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International audience; Bone is a dynamic tissue that remodels continuously in response to local mechanical and chemical stimuli. This process can also result in maladaptive ectopic bone in response to injury, yet pathological differences at the molecular and structural levels are poorly understood. A number of in vivo models exist but can often be too complex to allow isolation of factors which may stimulate disease progression. A self-structuring model of bone formation is presented using a fibrin gel cast between two calcium phosphate ceramic anchors. Femoral periosteal cells, seeded into these structures, deposit an ordered matrix that closely resembles mature bone in terms of chemistry (collagen:mineral ratio) and structure, which is adapted over a period of one year in culture. Raman spectroscopy and X-ray diffraction confirm that the mineral is hydroxyapatite associated with collagen. Second-harmonic imaging demonstrates that collagen is organized similarly to mature mouse femora. Remarkably, cells differentiated to the osteocyte phase are linked by canaliculi (as demonstrated with nano-computed tomography) and remained viable over the full year of culture. It is demonstrated that novel drugs can prevent ossification in constructs. This model can be employed to study bone formation in an effort to encourage or prevent ossification in a range of pathologies.

Histone acetyltransferases of the MYST family are recruited to chromatin by BRPF scaffolding proteins. We explored functional consequences and the therapeutic potential of inhibitors targeting acetyl-lysine dependent protein interaction domains (bromodomains) present in BRPF1-3 in bone maintenance. We report three potent and selective inhibitors: one (PFI-4) with high selectivity for the BRPF1B isoform and two pan-BRPF bromodomain inhibitors (OF-1, NI-57). The developed inhibitors displaced BRPF bromodomains from chromatin and did not inhibit cell growth and proliferation. Intriguingly, the inhibitors impaired RANKL-induced differentiation of primary murine bone marrow cells and human primary monocytes into bone resorbing osteoclasts by specifically repressing transcriptional programs required for osteoclastogenesis. The data suggest a key role of BRPF in regulating gene expression during osteoclastogenesis, and the excellent druggability of these bromodomains may lead to new treatment strategies for patients suffering from bone loss or osteolytic malignant bone lesions.

Tendons are known to adapt to their mechanical environment, however high frequency low magnitude (HFLM) loading regimes (10–50 Hz), which are effective in promoting bone anabolic effects, have not been investigated in controlled conditions in tendon. In vitro loading systems (IVLS) enable precise characterisation of the link between their controlled mechanical environment and cultured tissue biological response. We report a novel IVLS design using an applied magnetic field to produce time varying loading in cultured rat tail tendon fascicles (RTTF). The design was validated through magnetic flux, load cell and viability measurements, and we report the results of preliminary experiments testing the hypothesis that an HFLM loading regime will maintain the biochemical and mechanical properties of fresh RTTF in culture over 7 days. Tissue viability was maintained for 7 days under all loading conditions, and the average peak load applied to RTTFs using the IVLS at 20 Hz was 0.125 N. RTTFs cultured for 7 days with HFLM loading showed a trend for a higher tangent modulus than fresh tissue, and significantly higher modulus than unloaded RTTFs. GAG content of HFLM cultured RTTFs was not significantly changed from that of fresh RTTFs. This novel, validated IVLS will provide new knowledge of tendon mechanobiology and has already shown the potential of clinically relevant HFLM loading for influencing tendon biology.

Bromodomains are gaining increasing interest as drug targets. Commercially sourced and de novo synthesized substituted [1,2,4]triazolo[4,3-a]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.

Summary Methylation of lysine residues on histone tail is a dynamic epigenetic modification that plays a key role in chromatin structure and gene regulation. Members of the KDM5 (also known as JARID1) sub-family are 2-oxoglutarate (2-OG) and Fe2+-dependent oxygenases acting as histone 3 lysine 4 trimethyl (H3K4me3) demethylases, regulating proliferation, stem cell self-renewal, and differentiation. Here we present the characterization of KDOAM-25, an inhibitor of KDM5 enzymes. KDOAM-25 shows biochemical half maximal inhibitory concentration values of, Graphical Abstract, Highlights • KDOAM-25 is a selective inhibitor of KDM5A-D, the H3K4me3 lysine demethylases • KDOAM-25 inhibits the demethylation of H3K4me3 in an immunofluorescence assay • Increased KDM5B expression correlates with shorter survival in multiple myeloma • KDOAM-25-treated cells show an increase in H3K4me3 at transcription start sites, Tumber et al. report the discovery and characterization of KDOAM-25, a potent and selective inhibitor of the KDM5 H3K4me3 lysine demethylases. KDOAM-25 inhibits the cellular demethylation of H3K4me3 at transcription start sites and the proliferation of MM1S multiple myeloma cells.

In prostate cancer, two different types of histone lysine demethylases (KDM), LSD1/KDM1 and JMJD2/KDM4, are co-expressed and co-localize with the androgen receptor. We designed and synthesized hybrid LSD1/JmjC - "pan-KDM" - inhibitors 1-6, by coupling the skeleton of tranylcypromine 7, a known LSD1 inhibitor, with 4-carboxy-4'-carbomethoxy-2,2'-bipyridine 8 or 5-carboxy-8-hydroxyquinoline 9, two 2-oxoglutarate competitive templates developed for JmjC inhibition. Hybrid compounds 1-6 are able to simultaneously target both KDM families, and have been validated as potential antitumor agents in cells. Among them, compounds 2 and 3 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 non-cancer mesenchymal progenitor (MePR) cells, 2 and 3 induced little and no apoptosis, respectively, thus showing a cancer-selective inhibiting action.