Abstract
SET domain bifurcated protein 1 (SETDB1) is a human histone-lysine methyltransferase which is amplified in human cancers and was shown to be crucial in the growth of non-small and small cell lung carcinoma. In addition to its catalytic domain, SETDB1 harbors a unique tandem tudor domain which recognizes histone sequences containing both methylated and acetylated lysines, and likely contributes to its localization on chromatin. Using X-ray crystallography and NMR spectroscopy fragment screening approaches, we have identified the first small molecule fragment hits that bind to histone peptide binding groove of the Tandem Tudor Domain (TTD) of SETDB1. Herein, we describe the binding modes of these fragments and analogues and the biophysical characterization of key compounds. These confirmed small molecule fragments will inform the development of potent antagonists of SETDB1 interaction with histones.
Highlights
Post-translational modifications (PTMs) such as methylation, phosphorylation, acetylation, ubiquitination, and SUMOylation of histone proteins impact gene expression by altering chromatin compaction or recruiting effector proteins.1-3 Methylation of histones occurs predominantly at the lysine and arginine sidechains of histone 3 (H3K4, H3K9, H3K27, H3K36, H3K79 and H3R2) and histone 4 (H4K20 and H4R3)
Its overexpression is crucial to the growth of non-small and small cell lung cancer cell lines and has aCC-BY 4.0 International license
In the co-crystal structure of the fragment hit 1 with SET domain bifurcated protein 1 (SETDB1)-tandem tudor domain (TTD), the phenyl ring of the tetrahydroisoquinoline core occupies the aromatic cage formed by W358, W363 and F379 and the carbonyl group makes hydrogen bonding with D382 (Figure 3a)
Summary
Post-translational modifications (PTMs) such as methylation, phosphorylation, acetylation, ubiquitination, and SUMOylation of histone proteins impact gene expression by altering chromatin compaction or recruiting effector proteins. Methylation of histones occurs predominantly at the lysine and arginine sidechains of histone 3 (H3K4, H3K9, H3K27, H3K36, H3K79 and H3R2) and histone 4 (H4K20 and H4R3). We used a crystal soaking fragment screening approach to identify small-molecule ligands occupying the Kme and/or Kac pockets of SETDB1-TTD. As a major part of the H3K9me2K14ac peptide occupies and forms hydrogen bonding interactions in and around the Kac pocket, fragments binding in the Kac pocket are likely to have higher ligand efficiency and can potentially be optimized to improve affinity. To test this hypothesis, we selected and soaked 5 constrained analogues of known bromodomain inhibitors synthesized in our laboratory (Supplementary Figure 1). The Boc group was deprotected under acidic conditions and the resulting secondary amine was capped with acetyl chloride to give compound 4
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