Novel Quinazoline Derivatives Inhibit Splicing of Fungal Group II Introns

Figures

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  Figure 1. Experimental pipeline for discovery of new group II intron inhibitors.

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  Figure 2. General synthesis of quinazoline analogues (a) NaOMe, MeOH, 65 °C, 16 h, 78%. (b) DMF, SOCl2, 75 °C, 2 h, 91%. Purple hexagon indicates aryl and benzylamines.

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  Figure 3. Summary of the SAR data. The effect of substituents on the Ki for the in vitro splicing of the ai5γ intron and minimum inhibitory concentration (MIC) for C. parapsilosis. Three functional regions of parental scaffolds 1 and 3 [4-aminoquinazoline, phenyl (or benzyl), and pyridine moieties] are highlighted in green, blue, and orange, respectively. Highlighted regions of the compound analogues are modified relative to the parental compounds.

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  Figure 4. Inhibition of the ai5γ group II intron splicing by new inhibitors in vitro. (A) Schematic of the ai5γ intron splicing. (B) PAGE analysis of representative time courses of the ai5γ intron splicing in the absence (left) and in the presence (right) of compound 17. (C) Representative Ki curves were obtained by plotting the rate constants (kobs values) vs concentration of inhibitors. Data represent average of n = 2 independent experiments. Error bars are s.e.m. For comparison, the Ki values for the previously published active inhibitors intronistat A and intronistat B are 2.1 ± 0.2 and 0.36 ± 0.02 μM, respectively.33

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  Figure 5. Inhibition of the ai5γ intron splicing by 17 at 20 μM (red) and after dilution from 20 μM to 1 μM (black). Data represent average of n = 3 independent experiments, error bars are s.e.m.

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  Figure 6. C. parapsilosis COX1 exhibits a splicing defect in the presence of targeted intron inhibitors 16 and 17. C. parapsilosis was grown and treated with DMSO vehicle only, inactive compound (19), or active compound (16, 17). Relative levels of total and unspliced C. parapsilosis COX1 transcripts are indicated by RT-qPCR quantification of amplicons covering the exon (total) or intron-exon junction from the group IIB intron (unspliced). Mean values and s.e.m. from n = 3 independent experiments are shown with C. parapsilosis PGK1 as a standard. For comparison, in the presence of intronistat B, the levels of unspliced C. parapsilosis COX1 transcripts are 1.5-fold higher than those of total C. parapsilosis COX1.33