Additional file 3: Figure S2. of H3K27me3 demethylases regulate in vitro chondrogenesis and chondrocyte activity in osteoarthritis

siRNA validation qPCR on MSCs and validation chondrogenic gene expression following treatment with an additional siRNA against JMJD3 and UTX. MSC were treated with non-targeting siRNA control and two siRNA against JMJD3 (A) and UTX (B) at 2.5, 5 and 10 nM for 48 h. MTC = mock transfection control (no siRNA), NTC = no transfection control. (C) Targeting JMJD3 and UTX with additional siRNA. MSCs were pre-treated with siRNA_2 against JMJD3, UTX or non-targeting siRNA control prior to chondrogenic induction in transwell culture. RNA was extracted and cDNA synthesized at day 7 of chondrogenesis and expression of SOX9, ACAN COL2A1, COL10A1 and COL1A1 assessed by RT-qPCR (n = 4 patients, n =2 technical replicates per patient). Dashed line represents expression level following MSC treatment with non-targeting siRNA control. (TIFF 372 kb

Additional file 1: Figure S1. of H3K27me3 demethylases regulate in vitro chondrogenesis and chondrocyte activity in osteoarthritis

The results of treatment with GSK-5, the less active enantiomer of GSK-4, on MSCs undergoing chondrogenesis. (A-E) Assessment of gene expression at days 7 and 14 of MSC chondrogenesis to articular cartilage discs revealed following GSK-J4 and GSK-J5 treatment. (F) NANOG expression in monolayer MSCs treated for 24 h with GSK-J4 and GSK-J5. (G) PAI1 and JMJD3 expression were decreased in monolayer MSCs treated for 1 h with GSK-J4 and GSK-J5. (H) H3K27Me3 staining (green) in MSCs cultured for 24 h in control, chondrongenic or chondrogenic medium plus GSK-J4 or GSK-J5. Cell cytoskeleton/actin (phalloidin, red), nuclear staining (DAPI, blue). (TIFF 2818 kb

Human Lysine Demethylase JMJD2D (KDM4D); A Target Enabling Package

<p>There are 4 members of the Lysine Demethylase JMJD2 (KDM4) family. SGC Oxford has expressed, purified and crystallized the catalytic domains of JMJD2A, JMJD2B, JMJD2C and JMJD2D as part of the probe programme. Fragment screening and X-ray crystallography identified a large number of binders, some of which were progressed into a medicinal chemistry programme. Despite significant effort molecules with probe properties were not obtained. Consequently it has been decided to put the information generated into the public domain.</p

Human Lysine Demethylase JMJD2D (KDM4D); A Target Enabling Package

<p>There are 4 members of the Lysine Demethylase JMJD2 (KDM4) family. SGC Oxford has expressed, purified and crystallized the catalytic domains of JMJD2A, JMJD2B, JMJD2C and JMJD2D as part of the probe programme. Fragment screening and X-ray crystallography identified a large number of binders, some of which were progressed into a medicinal chemistry programme. Despite significant effort molecules with probe properties were not obtained. Consequently it has been decided to put the information generated into the public domain.</p

Human Lysine Demethylase JMJD1B (KDM3B); A Target Enabling Package

<p>There are 3 members of the Lysine Demethylase JMJD1 (KDM3) family, JMJD1A-C. SGC Oxford has expressed, purified and crystallized the catalytic domains of JMJD1A, JMJD1B and JMJD1C as part of the probe programme. Fragment screening and X-ray crystallography identified a large number of binders, some of which were progressed into a medicinal chemistry programme. Despite significant effort molecules with probe properties were not obtained. Consequently it has been decided to put the information generated into the public domain.</p

Identification and Development of 2,3-Dihydropyrrolo[1,2‑<i>a</i>]quinazolin-5(1<i>H</i>)‑one Inhibitors Targeting Bromodomains within the Switch/Sucrose Nonfermenting Complex

Bromodomain containing proteins PB1, SMARCA4, and SMARCA2 are important components of SWI/SNF chromatin remodeling complexes. We identified bromodomain inhibitors that target these proteins and display unusual binding modes involving water displacement from the KAc binding site. The best compound binds the fifth bromodomain of PB1 with a <i>K</i>_D of 124 nM, SMARCA2B and SMARCA4 with <i>K</i>_D values of 262 and 417 nM, respectively, and displays excellent selectivity over bromodomains other than PB1, SMARCA2, and SMARCA4

[1,2,4]Triazolo[4,3‑<i>a</i>]phthalazines: Inhibitors of Diverse Bromodomains

Bromodomains are gaining increasing interest as drug targets. Commercially sourced and de novo synthesized substituted [1,2,4]­triazolo­[4,3-<i>a</i>]­phthalazines are potent inhibitors of both the BET bromodomains such as BRD4 as well as bromodomains outside the BET family such as BRD9, CECR2, and CREBBP. This new series of compounds is the first example of submicromolar inhibitors of bromodomains outside the BET subfamily. Representative compounds are active in cells exhibiting potent cellular inhibition activity in a FRAP model of CREBBP and chromatin association. The compounds described are valuable starting points for discovery of selective bromodomain inhibitors and inhibitors with mixed bromodomain pharmacology

Pan-Histone Demethylase Inhibitors Simultaneously Targeting Jumonji C and Lysine-Specific Demethylases Display High Anticancer Activities

In prostate cancer, two different types of histone lysine demethylases (KDM), LSD1/KDM1 and JMJD2/KDM4, are coexpressed and colocalize with the androgen receptor. We designed and synthesized hybrid LSD1/JmjC or “pan-KDM” inhibitors <b>1</b>–<b>6</b> by coupling the skeleton of tranylcypromine <b>7</b>, a known LSD1 inhibitor, with 4-carboxy-4′-carbomethoxy-2,2′-bipyridine <b>8</b> or 5-carboxy-8-hydroxyquinoline <b>9</b>, two 2-oxoglutarate competitive templates developed for JmjC inhibition. Hybrid compounds <b>1</b>–<b>6</b> are able to simultaneously target both KDM families and have been validated as potential antitumor agents in cells. Among them, <b>2</b> and <b>3</b> increase H3K4 and H3K9 methylation levels in cells and cause growth arrest and substantial apoptosis in LNCaP prostate and HCT116 colon cancer cells. When tested in noncancer mesenchymal progenitor (MePR) cells, <b>2</b> and <b>3</b> induced little and no apoptosis, respectively, thus showing cancer-selective inhibiting action

Highly selective inhibition of histone demethylases by de novo macrocyclic peptides.

The JmjC histone demethylases (KDMs) are linked to tumour cell proliferation and are current cancer targets; however, very few highly selective inhibitors for these are available. Here we report cyclic peptide inhibitors of the KDM4A-C with selectivity over other KDMs/2OG oxygenases, including closely related KDM4D/E isoforms. Crystal structures and biochemical analyses of one of the inhibitors (CP2) with KDM4A reveals that CP2 binds differently to, but competes with, histone substrates in the active site. Substitution of the active site binding arginine of CP2 to N-ɛ-trimethyl-lysine or methylated arginine results in cyclic peptide substrates, indicating that KDM4s may act on non-histone substrates. Targeted modifications to CP2 based on crystallographic and mass spectrometry analyses results in variants with greater proteolytic robustness. Peptide dosing in cells manifests KDM4A target stabilization. Although further development is required to optimize cellular activity, the results reveal the feasibility of highly selective non-metal chelating, substrate-competitive inhibitors of the JmjC KDMs

Discovery and Optimization of Small-Molecule Ligands for the CBP/p300 Bromodomains

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 (<i>K</i>_d = 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