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, 17 (7), 2020-2027

Selectivity, Ligand Deconstruction, and Cellular Activity Analysis of a BPTF Bromodomain Inhibitor

Affiliations

Selectivity, Ligand Deconstruction, and Cellular Activity Analysis of a BPTF Bromodomain Inhibitor

Steven E Kirberger et al. Org Biomol Chem.

Abstract

Bromodomain and PHD finger containing protein transcription factor (BPTF) is an epigenetic protein involved in chromatin remodelling and is a potential anticancer target. The BPTF bromodomain has one reported small molecule inhibitor (AU1, rac-1). Here, advances made on the structure-activity relationship of a BPTF bromodomain ligand are reported using a combination of experimental and molecular dynamics simulations leading to the active enatiomer (S)-1. Additionally, a ligand deconstruction analysis was conducted to characterize important pharmacophores for engaging the BPTF bromodomain. These studies have been enabled by a protein-based fluorine NMR approach, highlighting the versatility of the method for selectivity, ligand deconstruction, and ligand binding. To enable future analysis of biological activity, cell growth analyses in a panel of cancer cell lines were carried out using CRISPR-Cas9 and (S)-1 to identify cell-based model systems that are sensitive to BPTF inhibition.

Conflict of interest statement

Conflicts of interest

There are no conflicts to declare.

Figures

Figure 1:
Figure 1:
Left) structure of racemic AU1 (rac-1). Right) Ribbon diagrams from the crystal structures of the bromodomain of BPTF (gray) overlaid with the bromodomain of 5FW BPTF (blue) with the fluorine of W2824 labeled in green. Bottom) Domain scheme for BPTF with PHD and bromodomain (BRD) labeled.
Figure 2:
Figure 2:
A) Ribbon structure of the acetylated lysine binding site of the bromodomain of BPTF with 5FW(2824) displayed. The fluorine atom is colored in cyan. N2881 is a key residue for binding of acetylated lysines on histone proteins B) PrOF NMR using the BPTF bromodomain labeled with 5FW at 2824 of rac-1 and both enantiomers against 5FW BPTF. Each ligand is in a two-fold excess of the protein. Both experiments with rac-1 and (S)-1 resulted in significant broadening of the fluorine resonance.
Figure 3:
Figure 3:
Structures of select rac-1 analogs. PrOF NMR titrations of all compounds can be found in the supporting information
Figure 4:
Figure 4:
Sample titration data of 9, a broadening and shifting of the 5FW BPTF resonance is observed. Full titration can be found in the Supporting Information.
Figure 5:
Figure 5:
Ligand deconstruction study of (S)-1, with the exception of F1, all fragments bind 5FW BPTF with similar ligand efficiencies, indicating binding contacts are dispersed throughout the molecule. Using PrOF NMR, a binding isotherm can be generated to quantify weak binding ligands. F6 is shown as a representative example. NB = No observable binding. Rac-1 ligand efficiency = 0.22
Figure 6:
Figure 6:
A) Conformation space network describing all of the binding poses found and their interconnectivity. Each node represents a ligand pose and nodes are colored according to the solvent accessible surface area of the ligand (blue/black = unbound, green/yellow = fully bound). The two starting poses, as well as the predicted most probable pose are labeled. B) Views of the most probable pose. A density isosurface was computed using 10 random ligand structures taken from most probable state (shown in transparent gray). Interacting residues are labeled.
Figure 7:
Figure 7:
Selectivity of (S)-1 toward four different bromodomains, BRD4(1), BRDT(1), PCAF, and PfGCN5. All experiments were carried out with 50 μM protein in the absence (black spectrum) or presence of 100 μM (S)-1 (red spectrum).
Figure 8:
Figure 8:
Sensitivity of cancer cell lines to (S)-1 and (R)-1. A) CRISPR-Cas9 BPTF-targeting. HepG2, MCF-7 and K562 cell lines were infected with all in one CRISPR-Cas9-GFP lentiviral particles, expressing sgRNAs targeting RPA3 gene (depletion control) and the BPTF gene. Fold change was calculated by comparing final measure of GFP to initial infection GFP. n=2 technical replicates. B) Cell viability analysis in cancer cell lines treated with 5 μM of either (S)-1 or (R)-1 for 72 h, following incubation with AlamarBlue for 4 h. Error bars demonstrate standard deviation across biological replicates.

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