Identification of a CARM1 Inhibitor with Potent In Vitro and In Vivo Activity in Preclinical Models of Multiple Myeloma.

CARM1 is an arginine methyltransferase with diverse histone and non-histone substrates implicated in the regulation of cellular processes including transcriptional co-activation and RNA processing. CARM1 overexpression has been reported in multiple cancer types and has been shown to modulate oncogenic pathways in in vitro studies. Detailed understanding of the mechanism of action of CARM1 in oncogenesis has been limited by a lack of selective tool compounds, particularly for in vivo studies. We describe the identification and characterization of, to our knowledge, the first potent and selective inhibitor of CARM1 that exhibits anti-proliferative effects both in vitro and in vivo and, to our knowledge, the first demonstration of a role for CARM1 in multiple myeloma (MM). EZM2302 (GSK3359088) is an inhibitor of CARM1 enzymatic activity in biochemical assays (IC50 = 6 nM) with broad selectivity against other histone methyltransferases. Treatment of MM cell lines with EZM2302 leads to inhibition of PABP1 and SMB methylation and cell stasis with IC50 values in the nanomolar range. Oral dosing of EZM2302 demonstrates dose-dependent in vivo CARM1 inhibition and anti-tumor activity in an MM xenograft model. EZM2302 is a validated chemical probe suitable for further understanding the biological role CARM1 plays in cancer and other diseases.

Aryl Pyrazoles as Potent Inhibitors of Arginine Methyltransferases: Identification of the First PRMT6 Tool Compound.

A novel aryl pyrazole series of arginine methyltransferase inhibitors has been identified. Synthesis of analogues within this series yielded the first potent, selective, small molecule PRMT6 inhibitor tool compound, EPZ020411. PRMT6 overexpression has been reported in several cancer types suggesting that inhibition of PRMT6 activity may have therapeutic utility. Identification of EPZ020411 provides the field with the first small molecule tool compound for target validation studies. EPZ020411 shows good bioavailability following subcutaneous dosing in rats making it a suitable tool for in vivo studies.

Comparison of 2 cell-based phosphoprotein assays to support screening and development of an ALK inhibitor.

Anaplastic lymphoma kinase (ALK) when expressed as a fusion protein with nucleophosmin (NPM) has been implicated as a driving oncogene in a subset of lymphomas. Recent reports of ALK expression in a number of other cancers have raised the possibility that an ALK inhibitor may benefit patients with these diseases as well. In a campaign to identify and develop a selective ALK inhibitor, 2 assays were devised to measure the phosphorylation of tyrosine residue 1604 of ALK (pY(1604) ALK). Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen(®)) and phosflow platforms were used to detect modulation of pY(1604) ALK to determine the relative potency of a set of small-molecule inhibitors. Prior to making use of these assays in diverse settings, the authors attempted to ensure their equivalence with a direct comparison of their performance. The pY(1604) ALK assays correlated well both with each other and with assays of ALK enzyme activity or ALK-dependent cell proliferation. The AlphaScreen(®) assay was amenable to automation and enabled rapid, high-throughput compound assessment in an NPM-ALK-driven cell line, whereas the phosflow assay enabled the authors to characterize the activity of compounds with respect to their impact on targeted enzymes and pathways. Results show that both AlphaScreen(®) and phosflow ALK assays exhibited diverse characteristics that made them desirable for different applications but were determined to be equally sensitive and robust in the detection of inhibition of pY(1604) ALK.

Nicotinic receptor-mediated regulation of the dopamine transporter in rat prefrontocortical slices following chronic in vivo administration of nicotine.

Low levels of dopaminergic activity in prefrontal cortex are thought to contribute to negative symptoms of schizophrenia. Negative symptoms are associated with the prefrontocortical area of the brain. Schizophrenic patients have a high rate of smoking, which by subjective as well as objective measures produces a cognitive benefit. We have previously shown that agonists at nicotinic receptors containing alpha4 and beta2 subunits can enhance amphetamine-stimulated [3H]dopamine ([3H]DA) release via the dopamine transporter (DAT) from slices of rat prefrontal cortex. This effect is selective for prefrontal cortex; the enhancement does not occur in striatum or nucleus accumbens. The enhancement is dependent upon activation of protein kinase C (PKC). In the current study, we show that the enhancement of amphetamine-stimulated [3H]DA release is maintained after 10 days of chronic nicotine treatment, delivered subcutaneously twice daily. There are no significant changes in the ability of prefrontocortical brain slices to take up [3H]DA in tissue prepared from nicotine-treated vs. saline-treated rats. Nicotinic receptors mediating enhancement of amphetamine-stimulated [3H]DA release are at least partially localized to nerve terminals, as an enhancement in release is also observed in synaptosomal preparations. Finally, the sensitivity of the nicotine enhancement in release to the PKC inhibitor chelerythrine is also seen in synaptosomal preparations, suggesting that the signaling mechanism activated through alpha4beta2 receptors is intact.