gram-negative bacteria. Compound 1771 [2-oxo-2-(5-phenyl-1,3,4-oxadiazol-2-ylamino)ethyl 2-naphtho[2,1-b]furan-1-ylacetate] blocked phosphatidylglycerol binding to LtaS and inhibited LTA synthesis in S. aureus and in Escherichia coli expressing ltaS. Compound 1771 inhibited the growth of antibiotic-resistant gram-positive bacteria and prolonged the survival of mice with lethal S. aureus challenge, validating LtaS as a target for the development of antibiotics.

Fig. 1.

Compound 1771 inhibits LTA biosynthesis in S. aureus. (A) S. aureus was grown in medium supplemented with either 1% DMSO (control) or two subinhibitory concentrations of 1771 for 1 h, and the optical density at 600 nm (OD₆₀₀ nm) was recorded. (B and C) Cultures shown in A were normalized to the same density, and cells were lysed to prepare extracts for separation on SDS/PAGE and Coomassie staining (B, Left) or immunoblot analyses with antibodies specific for polyglycerol-phosphate (B, Right; α-LTA) or LtaS (C, Upper; α-LtaS) and SrtA (C, Lower; α-SrtA). Native S. aureus LtaS is detected as both full-length and processed proteins (70 and 49 kDa, respectively). (D–I) Recombinant S. aureus LtaS_SA (D–F) or B. anthracis LtaS2_BA (G–I) were expressed in E. coli, and synthesis of polyglycerol-phosphate was detected by immunoblot. E. coli cultures induced (+) or noninduced (−) for expression of LtaS_SA (D) or LtaS2_BA (G) were grown in medium supplemented with either 1% DMSO (−) or 200 µM 1771 (+). Culture density measurements were used to normalize cell lysates that were separated by SDS/PAGE and analyzed by Coomassie staining (E and H, Left) or immunoblotting with antibodies against LTA (E and H, Right) and LtaS (F and I). Recombinant LtaS_SA is detected as a double band like the native protein produced in S. aureus, whereas only processed LtaS2_BA is detected in E. coli extracts. Molecular weight markers are indicated in kilodaltons.

Fig. 2.

Structural changes of Gram-positive bacteria treated with compound 1771. (A–D) Scanning electron micrographs of bacterial cultures grown without or with compound 1771. Bacteria were cultured in BHI medium supplemented with either 1% DMSO (− inhibitor) or subinhibitory concentrations of 1771 (+ inhibitor): (A) S. aureus RN4220 ±30 µM 1771 scanned at 10,000× (Upper) and 80,000× (Lower) magnification; (B) E. faecalis V583 cells ±25 µM 1771 scanned at 10,000× (Upper) and 24,000× (Lower); (C) E. faecium TX0016 cells ±20 µM 1771 scanned at 10,000× (Upper) and 20,000× (Lower); (D) B. anthracis Sterne ±5 µM 1771 scanned at 5,000× (Upper) or 10,000× (Lower). (E) Thin-section transmission electron micrographs of S. aureus reveal a thickening of the envelope with visible deformations in the presence of 30 or 40 μM compound 1771. (Upper) Electron micrographs of staphylococci in the midst of cell division. Brackets in the upper micrographs indicate the positions of enlarged image sections shown below. M, plasma membrane; P, peptidoglycan layer containing teichoic acids. Scale bars are indicated at the bottom left of each micrograph.

Fig. 3.

Compound 1771 inhibits eLtaS binding to and cleavage of phosphatidylglycerol (PG) in vitro. (A) Size-exclusion HPLC of 2 nmol eLtaS or SrtA on BioBasic SEC300 column preequilibrated with 20 nmol nitro-benzoxadiazole PG containing chains of 16 carbon atoms (NBD-PG_C16) reveals elution of the NBD-PG_C16·eLtaS complex with absorbance at 460 nm (blue trace), but not formation of a NBD-PG_C16·SrtA complex (green trace). Inclusion of 200 µM compound 1771 in the mobile HPLC phase abolished the elution of NBD-PG_C16·eLtaS complex (red trace). (B) Using NBD-PG_C6 with six carbon acyl chains cleavage of 2 nmol substrate by 2 nmol eLtaS was detectable after 6-h incubation at 37 °C. Chloroform extraction separated nonhydrolyzed NBD-PG_C6 [aqueous phase (AP)] from the hydrophobic reaction product nitro-benzoxadiazole diacylglycerol (NBD-DAG_C6), which segregated into the organic phase (OP). Both phases were analyzed by normal-phase HPLC on a diol column (blue traces). Elution profiles were monitored by fluorescence (excitation at 460 nm, emission at 534 nm). Identity of peak fractions was confirmed by mass spectrometry. Addition of 100 µM 1771 blocked NBD-DAG_C6 production by eLtaS (red traces). A control reaction incubated for 6 h without eLtaS did not contain detectable amounts of NBD-DAG_C6 (green traces).

Fig. 4.

Structure-activity relationships of compound 1771. (A) 3D models showing the molecular hydrophobicity of PG (Left) and compound 1771 (Right). Models were generated using Galaxy 3D Structure Generator v2011.02. Color coding for hydrophobic and hydrophilic areas are shown. (B) Structural formula of 1771 with the chemical designation 2-oxo-2-(5-phenyl-1,3,4-oxadiazol-2-ylamino)ethyl 2-naphtho[2,1-b]furan-1-ylacetate. The naphthofuranyl group (NF) is indicated with a gray line, and the remainder of the molecule is referred to as the R group. Growth inhibitory activity and structural formula of substructures with intact naphthofuranyl group (C) or intact R region (D). Growth inhibitory activities were measured by adding compounds to cultures of S. aureus RN4220 and displayed as mean with SDs of three independent experiments. Dose–response graphs were calculated by fitting data with variable slope sigmoidal function using GraphPad Prism 5. Corresponding IC₅₀ values are presented in Table S6.

Fig. 5.

Compound 1771 increased the time to death in a mouse model of S. aureus sepsis. Survival of cohorts of BALB/c mice (n = 15) challenged by i.v. injection with 1 × 10⁸ CFU S. aureus Newman. Animals were treated for 4 d starting 1 d before infection in 12-h intervals via i.p. injection with either compound buffer (Mock) or 32 mg/kg weight of compound 1771. Statistical significance was examined with the log-rank test: mock vs. 1771, P < 0.0001. Data are representative of two independent experiments.