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Small-molecule inhibitors that target
bromodomains outside
of the bromodomain and extra-terminal (BET) sub-family are lacking.
Here, we describe highly potent and selective ligands for the bromodomain
module of the human lysine acetyl transferase CBP/p300, developed
from a series of 5-isoxazolyl-benzimidazoles. Our starting
point was a fragment hit, which was optimized into a more potent and
selective lead using parallel synthesis employing Suzuki couplings,
benzimidazole-forming reactions, and reductive aminations.
The selectivity of the lead compound against other bromodomain
family members was investigated using a thermal stability assay, which
revealed some inhibition of the structurally related BET family members.
To address the BET selectivity issue, X-ray crystal structures of
the lead compound bound to the CREB binding protein (CBP) and the
first bromodomain of BRD4 (BRD4(1)) were used to guide the design
of more selective compounds. The crystal structures obtained revealed
two distinct binding modes. By varying the aryl substitution pattern
and developing conformationally constrained analogues, selectivity
for CBP over BRD4(1) was increased. The optimized compound is highly
potent (Kd = 21 nM) and selective, displaying
40-fold selectivity over BRD4(1). Cellular activity was demonstrated
using fluorescence recovery after photo-bleaching (FRAP) and a p53
reporter assay. The optimized compounds are cell-active and have nanomolar
affinity for CBP/p300; therefore, they should be useful in studies
investigating the biological roles of CBP and p300 and to validate
the CBP and p300 bromodomains as therapeutic targets.
Bromo and extra terminal (BET) proteins (BRD2, BRD3, BRD4 and BRDT) are transcriptional regulators required for efficient expression of several growth promoting and anti-apoptotic genes as well as for cell cycle progression. BET proteins are recruited to transcriptionally active chromatin via their two N-terminal bromodomains (BRDs), a protein interaction module that specifically recognizes acetylated lysine residues in histones H3 and H4. Inhibition of the BET-histone interaction results in transcriptional down-regulation of a number of oncogenes providing a novel pharmacological strategy for the treatment of cancer. Here we present a potent and highly selective dihydroquinazoline-2-one inhibitor, PFI-1 that efficiently blocks the interaction of BET BRDs with acetylated histone tails. Co-crystal 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 leukaemic cell lines and efficiently abrogates their clonogenic growth. Exposure of sensitive cell lines with PFI-1 results in G1 cell cycle arrest, down-regulation of MYC expression as well as induction of apoptosis and induces differentiation of primary leukaemic blasts. Intriguingly, cells exposed to PFI-1 showed significant down-regulation 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.
The posttranslational modification of chromatin through
acetylation
at selected histone lysine residues is governed by histone acetyltransferases
(HATs) and histone deacetylases (HDACs). The significance of this
subset of the epigenetic code is interrogated and interpreted by an
acetyllysine-specific protein–protein interaction with bromodomain
reader modules. Selective inhibition of the bromo and extra C-terminal
domain (BET) family of bromodomains with a small molecule is feasible,
and this may represent an opportunity for disease intervention through
the recently disclosed antiproliferative and anti-inflammatory properties
of such inhibitors. Herein, we describe the discovery and structure–activity
relationship (SAR) of a novel, small-molecule chemical probe for BET
family inhibition that was identified through the application of structure-based
fragment assessment and optimization techniques. This has yielded
a potent, selective compound with cell-based activity (PFI-1) that
may further add to the understanding of BET family function within
the bromodomains.
Significance
Protein methyltransferases constitute an emerging but undercharacterized class of therapeutic targets with diverse roles in normal human biology and disease. Small-molecule “chemical probes” can be powerful tools for the functional characterization of such enzymes, and here we report the discovery of (
R
)-PFI-2—a first-in-class, potent, highly selective, and cell-active inhibitor of the methyltransferase activity of SETD7 [SET domain containing (lysine methyltransferase) 7]—and two related compounds for control and chemoproteomics studies. We used these compounds to characterize the role of SETD7 in signaling, in the Hippo pathway, that controls cell growth and organ size. Our work establishes a chemical biology tool kit for the study of the diverse roles of SETD7 in cells and further validates protein methyltransferases as a druggable target class.
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