A-395

A-395 associates tightly to EED in a 1:1 binding model with a K_D value of 1.5 nM (shown by SPR). A-395 inhibits the trimeric PRC2 complex (EZH2–EED–SUZ12) with an IC₅₀ of 18 nM using a radioactivity-based assay. The structurally related, negative control, A-395N, was significantly less potent against PRC2 in this assay, with an IC₅₀ >50,000 nM. A-395 has no activity against any of the other protein lysine methyltransferases, arginine methyltransferases, or DNA methyltransferases tested (panel of 32 diverse proteins). In cellular assays, A-395 inhibits H3K27 methylation with IC50=90 nM (rhabdoid tumor cell line RD; 3 days). The negative control, A-395N, exhibits no activity in the cellular assays. In vivo activity of A-395 was tested in a DLBCL Pfeiffer xenograft model. Subcutaneous (s.c.) administration of compound in an extended-release vehicle was the only route that showed a sufficient exposure profile for effectiveness. Pharmacokinetic analysis suggested that s.c. delivery of A-395 at 300 mg/kg can maintain an unbound 90% effective concentration (EC₉₀) for at least 48 h.

This compound closely phenocopies EZH2 enzyme inhibitors, which are better-characterized and have greater in vivo utility. Therefore, this would be an alternative choice for EZH2 inhibitor-resistant mutant cells or to specifically probe the role of EED WD40-H3K27me3 interactions.

A-395 is a potent and selective inhibitor of EED/PRC2 interaction (SPR Kd=1.5 nM, 1:1 EED/A-395 binding; Ki=0.5 nM). Calculated physicochemical properties: MW=486.65, PSA=55.5, AlogP=3.37, clogD pH 7=1.74, HBDon=0, HBAcc=5, Nrot=5, Ligand Efficiency (LE)=0.371, Lipophilicity Ligand Efficiency (LipE)=7.655. A co-crystal structure is available (PDB code 5K0M, 1.83 Å).

It works well in cells and was reported to inhibit both H3K27me2 and H3K27me3 marks with IC50=390 nM, 90 nM, respectively, in human rhabdoid tumor cell line RD after 72 hours. A-395 inhibited proliferation in multiple myeloma cell lines with EC50s=1-2 uM after 10 days in RPMI 8226, MM.1s and L-363 cell lines. A-395 is highly selective and, thus, can be used to tease out the EED pharmacology in cells and its role in cancer and other diseases. It was selective against a panel of 32 diverse epigenetic methyltransferases including lysine, arginine, and DNA methyltransferases as well as against a survey of other epigenetic ‘reader domains’, including the WD-40-containing protein WDR5 (as measured by TSA) and against the chromodomain of PcG protein CBX7 (as measured by fluorescence polarization).

A-395 was tested in a xenograft model using the DLBCL Pfeiffer cells in SCID mice. A-395 was administered at a very high concentration (300 mg/kg subcutaneously, twice per week for 35 days) and showed significant reduction of the tumor volume up to 150 days as well as the H3K27m3 levels measured after 1 week compared with vehicle. Based on reported pharmacokinetic studies, subcutaneous delivery of A-395 at 300 mg/kg can maintain an unbound 90% effective concentration (EC90) for at least 48 hours. Based on the long lasting exposure after the subcutaneous administration, I suspect that A-395 may also be administered IV or IP with a proper formulation provided that the compound is soluble at the tested concentration. Development of an improved analog with good oral, IV or IP exposure would be beneficial. Selectivity profiling in a broad CEREP panel would help determine if any of the phenotypic or in vivo readout is partially due to some off-target activity. Also there is no evidence that the H3K27m3-mark reduction is responsible for the phenotypic and in vivo response.

A-395 is a very potent and selective inhibitor of EED/PRC2 interaction (SPR Kd 1.5 nM, 1:1 EED/A-395 binding; Ki 0.5 nM). Calculated physicochemical properties: MW 486.65, PSA 55.5, AlogP 3.37, clogD pH 7 1.74, HBDon 0, HBAcc 5, Nrot 5, Ligand Efficiency (LE) 0.371, Lipophilicity Ligand Efficiency (LipE) 7.655. Co-crystal structure is available (PDB code 5K0M, 1.83 Å). Other IDs: ZINC584904831, PubChem 123132213, 6PU in 5K0M PD.

It works well in cells and was reported to inhibit both H3K27me2 and H3K27me3 marks with IC50 values of 390 nM and 90 nM, respectively, in human rhabdoid tumor cell line RD after 72 hours. A-395 inhibited proliferation in multiple myeloma cell lines with EC50s of 1-2 uM after 10 days in RPMI 8226, MM.1s and L-363 cell lines. 

A-395 is highly very selective and thus can be used to tease out the EED pharmacology in cells its role in cancer and other diseases. It was selective a panel of 32 diverse epigenetic methyltransferases including lysine, arginine, and DNA methyltransferases as well as against a survey of other epigenetic ‘reader domains’, including the WD-40-containing protein WDR5, as measured by TSA and against the chromodomain of PcG protein CBX7 as measured by fluorescence polarization.

The xenograft model was done using the DLBCL Pfeiffer cells in SCID mice. A-395 was administered at a very high concentration 300 mpk s.c. (subcutaneously), twice per week for 35 days and showed significant reduction of the tumor volume up to 150 days as well as the H3K27m3 levels measured after 1 week compared to vehicle. Based on reported pharmacokinetic studies, s.c. delivery of A-395 at 300 mg/kg can maintain an unbound 90% effective concentration (EC90) for at least 48 hours. Based on the long lasting exposure after the subcutaneous administration, I suspect that A-395 may also be administered IV or IP with a proper formulation provided if the compound is soluble at the tested concentration. Development of an improved analog with good oral, IV or IP exposure would be beneficial. Selectivity profiling in a broad CEREP panel would help determine if any of the phenotypic or in vivo readout is partially due to some off-target activity. Also there seems to be no evidence that the H3K 27m3 mark reduction is responsible for the phenotypic and in vivo response.