PFI-1, a highly selective protein interaction inhibitor, targeting BET Bromodomains

Abstract

Bromo and extra terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) are transcriptional regulators required for efficient expression of several growth promoting and antiapoptotic genes as well as for cell-cycle progression. BET proteins are recruited on transcriptionally active chromatin via their two N-terminal bromodomains (BRD), a protein interaction module that specifically recognizes acetylated lysine residues in histones H3 and H4. Inhibition of the BET-histone interaction results in transcriptional downregulation of a number of oncogenes, providing a novel pharmacologic strategy for the treatment of cancer. Here, we present a potent and highly selective dihydroquinazoline-2-one inhibitor, PFI-1, which efficiently blocks the interaction of BET BRDs with acetylated histone tails. Cocrystal structures showed that PFI-1 acts as an acetyl-lysine (Kac) mimetic inhibitor efficiently occupying the Kac binding site in BRD4 and BRD2. PFI-1 has antiproliferative effects on leukemic cell lines and efficiently abrogates their clonogenic growth. Exposure of sensitive cell lines with PFI-1 results in G1 cell-cycle arrest, downregulation of MYC expression, as well as induction of apoptosis and induces differentiation of primary leukemic blasts. Intriguingly, cells exposed to PFI-1 showed significant downregulation of Aurora B kinase, thus attenuating phosphorylation of the Aurora substrate H3S10, providing an alternative strategy for the specific inhibition of this well-established oncology target.

Figure 1. Potency and selectivity of PFI-1

A.: Chemical structure of PFI-1. B.: Selectivity screening data of PFI-1 using temperature shift assays. Screened targets are highlighted in bold. Temperature shifts are indicated by red filled circles with increasing radii for higher Tm values as indicated in the figure. C.: Isothermal titration data measured on BRD4(1) (black) and BRD4(2) (red). Shown are heat effects for each injection and the normalized binding isotherms (insert) including the fitted function (solid line). D.: ALPHA-screen data measured using isolated bromodomains of BRD4(1) (red triangle) BRD4(2) (blue triangle) as well as the bromodomain of CREBBP (green circle). Fitted functions are show as solid lines.

Figure 2. PFI-1 co-crystal structure with BRD4(1) and BRD2(2)

A.: Structural overview of the PFI-1/BRD4(1) complex. PFI-1 is shown in ball and stick representation. Hydrogen bonds to the conserved asparagine (N140) are shown as dotted lines and water molecules as semi-transparent spheres. B.: 2FoFc omit electron density map contoured at 2σ around the PFI-1. C.: Superimposition of the PFI-1 complex with the di-acetylated K5_acK8_ac peptide complex(2) (upper panel) and the BRD4(1)/(+)-JQ1 complex(10) (lower panel). D.: Surface representation of the BRD4(1) acetyl-lysine binding site. Residues that are different between first and second bromodomains are labelled in red and conserved residues in black, respectively. The conserved asparagine is highlighted in blue. E.: Surface representation of the BRD2(2) acetyl-lysine binding site. Residues were labelled using the same colour code as in D.

Figure 3. FRAP data demonstrating dissociation of GFP-BRD4 from chromatin

A.: Nuclei of PFI-1 treated (upper panel) and untreated (lower panel) cells. The bleached area is indicated by a read polygon. B.: Time dependence of fluorescent recovery in the bleached area for DMSO, (+)-JQ1 and PFI-1 (1, 5 μM) treated cells. C.: Half times of fluorescence recovery of DMSO, (+)-JQ1 and PFI-1 (1, 5 μM) treated cells. The data shown represent the average values of 20 experiments.

Figure 4. Effects of PFI-1 on cell survival and clonogenic growth

A.: Dose response of cell survival of MV4;11 and K-562 cell lines in the presence of PFI-1 and (+)-JQ1 (B). C.: Effects of PFI-1 on clonogenic growth of MV4;11 and human CD34⁺ stem cells (HSC) and K-562 cells (D). E.: Bar diagram showing the number of colonies or cells (F) measured in clonogenic growth assays of leukaemic cells and HSCs. The measurements have been carried out in duplicates. However, due to variations of the colony numbers obtained from different donors only one set of data is shown for CD34⁺ stem cells.

Figure 5. Western Blots demonstrating induction of apoptosis and downregulation of c-Myc in MV4;11 but not K-562 cells

Shown are western blot data on the BET inhibitor sensitive cell line MV4;11 and the insensitive cell line K-562 (cleavage of pro-caspase 7, PARB activation, BAD phosphorylation and c-Myc downregulation) using cell extracts of PFI-1 treated cells with after 0, 24 and 48h incubation times.

Figure 6. Downregulation of Aurora B

A.: Western blot analysis of Aurora B expression in MV4;11 and K-562 cells treated with PFI-1 and (+)-JQ1. B: Immunohistochemistry showing reduction of phosphorylation of the Aurora B substrate H3S10. DNA is stained with DAPI. C.: Western blot analysis of phosphor H3S10 after PFI-1 and (+)-JQ1 treatment of MV4;11 and K-562 cells.

Figure 7. Synergy of BET and Aurora inhibition in vitro and in vivo and effects on HOXA9 expression

A: Cytotoxicity of MV4;11 cells using combinations of BET inhibitors (PFI-1, JQ1) and the pan-Aurora inhibitor VX-680. Values shown represent data from four independent experiments. B.: Staining for Aurora B and c-Myc of bone marrow extracted from JQ1 and vehicle treated BALL bearing mice. C.: Chromatin immunoprecipitation (ChIP) of BRD4 binding to the HOXA9 promoter, THP-1 cells were treated with either vehicle (DMSO) or PFI-1. D.: qRT-PCR of HOXA9 levels in K-562 and MV4;11 cells. E.: MLL-AF9 murine leukaemic blast cells treated with vehicle (DMSO) and PFI-1.