Probe		Negative control
GSK864		GSK990

GlaxoSmithKline has developed GSK864 [1], a potent and selective inhibitor of mutant IDH1, and has made this available as an SGC chemical probe.  GSK864 inhibits IDH1 mutants R132C/R132H/R132G with IC50 values of 9/15/17 nM, respectively, and is moderately selective over wild-type IDH1 and IDH2 mutants/wild-type. Treatment of R132C IDH1 mutant HT-1080 cells for 24 hours with GSK864 results in a dose-dependent reduction of 2-hydroxyglutarate (2-HG), which is not observed with GSK990, a structurally similar compound which is inactive as an IDH1 inhibitor. GSK864 has been shown to be selective in vitro for IDH1 over other classes of proteins (7TMs, ion channels, kinases) and chemoproteomic studies with GSK321, an analog of GSK864, confirm selective binding of IDH1 by this chemical series.  GSK864 has a pharmacokinetic profile suitable for in vivo studies.

Chemoproteomics: the selectivity of GSK864 for IDH1 was illustrated by conducting a chemoproteomics experiment with a closely related analog, GSK321 [1]. GSK321 was functionalized so that it could be immobilized to NHS-activated Sepharose beads which were then incubated with a lysate (protein concentration 5 mg/mL) from HT-1080 cells. The experimental plan included using GSK990 and vehicle as control bait. The proteins were eluted from the beads and then subjected to in-gel digestion and labelling with TMT reagents. LC/MS/MS identified over 300 proteins of which only one, IDH1 had IC50 < 200 nM. 

Probe		Negative control
MS023		MS094

Protein arginine methyltransferases (PRMTs) play a crucial role in a variety of biological processes [1]. Overexpression of PRMTs has been implicated in various human diseases including cancer [2-4]. To date, nine PRMTs have been identified and they are grouped into three categories: types I, II and III. Type I PRMTs catalyze mono- and asymmetric dimethylation of arginine residues and include PRMT1, 3, 4 (also known as CARM1 (Co-activator-associated arginine methyltransferase 1)), 6 and 8. It has been shown that knocking down the expression of PRMT1 or PRMT6 genes (using siRNA) significantly reduces the growth of bladder and lung cancer cells [5]. Consequently, selective small-molecule inhibitors of PRMTs have been pursued by both academia and pharmaceutical industry as chemical tools for testing biological and therapeutic hypotheses [6,7].

A collaboration between the Icahn School of Medicine at Mount Sinai and the SGC has resulted in MS023 [8], a chemical probe for type I PRMTs, and a structurally similar control compound, MS094. The in vitro and cell-based activity of MS023 is summarized in the table and the Western blots.

(A) MS023 inhibits PRMT1 methyltransferase activity in MCF7 cells. (B) MS094 does not inhibit PRMT1 methyltransferase activity in MCF7 cells. MCF7 cells were treated with MS023 (A) or MS094 (B) at indicated concentrations for 48 h and H4R3me2a levels were determined by Western blot. The graphs represent nonlinear fits of H4R3me2a signal intensities normalized to total histone H4. The results are MEAN ± SEM of two experiments done in triplicate. (C) MS023 inhibits PRMT6 methyltransferase activity in HEK293 cells. HEK293 cells were transfected with FLAG-tagged PRMT6 or its catalytically inactive mutant V86K/D88A (MUT) and treated with MS023 at indicated concentrations for 20 h. H3R2me2a levels were determined by Western blot. The graphs represent nonlinear fits of H3R2me2a signal intensities normalized to total histone H3. The results are MEAN ± SEM of 3 replicates.

Probe		Negative control
GSK591		SGC2096

Protein arginine methyltransferases (PRMTs) play a crucial role in a variety of biological processes [1]. Overexpression of PRMTs has been implicated in various human diseases including cancer [2-4]. To date, nine PRMTs have been identified and they are grouped into three categories: types I, II and III. Type II PRMTs, 5 and 9, catalyze symmetric dimethylation of arginine residues, however, PRMT5 is the predominant enzyme for this process. PRMT5 interacts with a number of binding partners that influence its substrate specificity. In particular, MEP50, a member of the WD40 family of proteins, is required for PRMT5 methyltransferase activity.

PRMT5 is reported to have a role in mantle cell lymphoma (MCL) as evidenced by its upregulation in patient samples [5,6]. Consequently, a chemical probe of PRMT5 would be a very useful tool for testing biological and therapeutic hypotheses. The first chemical probe of PRMT5 was co-developed by Epizyme and GlaxoSmithKline (GSK) [7]. An analog of this compound, EPZ015866/GSK3203591 [8], has been kindly donated to the SGC for distribution as GSK591. In an in vitro biochemical assay, GSK591 potently inhibits the PRMT5/MEP50 complex from methylating (histone) H4 with IC50 = 11 nM. In Z-138 cells, GSK591 inhibits the symmetric arginine methylation of SmD3 with EC50 = 56 nM. Further, GSK591 is selective for PRMT5 (up to 50 micromolar) relative to a panel of methyltransferases. 

A control compound, SGC2096, that is inactive up to 10 micromolar is also available from the SGC.

In addition, a biotinylated inhibitor, SGC3185 is available

Biotinylated inhibitor

SGC3185

In Vitro potency