Mutations in isocitrate dehydrogenases (IDHs) have a gain‐of‐function effect leading to R (−)‐2‐hydroxyglutarate ( R‐ 2HG) accumulation. By using biochemical, structural and cellular assays, we show that either or both R ‐ and S ‐2HG inhibit 2‐oxoglutarate (2OG)‐dependent oxygenases with varying potencies. Half‐maximal inhibitory concentration (IC50) values for the R ‐form of 2HG varied from approximately 25 μM for the histone N ε‐lysine demethylase JMJD2A to more than 5 mM for the hypoxia‐inducible factor (HIF) prolyl hydroxylase. The results indicate that candidate oncogenic pathways in IDH‐associated malignancy should include those that are regulated by other 2OG oxygenases than HIF hydroxylases, in particular those involving the regulation of histone methylation.

Lithium is the most effective mood stabilizer for the treatment of bipolar disorder, but it is toxic at only twice the therapeutic dosage and has many undesirable side effects. It is likely that a small molecule could be found with lithium-like efficacy but without toxicity through target-based drug discovery; however, therapeutic target of lithium remains equivocal. Inositol monophosphatase is a possible target but no bioavailable inhibitors exist. Here we report that the antioxidant ebselen inhibits inositol monophosphatase and induces lithium-like effects on mouse behaviour, which are reversed with inositol, consistent with a mechanism involving inhibition of inositol recycling. Ebselen is part of the National Institutes of Health Clinical Collection, a chemical library of bioavailable drugs considered clinically safe but without proven use. Therefore, ebselen represents a lithium mimetic with the potential both to validate inositol monophosphatase inhibition as a treatment for bipolar disorder and to serve as a treatment itself.

N⁶-Methyladenosine (m6A) is currently one of the most intensively studied post-transcriptional modifications in RNA. Due to its critical role in epigenetics and physiological links to several human diseases, it is also of tremendous biological and medical interest. The m6A mark is dynamically reversed by human demethylases FTO and ALKBH5, however the mechanism by which these enzymes selectively recognise their target transcripts remains unclear. Here, we report combined biophysical and biochemical studies on the specificity determinants of m6A demethylases, which led to the identification of an m6A-mediated substrate discrimination mechanism. Our results reveal that m6A itself serves as a ‘conformational marker’, which induces different conformational outcomes in RNAs depending on sequence context. This critically impacts its interactions with several m6A-recognising proteins, including FTO and ALKBH5. Remarkably, through the RNA-remodelling effects of m6A, the demethylases were able to discriminate substrates with very similar nucleotide sequences. Our findings provide novel insights into the biological functions of m6A modifications. The mechanism identified in this work is likely of significance to other m6A-recognising proteins.

Ferrous ion and 2-oxoglutarate (2OG) oxygenases catalyze the demethylation of N^ε-methylated lysine residues in histones. Here we report studies on the inhibition of the JMJD2 subfamily of histone demethylases, employing binding analyses by nondenaturing mass spectrometry (MS), dynamic combinatorial chemistry coupled to MS, turnover assays, and crystallography. The results of initial binding and inhibition assays directed the production and analysis of a set of N-oxalyl-d-tyrosine derivatives to explore the extent of a subpocket at the JMJD2 active site. Some of the inhibitors were shown to be selective for JMJD2 over the hypoxia-inducible factor prolyl hydroxylase PHD2. A crystal structure of JMJD2A in complex with one of the potent inhibitors was obtained; modeling other inhibitors based on this structure predicts interactions that enable improved inhibition for some compounds.

Cell-derived nanovesicles (CDNs) are an emerging class of biological drug delivery systems (DDS) that retain the characteristics of the cells they were derived from, without the need for further surface functionalization. CDNs are also biocompatible, being derived from natural sources and also take advantage of the enhanced permeability and retention effect due to their nanodimensions. Furthermore, CDNs derived from monocytes were shown to have an in vivo targeting effect, accumulating at the tumor site in a previous study conducted in a mouse tumor model. Here, we report a systematic approach pertaining to various loading methods of the chemotherapeutic drug doxorubicin into our CDNs and examine the differential cellular uptake of drug-loaded CDNs in cancerous (HeLa) and healthy (HEK293) cell lines. Lastly, we proved that the addition of doxorubicin-loaded CDNs to the HeLa and HEK293 co-cultures showed a clear discrimination toward cancer cells at the cellular level. Our results further reinforce the intriguing potential of CDNs as an alternative targeted strategy for anticancer therapy.