A Novel Spiro-Heterocyclic Compound Identified by the Silkworm Infection Model Inhibits Transcription in Staphylococcus aureus

Abstract

Synthetic compounds are a vital source of antimicrobial agents. To uncover therapeutically effective antimicrobial agents from a chemical library, we screened over 100,000 synthetic compounds for in vitro antimicrobial activity against methicillin-resistant Staphylococcus aureus and evaluated the in vivo therapeutic effectiveness of the hits in S. aureus-infected silkworms. Three antimicrobial agents exhibited therapeutic effects in the silkworm infection model. One of these, GPI0363, a novel spiro-heterocyclic compound, was bacteriostatic and inhibited RNA synthesis in S. aureus cells. GPI0363-resistant S. aureus strains harbored a point mutation in the gene encoding the primary sigma factor, SigA, of RNA polymerase, and this mutation was responsible for the resistance to GPI0363. We further revealed that GPI0363 could bind to SigA, inhibit promoter-specific transcription in vitro, and prolong the survival of mice infected with methicillin-resistant S. aureus. Thus, GPI0363 is an attractive candidate therapeutic agent against drug-resistant S. aureus infections.

Keywords: RNA polymerase; anti-staphylococcal; antimicrobial agent; chemical library; sigma factor; silkworm infection model; therapeutic activity.

Figure 1

In vitro and in vivo activities of GPI0363. (A) Screening strategy and chemical structure of GPI0363. Compounds that displayed in vitro activity against S. aureus MRSA4 by broth microdilution assay were screened using the silkworm infection assay. Silkworms (n = 3) were injected with S. aureus MSSA1 suspension (3 × 10⁷ CFU/larva) and immediately injected with 50 μL of 2 mM compounds. (B) In vivo activity of GPI0363 in the silkworm infection model. Silkworms were injected with S. aureus MSSA1 suspension (3 × 10⁷ CFU/larva) and immediately injected into the hemolymph with GPI0363 at different doses (n = 10). (C) Bacteriostatic activity of GPI0363. Exponentially growing S. aureus MSSA1 was treated with 20 μg/mL GPI0363, 5 μg/mL daptomycin, or vehicle. Culture aliquots were collected at different intervals as shown, diluted, and spread on agar plates, and cell viability was determined by counting the CFU per mL. Data are shown as mean ± SD of three independent experiments. Data were analyzed by one-way ANOVA using Dunnett's Multiple Comparison test and significant differences compared with no drug are indicated by asterisks (^***p ≤ 0.001).

Figure 2

Effect of GPI0363 on macromolecule biosynthesis. The effect of GPI0363 on macromolecule biosynthesis was examined by measuring the incorporation of the corresponding radiolabeled precursors. Aliquots were collected at different intervals as indicated, and the radioactivity of acid-insoluble fractions was measured by a liquid scintillation counter and is shown as counts per minute (CPM). (A) Effect on RNA synthesis. Data are shown as mean ± SD of three independent experiments and analyzed by one-way ANOVA using Dunnett's Multiple Comparison test and significant differences compared with no drug are indicated by asterisks (^*p ≤ 0.05, ^***p ≤ 0.001) (B) Effect on DNA synthesis. (C) Effect on peptidoglycan synthesis. (D) Effect on protein synthesis. Data from a single experiment are shown in figures (B–D). (E) Dose response of RNA synthesis inhibition: Aliquots were collected after 30 min, and radioactivity of the acid insoluble fractions was measured. Data represents mean ± SD of triplicates. Data were analyzed by one-way ANOVA using Dunnett's Multiple Comparison test and significant differences compared with no treatment are indicated by asterisks (^**p ≤ 0.01, ^***p ≤ 0.001).

Figure 3

Involvement of SigA in transcription inhibition by GPI0363. (A) sigA gene and the resistance marker used for the phage transduction experiments. (B) Correlation between mutation in the sigA gene and resistance to GPI0363 by phage transduction analysis. Wild-type sigA gene and mutated sigA gene were transduced into the GPI0363-resistant strain and wild-type RN4220 strain, respectively, by phage transduction. The genotype of the sigA gene and the susceptibility of the strains toward GPI0363 were determined. (C) GPI0363 inhibits promoter-specific transcription in vitro. Sau RNAP from wild-type strain and SigA^D201N mutant GPI0363-resistant strain were prepared as described in the Section Materials and Methods. Transcripts were extracted and electrophoresed on 7M urea and 6% polyacrylamide gel, and visualized by autoradiography. The promoter-specific transcript band is indicated by the arrowhead in the autoradiogram. (D)The intensities of the bands obtained were measured quantitatively using ImageJ software. The band intensity in the lanes containing the fraction and template was taken as 100%. Results are expressed as the average and range of the two independent experiments.

Figure 4

GPI0363 binds to SigA. Magnetic beads with (i) and without (ii) His-tagged SigA were incubated with GPI0363 pretreated with BSA; the bound fraction was washed, eluted, and analyzed by HPLC. The retention time of the peak appeared was confirmed to be GPI0363 by running reference GPI0363 (iii). Representative result of two independent experiments is shown.

Figure 5

Effect of GPI0363 on a mouse model of infection. Mice were infected with S. aureus USA300 by intravenous injection, followed 30 min later by intraperitoneal injection of 400 mg/kg GPI0363. Data were analyzed using the Gehan-Breslow-Wilcoxon test (^*p = 0.016) and log-rank test (^*p = 0.016).