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Finally, compound 7 was studied in more detail for its
HDAC isoform inhibition profile. While the nuclear ex-
tract containing a mixture of several HDAC isoenzymes
was inhibited in the submicromolar range (pIC50 = 6.4),
all tested isoenzymes were inhibited in the low micromo-
lar range (rHDAC1: pIC50 = 5.4; rHDAC3: pIC50 = 5.5;
rHDAC6: pIC50 = 5.9; rHDAC8: pIC50 = 4.8). There-
fore, the higher inhibitory activity of 7 in the nuclear
extract assay must be explained by another, not tested
HDAC isoform(s) or defined HDAC containing com-
plexes not represented in purified enzyme preparations.
The most potent inhibition was observed with HDAC6,
which is not present in the HeLa nuclear extract. Inhibi-
tion of isoenzymes HDAC1 and HDAC3 was similar,
while HDAC8 was significantly less potently inhibited.
HDAC inhibition in a cellular context through trithio-
carbonates was also demonstrated with 7 as a represen-
tative example by induction of histone H3
hyperacetylation at pIC50 = 5.7 in a high-content screen-
ing assay19 and inhibition of cellular HDAC enzymatic
activity in vital Hela cells with pIC50 = 5.3. These target
related data correlated well with the inhibition of prolif-
eration of several cancer cell lines (HeLa-cervical carci-
noma pIC50 = 5.6; A549-lung cancer: pIC50 = 5.2;
RKO-colon cancer: pIC50 = 5.7).
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We have demonstrated that trithiocarbonates are po-
tent, cellular active HDAC inhibitors. Based on the
inhibition of a HDAC isoenzyme mixture, a clear
SAR picture could be established for the cap-linker
and head groups. In enzyme kinetic studies with the
recombinant HDAC1 isoenzyme, a substrate competi-
tive mode-of-action of 1c was determined. This result
as well as the potent activity of chimeric SAHA ana-
logs 36 and the narrow SAR of the trithiocarbonate
motif makes it highly likely, that this motif complexes
Zn2+ in the active site of HDAC class I and II en-
zymes. Surprisingly and in contrast to the hydroxamate
and benzamide head groups, an extension of the head
group is compatible with HDAC inhibitory activity.
As exemplified by the phenyl or ethyl–phenyl residues
in 1f and 2e, even bulky moieties are potent submi-
cromolar inhibitors with reasonable cellular activity.
Defined modifications in the cap-linker moiety as
exemplified by analogs 7, 17, and 19 lead to a cellular
activity in the Alamar blue proliferation assay compa-
rable to that of SAHA (Table 3).
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Acknowledgments
12. Gu, W.; Nusinzon, I.; Smith, R. D., Jr.; Horvarth, C. M.;
Silverman, R. B. Bioorg. Med. Chem. 2006, 14, 3320.
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We thank M. Feth, J. Volz (physicochemistry) and H.
Wieland, H. Julius, and K. Fettis for excellent technical
assistance.
References and notes
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