Rational Design and Synthesis of Selective PRMT4 Inhibitors: A New Chemotype for Development of Cancer Therapeutics*.

Protein arginine N-methyl transferase 4 (PRMT4) asymmetrically dimethylates the arginine residues of histone H3 and nonhistone proteins. The overexpression of PRMT4 in several cancers has stimulated interest in the discovery of inhibitors as biological tools and, potentially, therapeutics. Although several PRMT4 inhibitors have been reported, most display poor selectivity against other members of the PRMT family of methyl transferases. Herein, we report the structure-based design of a new class of alanine-containing 3-arylindoles as potent and selective PRMT4 inhibitors, and describe key structure-activity relationships for this class of compounds.

A chemical probe of CARM1 alters epigenetic plasticity against breast cancer cell invasion.

CARM1 is a cancer-relevant protein arginine methyltransferase that regulates many aspects of transcription. Its pharmacological inhibition is a promising anti-cancer strategy. Here SKI-73 (6a in this work) is presented as a CARM1 chemical probe with pro-drug properties. SKI-73 (6a) can rapidly penetrate cell membranes and then be processed into active inhibitors, which are retained intracellularly with 10-fold enrichment for several days. These compounds were characterized for their potency, selectivity, modes of action, and on-target engagement. SKI-73 (6a) recapitulates the effect of CARM1 knockout against breast cancer cell invasion. Single-cell RNA-seq analysis revealed that the SKI-73(6a)-associated reduction of invasiveness acts by altering epigenetic plasticity and suppressing the invasion-prone subpopulation. Interestingly, SKI-73 (6a) and CARM1 knockout alter the epigenetic plasticity with remarkable difference, suggesting distinct modes of action for small-molecule and genetic perturbations. We therefore discovered a CARM1-addiction mechanism of cancer metastasis and developed a chemical probe to target this process.

Drugs that are small molecules have the potential to block the individual proteins that drive the spread of cancer, but their design is a challenge. This is because they need to get inside the cell and find their target without binding to other proteins on the way. However, small molecule drugs often have an electric charge, which makes it hard for them to cross the cell membrane. Additionally, most proteins are not completely unique, making it harder for the drugs to find the correct target. CARM1 is a protein that plays a role in the spread of breast cancer cells, and scientists are currently looking for a small molecule that will inhibit its action. The group of enzymes that CARM1 belongs to act by taking a small chemical group, called a methyl group, from a molecule called SAM, and transferring it to proteins that switch genes on and off. In the case of CARM1, this changes cell behavior by turning on genes involved in cell movement. Genetically modifying cells so they will not produce any CARM1 stops the spread of breast cancer cells, but developing a drug with the same effects has proved difficult. Existing drugs that can inhibit CARM1 in a test tube struggle to get inside cells and to distinguish between CARM1 and its related enzymes. Now, Cai et al. have modified and tested a CARM1 inhibitor to address these problems, and find out how these small molecules work. At its core, the inhibitor has a structure very similar to a SAM molecule, so it can fit into the SAM binding pocket of CARM1 and its related enzymes. To stop the inhibitor from binding to other proteins, Cai et al. made small changes to its structure until it only interacted with CARM1.Then, to get the inhibitor inside breast cancer cells, Cai et al. cloaked its charged area with a chemical shield, allowing it to cross the cell membrane. Inside the cell, the chemical shield broke away, allowing the inhibitor to attach to CARM1. Analysis of cells showed that this inhibition only affected the cancer cells most likely to spread. Blocking CARM1 switched off genes involved in cell movement and stopped cancer cells from travelling through 3D gels. This work is a step towards making a drug that can block CARM1 in cancer cells, but there is still further work to be done. The next stages will be to test whether the new inhibitor works in other types of cancer cells, in living animals, and in human patient samples.

The study of epigenetic mechanisms based on the analysis of histone modification patterns by flow cytometry.

Epigenetic regulation of genes involved in cell growth, survival, or differentiation through histone modifications is an important determinant of cancer development and outcome. The basic science of epigenetics uses analytical tools that, although powerful, are not well suited to the analysis of heterogeneous cell populations found in human cancers, or for monitoring the effects of drugs designed to modulate epigenetic mechanisms in patients. To address this, we selected three clinically relevant histone marks (H3K27me3, H3K9ac, and H3K9me2), modulated their expression levels by in vitro treatments to generate high and low expressing control cells, and tested the relative sensitivity of candidate antibodies to detect the differences in expression levels by flow cytoametry using a range of sample preparation techniques. We identified monoclonal antibodies to all three histone marks that were suitable for flow cytoametry. Staining intensities were reduced with increasing formaldehyde concentration, and were not affected by ionic strength or by alcohol treatment. A protocol suitable for clinical samples was then developed, to allow combined labeling of histone marks and surface antigens while preserving light scatter signals. This was applied to normal donor blood, and to samples obtained from 25 patients with leukemia (predominantly acute myeloid leukemia). Significant cellular heterogeneity in H3K9ac and H3K27me3 staining was seen in normal peripheral blood, but the patterns were very similar between individual donors. In contrast, H3K27me3 in particular showed considerable inter-patient heterogeneity in the leukemia cell populations. Although further refinements are likely needed to fully optimize sample staining protocols, "flow epigenetics" appears to be technically feasible, and to have potential both in basic research, and in clinical application.

Sgf29 binds histone H3K4me2/3 and is required for SAGA complex recruitment and histone H3 acetylation.

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation.

Discovery of a 2,4-diamino-7-aminoalkoxyquinazoline as a potent and selective inhibitor of histone lysine methyltransferase G9a.

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.