SAR exploration of the 2,4-diamino-6,7-dimethoxyquinazoline template led to the discovery of 8 (UNC0224) as a potent and selective G9a inhibitor. A high resolution X-ray crystal structure of the ...G9a−8 complex, the first cocrystal structure of G9a with a small molecule inhibitor, was obtained. The cocrystal structure validated our binding hypothesis and will enable structure-based design of novel inhibitors. 8 is a useful tool for investigating the biology of G9a and its roles in chromatin remodeling.
The lysine methyltransferase SETD8 is the only known methyltransferase that catalyzes monomethylation of histone H4 lysine 20 (H4K20). Monomethylation of H4K20 has been implicated in regulating ...diverse biological processes including the DNA damage response. In addition to H4K20, SETD8 monomethylates non-histone substrates including proliferating cell nuclear antigen (PCNA) and promotes carcinogenesis by deregulating PCNA expression. However, selective inhibitors of SETD8 are scarce. The only known selective inhibitor of SETD8 to date is nahuoic acid A, a marine natural product, which is competitive with the cofactor. Here, we report the discovery of the first substrate-competitive inhibitor of SETD8, UNC0379 (1). This small-molecule inhibitor is active in multiple biochemical assays. Its affinity to SETD8 was confirmed by ITC (isothermal titration calorimetry) and SPR (surface plasmon resonance) studies. Importantly, compound 1 is selective for SETD8 over 15 other methyltransferases. We also describe structure–activity relationships (SAR) of this series.
Sirtuins are NAD+-dependent protein deacetylases and are emerging as molecular targets for the development of pharmaceuticals to treat human metabolic and neurological diseases and cancer. To date, ...several sirtuin inhibitors and activators have been identified, but the structural mechanisms of how these compounds modulate sirtuin activity have not yet been determined. We identified suramin as a compound that binds to human SIRT5 and showed that it inhibits SIRT5 NAD+-dependent deacetylase activity with an IC50 value of 22 μM. To provide insights into how sirtuin function is altered by inhibitors, we determined two crystal structures of SIRT5, one in complex with ADP-ribose, the other bound to suramin. Our structural studies provide a view of a synthetic inhibitory compound in a sirtuin active site revealing that suramin binds into the NAD+, the product, and the substrate-binding site. Finally, our structures may enable the rational design of more potent inhibitors.
Protein arginine methyltransferases (PRMTs) represent an emerging target class in oncology and other disease areas. So far, the most successful strategy to identify PRMT inhibitors has been to screen ...large to medium-size chemical libraries. Attempts to develop PRMT inhibitors using receptor-based computational methods have met limited success. Here, using virtual screening approaches, we identify 11 CARM1 (PRMT4) inhibitors with ligand efficiencies ranging from 0.28 to 0.84. CARM1 selective hits were further validated by orthogonal methods. Two structure-based rounds of optimization produced 27 (SGC2085), a CARM1 inhibitor with an IC50 of 50 nM and more than hundred-fold selectivity over other PRMTs. These results indicate that virtual screening strategies can be successfully applied to Rossmann-fold protein methyltransferases.
Histone deacetylases (HDACs) are protein deacetylases that play a role in repression of gene transcription and are emerging targets in cancer therapy. Here, we characterize the structure and ...enzymatic activity of the catalytic domain of human HDAC7 (cdHDAC7). Although HDAC7 normally exists as part of a multiprotein complex, we show that cdHDAC7 has a low level of deacetylase activity which can be inhibited by known HDAC inhibitors. The crystal structures of human cdHDAC7 and its complexes with two hydroxamate inhibitors are the first structures of the catalytic domain of class IIa HDACs and demonstrate significant differences with previously reported class I and class IIb-like HDAC structures. We show that cdHDAC7 has an additional class IIa HDAC-specific zinc binding motif adjacent to the active site which is likely to participate in substrate recognition and protein-protein interaction and may provide a site for modulation of activity. Furthermore, a different active site topology results in modified catalytic properties and in an enlarged active site pocket. Our studies provide mechanistic insights into class IIa HDACs and facilitate the design of specific modulators.
Protein lysine methyltransferase G9a, which catalyzes methylation of lysine 9 of histone H3 (H3K9) and lysine 373 (K373) of p53, is overexpressed in human cancers. Genetic knockdown of G9a inhibits ...cancer cell growth, and the dimethylation of p53 K373 results in the inactivation of p53. Initial SAR exploration of the 2,4-diamino-6,7-dimethoxyquinazoline template represented by 3a (BIX01294), a selective small molecule inhibitor of G9a and GLP, led to the discovery of 10 (UNC0224) as a potent G9a inhibitor with excellent selectivity. A high resolution X-ray crystal structure of the G9a−10 complex, the first cocrystal structure of G9a with a small molecule inhibitor, was obtained. On the basis of the structural insights revealed by this cocrystal structure, optimization of the 7-dimethylaminopropoxy side chain of 10 resulted in the discovery of 29 (UNC0321) (Morrison K i = 63 pM), which is the first G9a inhibitor with picomolar potency and the most potent G9a inhibitor to date.
Selective inhibitors of protein lysine methyltransferases, including SET domain-containing protein 8 (SETD8), are highly desired, as only a fraction of these enzymes are associated with high-quality ...inhibitors. From our previously discovered SETD8 inhibitor, we developed a more potent analog and solved a cocrystal structure, which is the first crystal structure of SETD8 in complex with a small-molecule inhibitor. This cocrystal structure allowed the design of a covalent inhibitor of SETD8 (MS453), which specifically modifies a cysteine residue near the inhibitor binding site, has an IC50 value of 804 nM, reacts with SETD8 with near-quantitative yield, and is selective for SETD8 against 28 other methyltransferases. We also solved the crystal structure of the covalent inhibitor in complex with SETD8. This work provides atomic-level perspective on the inhibition of SETD8 by small molecules and will help identify high-quality chemical probes of SETD8.
G9a is a histone lysine methyltransferase responsible for the methylation of histone H3 lysine 9. The discovery of A-366 arose from a unique diversity screening hit, which was optimized by ...incorporation of a propyl-pyrrolidine subunit to occupy the enzyme lysine channel. A-366 is a potent inhibitor of G9a (IC50: 3.3 nM) with greater than 1000-fold selectivity over 21 other methyltransferases.
In plants, the histone H3.1 lysine 27 (H3K27) mono-methyltransferases ARABIDOPSIS TRITHORAX RELATED PROTEIN 5 and 6 (ATXR5/6) regulate heterochromatic DNA replication and genome stability. Our ...initial studies showed that ATXR5/6 discriminate between histone H3 variants and preferentially methylate K27 on H3.1. In this study, we report three regulatory mechanisms contributing to the specificity of ATXR5/6. First, we show that ATXR5 preferentially methylates the R/F-K*-S/C-G/A-P/C motif with striking preference for hydrophobic and aromatic residues in positions flanking this core of five amino acids. Second, we demonstrate that post-transcriptional modifications of residues neighboring K27 that are typically associated with actively transcribed chromatin are detrimental to ATXR5 activity. Third, we show that ATXR5 PHD domain employs a narrow binding pocket to selectively recognize unmethylated K4 of histone H3. Finally, we demonstrate that deletion or mutation of the PHD domain reduces the catalytic efficiency (kcat/Km of AdoMet) of ATXR5 up to 58-fold, highlighting the multifunctional nature of ATXR5 PHD domain. Overall, our results suggest that several molecular determinants regulate ATXR5/6 methyltransferase activity and epigenetic inheritance of H3.1 K27me1 mark in plants.
Discovery of a Dual PRMT5–PRMT7 Inhibitor Smil, David; Eram, Mohammad S; Li, Fengling ...
ACS medicinal chemistry letters,
04/2015, Letnik:
6, Številka:
4
Journal Article
Recenzirano
Odprti dostop
The protein arginine methyltransferases PRMT7 and PRMT5, respectively, monomethylate and symmetrically dimethylate arginine side-chains of proteins involved in diverse cellular mechanisms, including ...chromatin-mediated control of gene transcription, splicing, and the RAS to ERK transduction cascade. It is believed that PRMT5 and PRMT7 act in conjunction to methylate their substrates, and genetic deletions support the notion that these enzymes derepress cell proliferation and migration in cancer. Using available structures of PRMT5, we designed DS-437, a PRMT5 inhibitor with an IC50 value of 6 μM against both PRMT5 and PRMT7 that is inactive against 29 other human protein-, DNA-, and RNA-methyltransferases and inhibits symmetrical dimethylation of PRMT5 substrates in cells. This compound behaves as a cofactor competitor and represents a valid scaffold to interrogate the potential of the PRMT5–PRMT7 axis as a target for therapy.
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