ACS Medicinal Chemistry Letters
Letter
arginine methyltransferases (PRMT3, PRMT4, PRMT5, and
PRMT7).
good bioavailability of 65.6 4.3% was observed, leading to
EPZ020411 unbound blood concentration remaining above the
PRMT6 biochemical IC50 value for more than 12 h.
In conclusion, we report here the first potent, selective, small
molecule PRMT6 inhibitors (EPZ020411 and 17−21). Further
characterization of EPZ020411 showed it to have good
bioavailability following subcutaneous dosing in rats making it
a suitable tool for potential in vivo target validation studies.
A 2.1 Å resolution crystal structure of the tool compound,
EPZ020411, with SAH and PRMT6 is shown in Figure 1c,d
(4Y30). The interactions between EPZ020411 and PRMT6 are
quite similar to those observed for compound 1 with the
majority of the interactions occurring with the diamine side-
chain and the pyrazole core. Since the R3 substituent is a
methyl instead of a hydrogen in this case, the hydrogen bonds
to both the Glu155 backbone carbonyl and water are lost, but
the methyl picks up additional van der Waals interactions that
are not present when R3 is a hydrogen atom. The entirety of
the oxygen linked alkyl side chain is not well ordered in the
structure with electron density not observed for the terminal
tetrahydropyran group.
ASSOCIATED CONTENT
■
S
* Supporting Information
ADME/PK methods, H3R2 cell assay conditions, biochemical
assay conditions, crystallography methods and data, and
synthesis and data for EPZ020411. This material is available
The PRMT6 cellular activity of compounds 15 and
EPZ020411 was tested in an engineered model in which
PRMT6 was transiently expressed in an A375 cell line
background. Notably, compound treatment did not affect
levels of PRMT6 expression (Supplemental Figure 3). Selective
methylation of the PRMT6 substrate H3R2 was robustly
induced upon 48 h of PRMT6 expression as shown in Figure
2A. Treatment with EPZ020411 resulted in a dose-dependent
AUTHOR INFORMATION
■
Corresponding Author
Notes
The authors declare the following competing financial
interest(s): All authors are stockholders of Epizyme, Inc.
decrease in H3R2 methylation (IC50 = 0.637
0.241 μM),
while treatment with the PRMT6-inactive compound 15 did
not generate an IC50 at concentrations up to 20 μM (Figure 2).
In order to determine the cellular activity of EPZ020411 against
PRMT1, monomethyl R*GG was quantified, which has
previously been demonstrated to be selectively modulated by
PRMT1 and not PRMT6.17 As seen in Supplemental Figure 4,
EPZ020411 had a >10-fold less potent effect on this PRMT1-
specific methylmark than was seen on the PRMT6-mediated
H3R2 methylmark, consistent with the biochemical potencies
of EPZ020411 on these two enzymes.
EPZ020411 had a free fraction of 0.51 0.01 and 0.52
0.02 in rat and human plasma, respectively. The compound
showed poor permeability in the parallel artificial membrane
permeation assay (PAMPA; 0.1 × 10−6 cm/s), in line with the
observation of low bioavailability after oral dosing in rats (<5%,
data not shown). Pharmacokinetic (PK) studies in rats were
also performed by intravenous (i.v.) bolus and subcutaneous
(s.c.) administration. Blood PK parameters derived from
noncompartmental analysis are displayed in Table 3. Male
Sprague−Dawley rats administered a single dose of EPZ020411
at 1 mg/kg by i.v. bolus showed a moderate clearance (CL) of
19.7 1.0 mL/min/kg, with a volume of distribution at steady
state (Vss) of 11.1 1.6 L/kg, translating to a mean terminal
half-life (t1/2) of 8.54 1.43 h. Following 5 mg/kg s.c. dosing, a
ABBREVIATIONS
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CL, clearance; DMSO, dimethyl sulfoxide; i.v., intraveneous;
Ni-NTA, nickel-nitrilotriacetic acid; PAMPA, parallel artificial
membrane permeation assay; PK, pharmacokinetic; PRMT,
protein arginine methyltransferase; SAH, S-adenosylhomocys-
teine; SAM, S-adenosylmethionine; s.c., subcutaneous; Vss,
volume of distribution at steady state
REFERENCES
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Table 3. Pharmacokinetic Parameters for EPZ020411
Following i.v. and s.c. Bolus Administration to Sprague-
Dawley Rats; Expressed as Mean SD, n = 3
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CL (mL/min/kg)
Vss (L/kg)
19.7 1.0
11.1 1.6
8.54 1.43
t1/2 (h)
9.19 1.60
0.444
(8) Kleinschmidt, M. A.; de Graaf, P.; van Teeffelen, H. A.; Timmers,
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Focke, F.; Gehrig, P.; Covic, M.; Hassa, P. O.; Schar, P.; Hubscher, U.;
tmax (h)
Cmax (ng/mL)
AUC0‑τ (h·ng/mL)
AUC0‑inf (h·ng/mL)
F (%)
844 306
2456 135
2775 181
65.6 4.3
745 34
846 45
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ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX