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A public-private partnership that supports the discovery of new medicines through open access research.
TP-064 A Chemical Probe For PRMT4This probe is available from Tocris, Sigma and Cayman Chemical. The control may be requested by clicking here. For any inquiries please contact proberequests@thesgc.org.group newOverview TP-064 TP-064N (negative control) A collaboration between Takeda and the SGC has resulted in the discovery of TP-064, the first potent, selective and cell active chemical probe for PRMT4. The in vitro activity of TP-064 includes inhibition of PRMT4 with IC50 < 10nM for methylation of H3 (1-25) and greater than 100-fold selectivity over other histone methyltransferases and non-epigenetic targets. In cellular assays, TP-064 inhibits the methylation of MED12 with IC50 = 43 nM. A closely related compound, TP-064N, exhibits no activity in the biochemical and cellular assays, and is an ideal control compound for cellular studies. Properties TP-064 TP-064N (negative control) Physical and chemical properties for TP-064 Molecular weight 458.3 Molecular formula C28H34N4O2 IUPAC name (methyl-((2-(1-(2-methylamino-ethyl)-piperidin-4-yl)-pyridin-4-yl)-methyl)-amino)-(3-phenoxy-phenyl)-methanone MollogP 3.422 PSA 47.71 No. of chiral centres 0 No. of rotatable bonds 10 No. of hydrogen bond acceptors 6 No. of hydrogen bond donors 1 Physical and chemical properties for TP-064N (Negative Control) Molecular weight 459.3 Molecular formula C28H33N3O3 IUPAC name (((2-(1-(2-methoxy-ethyl)-piperidin-4-yl)-pyridin-4-yl)-methyl)-methyl-amino)-(3-phenoxy-phenyl)-methanone MollogP 4.051 PSA 44.24 No. of chiral centres 0 No. of rotatable bonds 10 No. of hydrogen bond acceptors 6 No. of hydrogen bond donors 0 SMILES: TP-064: CNCCN1CCC(CC1)c1cc(CN(C)C(c2cccc(c2)Oc2ccccc2)=O)ccn1 TP-064N: CN(Cc1ccnc(c1)C1CCN(CC1)CCOC)C(c1cccc(c1)Oc1ccccc1)=O InChI: TP-064: InChI=1S/C28H34N4O2/c1-29-15-18-32-16-12-23(13-17-32)27-19-22(11-14-30-27)21-31(2)28(33)24-7-6-10-26(20-24)34-25-8-4-3-5-9-25/h3-11,14,19-20,23,29H,12-13,15-18,21H2,1-2H3 TP-064N: InChI=1S/C28H33N3O3/c1-30(28(32)24-7-6-10-26(20-24)34-25-8-4-3-5-9-25)21-22-11-14-29-27(19-22)23-12-15-31(16-13-23)17-18-33-2/h3-11,14,19-20,23H,12-13,15-18,21H2,1-2H3 InChIKey: TP-064: VUIITYLFSAXKIQ-UHFFFAOYSA-N TP-064N: IQCHVVCQDZOLHI-UHFFFAOYSA-N Selectivity Profile Selectivity of TP-064N at 10 μM (x) and 1 μM (x) and of TP-064 at 10 μM (x) and 1 μM (x) for PRMT1, 3, 4, 5, 6, 7, 8, and 9 as well as for 24 histone and DNA methyltransferases was assessed. Dose response data are presented in the top panel as IC50s (μM). Cell-based Assay Data TP-064 inhibits the dimethylation of PRMT4 substrates. HEK293 cells were treated with indicated concentrations of TP-064 for 3 days and dimethylation levels of BAF155 and MED12 in whole cell extracts were analyzed by western blotting. (B) Quantitation of data in (A). Graphs represent nonlinear curve fits of dimethyl-BAF155 and dimethyl-MED12 signal intensities normalized to total BAF155 or MED12, respectively. Data represent mean ± SEM of two independent experiments prepared in triplicate. TP-064 (blue) inhibits PRMT4 activity with an IC50 value of < 10 nM under balanced conditions. TP-064N (red) has no effect on PRMT4 activity up to 100 nM. The binding of TP-064 to PRMT4 was confirmed by DSLS with stabilization at about 6°C. No binding was observed with TP-064N SPR analysis of the TP-064 binding to PRMT4 in the presence of 50 μM SAM. A representative sensorgram (black dots) is shown with the kinetic fit (solid green). A Kd value of 7.1 ± 1.8 nM, with kon = 1.1 ± 0.1 × 105 M−1 s−1 and koff = 0.7 ± 0.1 × 10−3 s−1, was obtained from triplicate experiments. The steady state response (black circles) and 1:1 binding model fitting (red dashed line) is presented. Mechanism of action of TP-064 was assessed by determining IC50 of both substrates values at various concentrations. PK PropertiesPlasma concentration of TP-064 after i.p.administration to Icr-scid mice Test compound TP-064 Animal C.B-17/Icr-scid mouse, female, 7W, fed Formulation 10% DMSO/10% Cremophor EL /10% PropyleneGlycol (PG)/Distilled Water Administration i.p., 30 or 100 mg/kg as free body Test items Plasma Sampling time 0.25, 0.5, 1, 2, 4, 8, 24 hours after administration Pharmacokinetic parameters of TP-064 Co-crystal structures Please wait whilst the interactive viewer is loaded! Main features 1. Overall structure of TP-064 with PRMT4 showing dimeric crystal structure 2. Close-up of TP-064 binding pocket 3. Overlay of peptide structure 4. TP-064 binding interactions ReferencesKazuhide Nakayama, Magdalena M. Szewczyk, Carlo dela Sena, Hong Wu, Aiping Dong, Hong Zeng, Fengling Li, Renato Ferreira de Freitas, Mohammad S. Eram, Matthieu Schapira, Yuji Baba, Mihoko Kunitomo, Douglas R. Cary, Michiko Tawada, Akihiro Ohashi, Yasuhiro Imaeda, Kumar Singh Saikatendu, Charles E. Grimshaw, Masoud Vedadi, Cheryl H. Arrowsmith, Dalia Barsyte-Lovejoy, Atsushi Kiba, Daisuke Tomita and Peter J. Brown TP-064, a potent and selective small molecule inhibitor of PRMT4 for multiple myeloma. Oncotarget 9: 18480-93 (2018)