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.

Absence seizures are a common seizure type in children with genetic generalized epilepsy and are characterized by a temporary loss of awareness, arrest of physical activity, and accompanying spike-and-wave discharges on an electroencephalogram. They arise from abnormal, hypersynchronous neuronal firing in brain thalamocortical circuits. Currently available therapeutic agents are only partially effective and act on multiple molecular targets, including γ-aminobutyric acid (GABA) transaminase, sodium channels, and calcium (Ca(2+)) channels. We sought to develop high-affinity T-type specific Ca(2+) channel antagonists and to assess their efficacy against absence seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model. Using a rational drug design strategy that used knowledge from a previous N-type Ca(2+) channel pharmacophore and a high-throughput fluorometric Ca(2+) influx assay, we identified the T-type Ca(2+) channel blockers Z941 and Z944 as candidate agents and showed in thalamic slices that they attenuated burst firing of thalamic reticular nucleus neurons in GAERS. Upon administration to GAERS animals, Z941 and Z944 potently suppressed absence seizures by 85 to 90% via a mechanism distinct from the effects of ethosuximide and valproate, two first-line clinical drugs for absence seizures. The ability of the T-type Ca(2+) channel antagonists to inhibit absence seizures and to reduce the duration and cycle frequency of spike-and-wave discharges suggests that these agents have a unique mechanism of action on pathological thalamocortical oscillatory activity distinct from current drugs used in clinical practice.

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...

Calsenilin/potassium channel-interacting protein (KChIP)3/ downstream regulatory element sequence antagonist modulator (DREAM) is a neuronal calcium-binding protein that has been shown to have multiple functions in the cell, including the regulation of presenilin processing, repression of transcription and modulation of A-type potassium channels. To gain a better understanding of the precise role of calsenilin in specific cellular compartments, an interactor hunt for proteins that bind to the N-terminal domain of calsenilin was carried out. Using a yeast two-hybrid system and co-immunoprecipitation studies, we have identified the transcriptional co-repressor C-terminal binding protein (CtBP)2 as an interactor for calsenilin and have shown that the two proteins can interact in vivo. In coimmunoprecipitation studies, calsenilin also interacted with CtBP1, a CtBP2 homolog. Our data also showed a calsenilindependent increase in c-fos protein levels in CtBP knockout fibroblasts, suggesting that CtBP may modulate the transcriptional repression of c-fos by calsenilin. Furthermore, the finding that histone deacetylase protein and activity were associated with the calsenilin-CtBP immunocomplex suggests a mechanism by which calsenilin-CtBP may act to repress transcription. Finally, we demonstrated that calsenilin and CtBP are present in synaptic vesicles and can interact in vivo.