Journal: bioRxiv

Article Title: Targeting fungal BET bromodomains as a pan - Candida antifungal strategy

doi: 10.1101/2023.02.03.527073

Figure Lengend Snippet: ( a ) Domain boundaries of Cg BDF1 BDs. ( b ) Phylogeny of fungal and human BET BDs. ( c ) Summary of binding intensities of WT and mutant forms (Y166F or Y343F) of Cg BDF1 BD1 and BD2 to an array of acetylated H3 and H4 peptides. Binding intensities are normalized to a positive control signal, obtained from the interaction of an anti-c-myc antibody with a c-myc control peptide included on the array. ( d ) Pull-down assay. Immobilized H4ac0 and H4ac4 peptides were incubated with GST-tagged Cg Bdf1 BDs or with the corresponding Y/F mutants. After washing with buffers containing either 150, 300 or 500 mM NaCl, bound proteins were eluted and visualized by western blotting with an anti-GST antibody. ( e ) Colony formation assays showing the effect of Bdf1 repression or Bdf1 BD inactivation on C. glabrata growth.

Article Snippet: GST-tagged human Brd4 BD2 (residues 349–460) was purchased from Reaction Biology Corp. Human Brd4 BD1 (residues 22–204), CaBdf1 BD1 (residues 193–327) and BD2 (residues 361–501), CgBdf1 BD1 (residues 120–248) and BD2 (residues 289–411) and CauBdf1 BD1 (residues 127–257) and BD2 (residues 287–416) were cloned into a pGEX4t1 vector as GST-tagged fusion proteins.

Techniques: Binding Assay, Mutagenesis, Positive Control, Pull Down Assay, Incubation, Western Blot

Journal: bioRxiv

Article Title: Targeting fungal BET bromodomains as a pan - Candida antifungal strategy

doi: 10.1101/2023.02.03.527073

Figure Lengend Snippet: ( a ) Overview of the HTRF assay. A GST-tagged BET BD is bound to an anti-GST antibody coupled to the donor fluorophor (Lumi4-Terbium cryptate). A biotinylated H4ac4 peptide is bound to streptavidin coupled to the acceptor fluorophore (D2 dye). The binding of GST-BD to H4ac4 brings the donor and acceptor into close proximity, allowing for FRET. Following donor excitation, fluorescence emitted by the acceptor is detected following a delay between the excitation pulse and the time-gated measurement window. The presence of a BET inhibitor disrupts the binding interaction, resulting in reduced HTRF signal. ( b ) HTRF assays performed on Cg Bdf1 BD1 and BD2 (closed purple and open magenta circles, respectively) and human Brd4 BD1 and BD2 (closed dark green and open light green circles, respectively) in the presence of the indicated compound. IC 50 values are listed next to each graph. Data represent the mean and s.d. values from three independent experiments. ( c ) Potency and selectivity of BETi compounds. Top. Summary of p IC 50 values showing the potency of compounds against Brd4 and Cg Bdf1 BDs. Symbol definitions are as in (b). Bottom. Summary of Δp IC 50 values showing the selectivity of inhibitors towards the human BDs. Closed blue (BD1) and open cyan (BD2) circles represent the difference in p IC 50 value between corresponding Brd4 and Cg Bdf1 BDs. ( d ) The four BET inhibitors do not affect C. glabrata growth, even when Bdf1 BD1 or BD2 is deleted. Expression of endogenous BDF1 was suppressed by a 23h incubation with methionine and cysteine and hence only the WT or mutant Bdf1 protein encoded by the plasmid was expressed. Strains were then incubated with BETi compounds at 10 μM for 24h and viability was assessed. Data represent the mean and s.d. values from three independent experiments.

Article Snippet: GST-tagged human Brd4 BD2 (residues 349–460) was purchased from Reaction Biology Corp. Human Brd4 BD1 (residues 22–204), CaBdf1 BD1 (residues 193–327) and BD2 (residues 361–501), CgBdf1 BD1 (residues 120–248) and BD2 (residues 289–411) and CauBdf1 BD1 (residues 127–257) and BD2 (residues 287–416) were cloned into a pGEX4t1 vector as GST-tagged fusion proteins.

Techniques: HTRF Assay, Binding Assay, Fluorescence, Expressing, Incubation, Mutagenesis, Plasmid Preparation