The new compounds were tested by fluorograph at 50 and 20 μM against CARM1 using PABP1 as a substrate,20
and at 50 μM against PRMT1 (substrate: the heterogeneous nuclear ribonucleoprotein NPL3)21
and SET7 (substrate: histone H3), to assess their potency and selectivity (). At 20 μM, the 4-methyl- and the 2-, 3-, and 4-methoxybenzyl analogues of 7g
) as well as the 3-phenylpropyl-piperidone 8k
showed no effect against the PABP1 methylation, thus the methoxy-containing compounds were excluded by IC50
Inhibitory activities of compounds 8a-l against CARM1 using PABP1 as a substrate, PRMT1 using NPL3 as a substrate, and SET7 using histone H3 as a substrate. The concentration of the compounds used in each in vitro methylation assay is shown.
values for compounds 7g
were determined against CARM1 using PABP1 as a substrate, and against PRMT1 and SET7 using NPL3 and histone H3 as substrates, respectively (). The corresponding IC50
curves are reported in Supporting Information
. All the tested compounds displayed low micromolar activity against CARM1, the insertion of methyl as well as chloro substituents at the N1-benzyl moiety having only modulator effects on enzyme inhibition. The preferred position to introduce a methyl group at the benzyl portion seems to be the ortho position (compound 8a
), while for chlorine insertion the benzyl meta position afforded the highest inhibitory activity (compound 8e
), similar to that of the lead compound 7g
IC50 values of 7g and 8a-f,j-l against CARM1, PRMT1, and SET7.
All the tested compounds were selective towards CARM1, they showing very low (if any) activity against PRMT1 and SET7. Among them, we selected 7g, 8e, and 8l for further experiments: 7g and 8e were the most potent inhibitors of CARM1 with PABP1 as a substrate (see ), while 8l was the only analogue carrying a structural diversity, the carbonyl group at the substituent at N1, that could influence someway its binding with the enzyme and its inhibitory behavior.
First, we repeated the CARM1 assay testing 7g, 8e
, and 8l
at 100 μM by fluorograph and using four different CARM1 substrates: PABP1, CA150,22
the spliceosome protein SmB,22
and histone H3 ().
Figure 3 Inhibitory activity of 7g, 8e, and 8l against CARM1 using PABP1, CA150, SMB, and histone H3 as substrates, and against a panel of PRMTs (PRMT1, PRMT3, PRMT5, and PRMT6) using indicated histone and/or non-histone substrates. The fluorographs are shown (more ...)
All the three tested compounds strongly inhibited the CARM1 activity on the various substrates; among these, CA150 was the most sensitive whereas the use of histone H3 yielded the lowest CARM1 inhibition. To check the real selectivity of 7g, 8e
, and 8l
against various PRMTs, we tested them at 100 μM against i) PRMT1 using NPL3 and histone H4 as a nonhistone and histone substrate, respectively, ii) PRMT3 using NPL3 and the ribosome protein rpS223
as substrates, iii) PRMT5 and histone H4 as a substrate, iv) PRMT6 using NPL3 and histone H3 as substrates ().
In addition, 7g, 8e, and 8l were tested at 100 μM against a panel of HKMTs, namely SET7 (substrate: H3), DOTL1 (substrate: nucleosome), Suv39H1 (substrate: H3), and G9a (substrate: H3) (). Against PRMTs, 7g and 8e were able to inhibit to some extent PRMT3, and 8e and 8l showed high inhibition of PRMT5 at 100 μM, nevertheless in all cases the observed inhibition values were weaker than those observed with CARM1 when used at the same concentration (see ). No significant activity at 100 μM was registered for 7g, 8e, and 8l against the tested HKMTs (see ).
Inhibitory activities of 7g, 8e, and 8l against a panel of HKMTs (SET7, DOTL1, Suv39H1, and G9a) using the indicated histone and/or non-histone substrates.
Known CARM1 substrates such as PABP1 are hypermethylated in vivo and this methylation is very stable. To test the efficacy of potential PRMT inhibitors in cell may require days of treatment, while waiting for the methylated substrates to turn-over. Under these conditions, compounds with pleiotropic effects would be difficult to investigate in a cell-based assay. To reduce the exposure time of the compound to cells, and bypass this problem, we developed an Flag-tagged PABP1 inducible cell line obtained by engineering a tetracycline-controlled transrepressor protein (TetR) in human embryonic kidney HEK293 cells.24
The TetR protein binds to tet operator (tetO) sequences in absence but not in the presence of tetracycline, silencing the transcriptional activities at the promoter.
We can thus easily distinguish between the endogenous PABP1 and the induced Flag-tagged PABP1 due to its slower migration by SDS-PAGE. We tested 7g, 8e
, and 8l
in this reporter system. Upon addition of tetracycline, Flag-PABP1 is induced in HEK293 cells in the presence of the indicated compound, and its methylation status can be detected by the use of a methyl-specific PABP1 antibody generated in our lab.24
In this reporter system, only 7g
was able to inhibit Flag-PABP1 methylation (Figure S4 in Supporting Information
There is increasing evidence of the involvement of CARM1 in hormone responsive cancers such as prostate cancer. Thus, we determined the effect of 7g, 8e, and 8l on prostate-specific antigen (PSA) promoter in human prostate adenocarcinoma LNCaP cells by using PSA luciferase assay, relative to a CMV-Renilla control (, top panel). In particular, we transfected PSA reporter into LNCaP cells, and then we treated the cells with increasing concentration of 7g, 8e, or 8l for two days.
Figure 5 Effects of increasing concentrations of 7g, 8e, and 8l on PSA promoter activity by luciferase assay in LNCaP cells, relative to a CMV-Renilla control (top panel), and on cell viability based on quantitation of the ATP present, which is an indicator of (more ...)
As seen in , a dose-dependent decrease of the reporter activity was observed with 7g and 8e up to 8-10 μM, while 8l was effective only at 30 μM. In parallel, we measured the cell viability through Cell Titer-Glo (CTG), based on quantitation of the ATP present (, bottom panel). This was done to confirm that the observed PSA effects were the results of CARM1 inhibition, and to rule out involvement of other targets and/or cell death. 7g and 8l displayed no or little effects on cell viability, at concentrations that impacted the luciferase assay.
In conclusion, we have reported on the ability of the 1-substituted-3,5-bis(3-bromo-4- hydroxybenzylidene)piperidin-4-ones 7g and 8a-l to selectively inhibit CARM1 activity. Compounds 7g, 8e, and 8l were able to inhibit CARM1-mediated methylation of different substrates (PABP1, CA150, SmB, and H3) up to single-digit micromolar level, displaying low inhibitor activity (if any) against a panel of different PRMTs or HKMTs. In human prostate cancer LNCaP cells, 7g showed a significant dose-dependent reduction of the PSA promoter activity, at concentration that did not affect cell viability.