Largazole Transformed Mammary Epithelial Cells (MDAMB-231) GI 50 : 7.7nM LC 50 : 117nM Nontransformed Mammary Epithelial Cells (NMuMG) GI 50 : 122nM LC 50 : 272nM Fibroblastic osteosarcoma cells (U2OS) GI 50 : 55nM LC 50 : 94nM Nontransformed Fibroblast (NIH3T3) GI 50 : 480nM LC 50 : >8µM Colon Cancer Line (HT29)* GI 50 : 12nM LC 50 : 22nM Neuroblastoma (IMR-32)* GI 50 : 16nM LC 50 : 22µM Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H. Journal of the American Chemical Society 2008, 130, i, K.; Paul, V. J.; Luesch, H. Journal of the American Chemical Society 2008, 130, 1806 S N H O O O S N S N NH O O
Ying, Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H. Journal of the American Chemical Society 2008, 130, 8455*Taori, K.; Paul, V. J.; Luesch, H. Journal of the American Chemical Society 2008, 130, 1806
Thiol AnaloguesHCT-116 growth inhibition (nM)
HCT-116 HDAC cellular assay (nM)
HeLa nuclear extracts HDAC’s (nM)
44±10 51±3 37±11
38±5 209±15 42±29
33±2 50±18 52±27
S NH
O O OSN
S
NNH
O
O
S NH
O O OSN
S
NNH
O
O
HS NH
O OSN
S
NNH
O
O
Ying, Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H. Journal of the American Chemical Society 2008, 130, 8455
Analogues with no ThiolHCT-116 growth inhibition (nM)
HCT-116 HDAC cellular assay (nM)
HeLa nuclear extracts HDAC’s (nM)
> 10000 > 10000 > 10000
> 10000 > 10000 > 10000HO N
H
O OSN
S
NNH
O
O
NH
O OSN
S
NNH
O
O
Ying, Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H. Journal of the American Chemical Society 2008, 130, 8455
• HCT-116 cell line growth inhibition experiment
• HDAC assays were performed with cell permeable fluorescent substrate
• Cytotoxicity for HT-116 cells not reported
Ying, Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H. Journal of the American Chemical Society 2008, 130, 8455
Importance of Thiol
• Need thiol to chelate Zn2+
• Analogue is active if hydrolysis to thiol is possible• Structural similarity to FK228
Ying, Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H. Journal of the American Chemical Society 2008, 130, 8455
Importance of Octanoyl Residue
• Free thiol is better at inhibiting HDAC• Octanoyl moeity increases cell permeability• Antiproliferation studies– Largazole (Red)• IC50: 45-315 nM
– Free Thiol (Blue)• IC50: 360-2600 nM
Bowers, A.; West, N.; Taunton, J.; Schreiber, S. L.; Bradner, J. E.; Williams, R. M. Journal of the American Chemical Society 2008, 130, 11219
• Proliferation studies of various malignant human melanoma cell lines
• Cell viability assay based on ATP content
Bowers, A.; West, N.; Taunton, J.; Schreiber, S. L.; Bradner, J. E.; Williams, R. M. Journal of the American Chemical Society 2008, 130, 11219
Features of Efficient HDAC inhibitors
• Hydrophobic region that binds the rim of the active site
• a coordinating group (warhead) to chelate Zn2+ at the bottom of the channel
• a five- to seven-atom linker from the hydrophobic region to the coordinating group
Ying, Y.; Liu, Y.; Byeon, S. R.; Kim, H.; Luesch, H.; Hong, J. Organic Letters 2008, 10, 4021
Linker Lengthn HCT-116 growth inhibition
(nM)HeLa nuclear extracts HDAC’s (nM)
1 >10000 >20000
2 6.8±0.6 32±13
3 620±50 7600±900
4 2500±600 4100±430
NH
O OSN
S
NNH
O
Sn-C7H15
On
O
Linker size of 4 is optimized for HDAC inhibition
Ying, Y.; Liu, Y.; Byeon, S. R.; Kim, H.; Luesch, H.; Hong, J. Organic Letters 2008, 10, 4021
Valine Residue and C17 VariationHCT-116 growth inhibition (nM)
HeLa nuclear extracts HDAC’s (nM)
Largazole 6.8±0.6 32±13
21±2 72±21
3900±450 >20000C7H15 S N
H
O O OSN
S
NNH
O
O
-Valine Residue can be replaced-Stereochemistry at C17 center is important
C7H15 S NH
O O OSN
S
NNH
O
O
Ying, Y.; Liu, Y.; Byeon, S. R.; Kim, H.; Luesch, H.; Hong, J. Organic Letters 2008, 10, 4021
MDA-MB231 (nM) HME (nM)
Largazole 71 >600
Terminal olefin (no linker) >600 >600
>600 >600
>600 >600
>600 >600
S NH
O OH OSN
S
NNH
MeO2C
O
O NH
O O OSN
S
NNH
O
O
6
NH
O O OSN
S
NNH
O
O
6
-Breast cancer cell line vs. normal mammary epithelial cells-Crystal Violet Dye
Nasveschuk, C. G.; Ungermannova, D.; Liu, X.; Phillips, A. J. Organic Letters 2008, 10, 3595
Linker Analogues
• None of these analogues were active up to 5µM.– Human Epithelial Carcinoma Cell Line (A432)– Preadipocyte Cell Line (3T3L1)
• No free thiol can be formed.• Synthetic Largazole showed weak selectivity for the cancer cell line GI50 = 49 vs
127nM• Largazole Thiol showed weaker potency but better selectivity GI50 = 126 vs
1200nM
NH
O OSN
S
NNH
O
O
NH
O OSN
S
NNH
O
Br
O
O NH
O O OSN
S
NNH
O
O
Seiser, T.; Kamena, F.; Cramer, N. Angewandte Chemie International Edition 2008, 47, 6483
S-acetyl, disulfide analoguesLargazole Free Thiol
60nM 38nM 50nM 70nM
S NH
O O OSN
S
NNH
O
O
SS N
H
O OSN
S
NNH
O
N
O
-ED50 values from reporter gene assay in HEK293 cell promoted by cytomegalovirus (CMV) (nM)-Ability of being hydrolized to free thiol
Numajiri, Y.; Takahashi, T.; Takagi, M.; Shin-ya, K.; Doi, T. Synlett 2008, 2008, 2483
• Cytomegalovirus promoter• Induction of promoter by largazole analogues• Measured Phosphatase units to calculate ED50
values
Numajiri, Y.; Takahashi, T.; Takagi, M.; Shin-ya, K.; Doi, T. Synlett 2008, 2008, 2483
Isostere AnaloguesHDAC 1 HDAC 2 HDAC 3 HDAC 6
Largazole 40 42 96 >1000
Free Thiol 0.1 0.8 1 40
>3000 >3000 >3000 >3000
0.9 4 4 1500
S NH
O NH OSN
S
NNH
O
O
HS NH
NH OSN
S
NNH
O
O
-Purified Human HDAC’s IC50 values in nM-Probably due to changed conformational space on nitrogen vs. oxygen
Bowers, A. A.; Greshock, T. J.; West, N.; Estiu, G.; Schreiber, S. L.; Wiest, O.; Williams, R. M.; Bradner, J. E. Journal of the American Chemical Society 2009, 131, 2900
IC50 of Human HDACs of Other Analogues
HDAC1 (µM) HDAC2 (µM) HDAC3 (µM) HDAC6 (µM)
Largazole Thiol 0.0012 0.0035 0.0034 0.049
Enantiomer 1.2 3.1 1.9 2.2
C-2 Epimer 0.030 0.082 0.084 0.68
0.11 0.80 0.58 13
>30 >30 >30 >30C7H15 S N
H
O O OSN
S
NN
O
O
NH
O OSN
S
NNH
O
O
CO2H
Bowers, A. A.; West, N.; Newkirk, T. L.; Troutman-Youngman, A. E.; Schreiber, S. L.; Wiest, O.; Bradner, J. E.; Williams, R. M. Organic Letters 2009, 11, 1301
• Pyridine substitution of thiazol resulted in increased potency
• Methyl group in C-7 position is not important• Linker analogues did not result in increased
potency• Enantiomer potency decreased by 3 orders of
magnitude
• Also synthesized but not tested
NH
O OSN
S
NNH
O
O
HS
O
NH
O OSN
S
NNH
O
O
HS
O
Bowers, A. A.; West, N.; Newkirk, T. L.; Troutman-Youngman, A. E.; Schreiber, S. L.; Wiest, O.; Bradner, J. E.; Williams, R. M. Organic Letters 2009, 11, 1301
C-7 demethylationHDAC1 HDAC2 HDAC3 HDAC6 HCT-116
Largazole 0.0137 0.190 0.245 11.5 0.025±0.0004
2.0 18.5 16.8 14.7 24.1±3.0
NA NA NA NA >100
Macrocycle dimer NA 51.8 22.6 NA 26.8±3.7
S NH
O O OSN
S
NNH
O
O
S NH
O O OSN
S
NNH
O
O
-Purified Human HDAC’s. IC50 Values in µM-C-7 demethylation decreases potency-C17 stereochemistry is crucial-Macrocycle dimer was surprisingly active in HDAC 2 and 3 but not 1
-Purified Recombinant HDAC’s-Size of substituent on C-7 position does not affect activity
Souto, J. A.; Vaz, E.; Lepore, I.; Poppler, A.-C.; Franci, G.; Alvarez, R.; Altucci, L.; de Lera, A. n. R. Journal of Medicinal Chemistry 2010, 53, 4654
HDAC inhibition (nM) MCF-7 inhibition (nM)
Largazole 572±29 5±1
Free Thiol 0.043±0.026 277±130
17.2±2.3 377±62
0.17±0.05 2458±1135
3.15±0.35 >10000
0.99±0.07 5902±1698
HS NH
O OSN
S
NNH
O
O
HS NH
O OSN
S
NNH
O
O
HS NH
O OSN
NH
O
O NHO
HS NH
O OSN
S
NNH
O
O
Ph
Bene
lkeb
ir, H
.; M
arie
, S.;
Hay
den,
A. L
.; Ly
le, J
.; Lo
adm
an, P
. M.;
Crab
b, S
. J.;
Pack
ham
, G
.; G
anes
an, A
. Bio
orga
nic
& M
edic
inal
Che
mist
ry, I
n Pr
ess,
Cor
rect
ed P
roof
• Valine, glycine, and thiazoline groups could be replaced with slight decrease in activity
• Cell growth inhibition does not correlate necessarily with HDAC inhibition
• Half life of compounds is a concern• No specification on what HDAC was used
Benelkebir, H.; Marie, S.; Hayden, A. L.; Lyle, J.; Loadman, P. M.; Crabb, S. J.; Packham, G.; Ganesan, A. Bioorganic & Medicinal Chemistry, In Press, Corrected Proof
