Selective inhibition of protein methyltransferases is a promising new approach to drug discovery. An attractive strategy towards this goal is the development of compounds that selectively inhibit binding of the cofactor, S-adenosylmethionine, within specific protein methyltransferases. Here we report the three-dimensional structure of the protein methyltransferase DOT1L bound to EPZ004777, the first S-adenosylmethionine-competitive inhibitor of a protein methyltransferase with in vivo efficacy. This structure and those of four new analogues reveal remodelling of the catalytic site. EPZ004777 and a brominated analogue, SGC0946, inhibit DOT1L in vitro and selectively kill mixed lineage leukaemia cells, in which DOT1L is aberrantly localized via interaction with an oncogenic MLL fusion protein. These data provide important new insight into mechanisms of cell-active S-adenosylmethioninecompetitive protein methyltransferase inhibitors, and establish a foundation for the further development of drug-like inhibitors of DOT1L for cancer therapy.

Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade.

PRMT3, a protein arginine methyltransferase, has been shown to influence ribosomal biosynthesis by catalyzing the dimethylation of the 40S ribosomal protein S2. Although PRMT3 has been reported to be a cytosolic protein, it has been shown to methylate histone H4 peptide (H4 1-24) in vitro. Here, we report the identification of a PRMT3 inhibitor (1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-cyclohexenylethyl)urea; compound 1) with IC50 value of 2.5 μM by screening a library of 16,000 compounds using H4 (1-24) peptide as a substrate. The crystal structure of PRMT3 in complex with compound 1 as well as kinetic analysis reveals an allosteric mechanism of inhibition. Mutating PRMT3 residues within the allosteric site or using compound 1 analogs that disrupt interactions with allosteric site residues both abrogated binding and inhibitory activity. These data demonstrate an allosteric mechanism for inhibition of protein arginine methyltransferases, an emerging class of therapeutic targets.

Recently a major research effort has been focused on the development of anticancer drugs by targeting the components of a biochemical pathway to induce apoptosis in cancerous cells. Some of the natural products (e.g. paclitaxel) have been proven to be useful in inducing apoptosis in cancer cells with limited specificity. Pancratistatin, a natural product isolated and characterized over a decade ago, has been shown to be cytostatic and antineoplastic. We investigated the specificity and biochemical mechanism of action of pancratistatin. Pancratistatin seemed to show more specificity than VP-16 or paclitaxel as an efficient inducer of apoptosis in human lymphoma (Jurkat) cells, with minimal effect on normal nucleated blood cells. Caspase-3 activation and exposure of phosphatidyl serine on the outer leaflet of the plasma membrane were earlier events than the generation of ROS and DNA fragmentation observed following pancratistatin treatment. This indicates a possible involvement of caspase-3 and plasma membrane proteins in the induction phase of apoptosis. Our results indicate that pancratistatin does not cause DNA double-strand breaks or DNA damage prior to the execution phase of apoptosis in cancer cells. Parallel experimentation with VP-16, a currently used medication for cancer treatment, indicated that VP-16 causes substantial DNA damage in normal non-cancerous blood cells, while pancratistatin does not cause any DNA double-strand breaks or DNA damage in non-cancerous cells. Taken together, our finding that pancratistatin induces apoptosis in cancer cells using non-genomic targets, and more importantly does not seem to have any affect non-cancerous cells, presents a significant platform to develop non-toxic anticancer therapies.

ABSTRACT:The STAT3 gene is abnormally active in glioblastoma (GBM) and is a critically important mediator of tumor growth and therapeutic resistance in GBM. Thus, for poorly treated brain cancers such as gliomas, astrocytomas, and glioblastomas, which harbor constitutively activated STAT3, a STAT3-targeting therapeutic will be of significant importance. Herein, we report a most potent, small molecule, nonphosphorylated STAT3 inhibitor, 31 (SH-4-54) that strongly binds to STAT3 protein (K D = 300 nM). Inhibitor 31 potently kills glioblastoma brain cancer stem cells (BTSCs) and effectively suppresses STAT3 phosphorylation and its downstream transcriptional targets at low nM concentrations. Moreover, in vivo, 31 exhibited blood−brain barrier permeability, potently controlled glioma tumor growth, and inhibited pSTAT3 in vivo. This work, for the first time, demonstrates the power of STAT3 inhibitors for the treatment of BTSCs and validates the therapeutic efficacy of a STAT3 inhibitor for GBM clinical application.