SUMMARY Mislocated enzymatic activity of DOT1L has been proposed as a driver of leukemogenesis in mixed lineage leukemia (MLL). The characterization of EPZ004777, a potent, selective inhibitor of DOT1L is reported. Treatment of MLL cells with the compound selectively inhibits H3K79 methylation and blocks expression of leukemogenic genes. Exposure of leukemic cells to EPZ004777 results in selective killing of those cells bearing the MLL gene translocation, with little effect on non-MLL-translocated cells. Finally, in vivo administration of EPZ004777 leads to extension of survival in a mouse MLL xenograft model. These results provide compelling support for DOT1L inhibition as a basis for targeted therapeutics against MLL.

Protein arginine methyltransferase-5 (PRMT5) is reported to have a role in diverse cellular processes, including tumorigenesis, and its overexpression is observed in cell lines and primary patient samples derived from lymphomas, particularly mantle cell lymphoma (MCL). Here we describe the identification and characterization of a potent and selective inhibitor of PRMT5 with antiproliferative effects in both in vitro and in vivo models of MCL. EPZ015666 (GSK3235025) is an orally available inhibitor of PRMT5 enzymatic activity in biochemical assays with a half-maximal inhibitory concentration (IC50) of 22 nM and broad selectivity against a panel of other histone methyltransferases. Treatment of MCL cell lines with EPZ015666 led to inhibition of SmD3 methylation and cell death, with IC50 values in the nanomolar range. Oral dosing with EPZ015666 demonstrated dose-dependent antitumor activity in multiple MCL xenograft models. EPZ015666 represents a validated chemical probe for further study of PRMT5 biology and arginine methylation in cancer and other diseases.

ABSTRACT:The recent publication of a potent and selective inhibitor of protein methyltransferase 5 (PRMT5) provides the scientific community with in vivo-active tool compound EPZ015666 (GSK3235025) to probe the underlying pharmacology of this key enzyme. Herein, we report the design and optimization strategies employed on an initial hit compound with poor in vitro clearance to yield in vivo tool compound EPZ015666 and an additional potent in vitro tool molecule EPZ015866 (GSK3203591). KEYWORDS:Methyltransferase, PRMT5, property based optimization, structure guided design T he mammalian protein arginine methyltransferases are a group of nine enzymes that perform N G -mono methylation-, asymmetric-, or symmetric dimethylation of arginine residues on a range of nuclear and cytoplasmic protein substrates.1 One member of this group, PRMT5, is capable of performing methylation of up to two methyl groups and is currently believed to be the predominant enzyme for symmetric dimethylation. PRMT5 may play an important role in tumorigenesis and is upregulated in several human malignancies.2−8 The mechanism behind the cell-transforming capabilities of PRMT5 has been postulated to have roles in cell death, cell-cycle progression and cell growth, and proliferation and is still under investigation.9 Whether PRMT5 drives tumorigenesis by direct signal transduction, regulating gene expression, or by some other mechanism is generally unknown, although recent studies highlight a dependency on PRMT5 as part of the spliceosomal machinery with Sm proteins, particularly for MYC-driven tumors. 10EPZ015666 has recently been characterized as a potent inhibitor and in vivo tool compound of PRMT5.11 This compound is the first inhibitor to be described with a well characterized correlation between inhibition of PRMT5 enzyme and reduction of known substrate products including symmetric-dimethylated SmD3, coupled with a corresponding effect on tumor growth inhibition. In addition, structural biology studies highlighted a unique cation−π binding mode involving the tetrahydroisoquinoline (THIQ) containing chemical series as exemplified in the EPZ015666:PRMT5:-MEP50 cocrystal complex (PDB Codes: 4X60, 4X61). Herein we describe the medicinal chemistry optimization (Figure 1) in the development of tool compound EPZ015666.Compound 1 was recently described 11 as a hit identified from a 370 K member diversity high throughput screening (HTS) campaign, with modest inhibitory activity against PRMT5. Scheme 1 shows the synthetic route employed for the optimization of 1, as it enabled identification of SAR around the THIQ group at the penultimate step. Retaining the cyclopentylamino motif, a range of amines were used to open epoxide intermediate 5, providing the amino alcohol derivatives shown after boc-deprotection. No increase in activity was observed from this set, however, in comparison with the original hit compound (Scheme 1B). The contribution of the THIQ mediated cation−π interaction is apparent from this early data set with a number of non-THIQ co...

Extracelluar signal‐regulated kinase (ERK) pathway activation has been demonstrated following convulsant stimulation; however, little is known about the molecular targets of ERK in seizure models. Recently, it has been shown that ERK phosphorylates Kv4.2 channels leading to down‐regulation of channel function, and substantially alters dendritic excitability. In the kainate model of status epilepticus (SE), we investigated whether ERK phosphorylates Kv4.2 and whether the changes in Kv4.2 were evident at a synaptosomal level during SE. Western blotting was performed on rat hippocampal whole cell, membrane, synaptosomal, and surface biotinylated extracts following systemic kainate using an antibody generated against the Kv4.2 ERK sites and for Kv4.2, ERK, and phospho‐ERK. ERK activation was associated with an increase in Kv4.2 phosphorylation during behavioral SE. During SE, ERK activation and Kv4.2 phosphorylation were evident at the whole cell and synaptosomal levels. In addition, while whole‐cell preparations revealed no alterations in total Kv4.2 levels, a decrease in synaptosomal and surface expression of Kv4.2 was evident after prolonged SE. These results demonstrate ERK pathway coupling to Kv4.2 phosphorylation. The finding of decreased Kv4.2 levels in hippocampal synaptosomes and surface membranes suggest additional mechanisms for decreasing the dendritic A‐current, which could lead to altered intrinsic membrane excitability during SE.

Patients with non-Hodgkin lymphoma (NHL) are treated today with a cocktail of drugs referred to as CHOP (Cyclophosphamide, Hydroxyldaunorubicin, Oncovin, and Prednisone). Subsets of patients with NHL of germinal center origin bear oncogenic mutations in the EZH2 histone methyltransferase. Clinical testing of the EZH2 inhibitor EPZ-6438 has recently begun in patients. We report here that combining EPZ-6438 with CHOP in preclinical cell culture and mouse models results in dramatic synergy for cell killing in EZH2 mutant germinal center NHL cells. Surprisingly, we observe that much of this synergy is due to Prednisolone – a glucocorticoid receptor agonist (GRag) component of CHOP. Dramatic synergy was observed when EPZ-6438 is combined with Prednisolone alone, and a similar effect was observed with Dexamethasone, another GRag. Remarkably, the anti-proliferative effect of the EPZ-6438+GRag combination extends beyond EZH2 mutant-bearing cells to more generally impact germinal center NHL. These preclinical data reveal an unanticipated biological intersection between GR-mediated gene regulation and EZH2-mediated chromatin remodeling. The data also suggest the possibility of a significant and practical benefit of combining EZH2 inhibitors and GRag that warrants further investigation in a clinical setting.