GPI0363 inhibits the interaction of RNA polymerase with DNA in Staphylococcus aureus

Abstract

We previously reported a therapeutically effective spiro-heterocyclic compound, GPI0363, that inhibits the transcription of Staphylococcus aureus via the primary sigma factor of RNA polymerase, SigA. Here, we demonstrated that GPI0363 shares no cross-resistance with the clinically used RNA polymerase inhibitors rifampicin and fidaxomicin. Furthermore, we found that GPI0363 bound to SigA of both GPI0363-susceptible and resistant strains, and inhibited the interaction of the RNA polymerase holoenzyme with DNA. In addition, the gene expression patterns following GPI0363 treatment were different from those following rifampicin treatment. These findings suggest that GPI0363 has a unique mechanism of action and can serve as a promising lead molecule to develop staphylococcal RNA polymerase inhibitors.

Fig. 1. Transcription inhibition by GPI0363. (a) Structure of GPI0363. (b) Effect of GPI0363 on holoenzyme formation. S. aureus SigA was treated with GPI0363 (1.25, 0.62, 0.31, 0.15, 0.078 mg mL⁻¹) before incubation with E. coli RNAP core enzyme followed by in vitro transcription. The transcripts were extracted, electrophoresed, and visualized by autoradiography. (c) Effect of GPI0363 on promoter-specific transcription before and after S. aureus RNAP (Sau RNAP) was incubated with DNA. Sau RNAPs from the wild-type and GPI0363-resistant mutant were treated with GPI0363 (0.3 mg mL⁻¹) before or after incubation with template DNA followed by in vitro transcription. The transcripts were extracted, electrophoresed, and visualized by autoradiography. Data represent mean ± SD of three independent experiments and were analyzed by Student's t-test using Prism for Mac OS X, version 5.0d (GraphPad Software). Significant differences are indicated by asterisks (***p = 0.002). Band intensities were measured by ImageJ v1.47 by taking the band intensity without GPI0363 as 100%. Gel images are shown in ESI Fig. 3, 5 and 6.

Fig. 2. Sequence alignment of region 2 of SigA. The protein sequences were obtained from NCBI and aligned using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). The regions were assigned according to Vassylyev et al. *Region 2.5 was later named region 3.0¹⁵. The accession number of proteins used for the analysis are: Eco (E. coli): AAC76103.1; Bsu (B. subtilis): CAB14450.2; Sau (S. aureus): BAF67736.1; Tth (Thermus thermophilus): BAD70355.1. The D201 position is indicated by an arrow.

Fig. 3. Differential expression of genes by RNA-seq analysis after treatment with GPI0363 and Rifampicin. (a) Venn diagram showing the number of genes commonly downregulated and (b) upregulated. (c) Functional categorization of significantly upregulated and downregulated genes after antibiotic treatment.