PhD Defense

August 02, 2007

Molecular Design of
Histone Deacetylase Inhibitors with
Aromatic Ring Arrangement

Gururaj Mahadev Shivashimpi
Student Number: 04897021-1
Graduate School of Life Science and Systems Engineering
Kyushu Institute of Technology, Kitakyushu, Japan

Basis of the present work

HDAC8

A family of enzymes known as histone deacetylases (HDACs) helps to regulate
how and when our genome blueprint is transcribed and translated into protein.

HDAC enzymes have proven to be exciting and promising novel targets for the
treatment of solid tumors and hematological cancers.

Inhibitors of these enzymes are proposed to function through their ability to modify
the acetylation of histone tails, modifying the expression of oncogenes and tumor
suppressor genes, and rescuing normal cell growth and differentiation.

Small-molecule inhibitors of these enzymes are thus under intense investigation
as cancer therapeutics, as evidenced by extensive research in this field.

Kyushu Institute of Technology

• Introduction
• Molecular design of histone deacetylase Inhibitors by aromatic ring
shifting
– Introduction
– Aromatic ring shifting in chlamydocin framework
– Variation of spacer length of L-Phe in chlamydocin
– Conformational changes due to aromatic ring shifting
• Molecular design of non-peptide inhibitors
– Introduction
– SAHA analogues
– TSA like non-peptides
arrangement on cyclic dipeptide hydroxamic acids
– Introduction
– Diketopiperazine hydroxamic acids containing iminoacids
– Diketopiperazine hydroxamic acids containing α, α,-dialkylated
amino acids
• Conclusions
• References

Introduction
Schematic structure of nucleosome

Major post translational
modifications

A Acetylation

M Methylation

Ub Ubiquitination

P Phosphorylation

Mai, A., Massa, S., et al. Med. Res. Rev. 2005, 25, 261–309.

The nucleosome, the fundamental unit of chromatin, consists of a histone octamer wrapped
with 146 base pairs of DNA . The histone octamer composed each of the four core histones
H2A, H2B,H3 and H4. The basic N-terminal histone tails protrude from the core nucleosome.


Histone tail acetylation and bromodomain

de la Cruz, X., et al. Bioessays 2005, 27, 164-175.


Structure and functions of bromodomain

• It adopts typical left handed four helix bundle shape

(NMR studies).

• Interaction Ac-Lys occurs at hydrophobic pocket

• It plays important role in

- chromatin acetylation through HAT recruitment

- organizing chromatin domains

- recognizing acetylated non-histone proteins

Figure. Structure of bromodomain

Dhalluin, C., et al. Nature 1999, 399, 491-496.


Histone deacetylases

HDAC enzymes deacetylate the ε-amino groups of lysine residues in the N-terminal tails
of core histones leading to a compacted chromatin structure due to strong interaction of ε-
amino lysine residues with DNA.
- Gene transcription repressed by altering the accessibility of transcription
factors to DNA.
- Inappropriate recruitment of HDAC enzymes by oncogenic proteins may
alter gene expression in favor of arrested differentiation, and/or excessive proliferation.
- Inhibition of these HDACs leads to the accumulation of acetylated histones to
activate transcription causing inhibition of tumor cell growth and induction of apoptosis.


HDAC isoforms

Zn+2 ion
dependent
enzymes

NAD
dependent Sir2
family


Historical opening: Structure of A. aeolicius HDLP active site
bound to trichostatin A

Finnin, M. S., Donigian, J. R., et al. Nature 1999, 401, 188-193.


Proposed catalytic mechanism for the deacetylation of Ac-Lys

Finnin, M. S., Donigian, J. R., et al. Nature 1999, 401, 188-193.


Non-peptide HDAC inhibitors with different zinc ligands

Naturally occurring inhibitors Synthetic inhibitors

Miller, T. A., et al. J. Med. Chem. 2003, 46, 5097-5116.

Naturally occurring cyclic tetrapeptide inhibitors

Containing epoxy ketone moiety Containing ketone moiety

Trapoxin A (n = 2), Trapoxin B (n = 1) FR235222

Tan-1746
Chlamydocin

Apicidin

Cyl 1 (n= 1), Cyl 2 (n= 2)


How to design an HDAC inhibitor?

Three major features
- terminal group to bind the zinc in the
active site of enzymes

- linker unit, residing in the channel

- capping group that principally occupies
the external entrance to the channel of
the enzyme

Structure of histone deacetylase-like
protein (HDLP) co-crystallized with TSA *

*Finnin, M. S., Donigian, J. R., et al.
Nature 1999, 401, 188-193.


Cyclic tetrapeptide based synthetic HDAC inhibitors

Cancer Res. 2001, 61, 4459-4466. Bioorg. Med. Chem. Lett. 2004, 14, 2427-2431.

Org. Lett. 2003, 05, 5079-5082. Bioorg. Med. Chem. Lett. 2004, 14, 5343-5346.


Purpose of present study

X-ray crystal study reveals,
favorable interaction of cap group of inhibitor and HDAC enzyme
may afford better binding affinity

Molecular design of inhibitor by arranging aromatic rings in cap group


• Introduction
• Molecular design of histone deacetylase inhibitors by aromatic ring
shifting
– Introduction
– Introduction
– SAHA analogues
– Introduction
amino acids
• Conclusions
• References

Chlamydocin scaffold

Cyclic imino
acid site
O Spacing chain

O N HN X
NHHN
NHHN O Zn2+ ligand
toward reactive site
Aromatic group
site O

Aib site


Aromatic ring replacement in chlamydocin framework
(Work carried out by Dr. Preeti and Ms. Li)

L-Ala

L-Leu

Design of cyclic tetrapeptides

cyclo(-L-Am7(S2Py)-Aib-L-Ala-D-Pro-)
L-Ile
cyclo(-L-Am7(S2Py)-Aib-L-Leu-D-Pro-)

cyclo(-L-Am7(S2Py)-Aib-L-Ile-D-Pro-)

cyclo(-L-Am7(S2Py)-Aib-L-Nle-D-Pro-)

L-Nle


Assay methods
HDAC Inhibitory Activity (Cell free assay) Excitation = 380 nm,
Emission = 460 nm
Enzyme/buffer 18 µL
Inhibitor/DMSO 1 µL Trypsin/buffer 30 µL
Substrate/DMSO 1 µL
30 min., 370C 15 min., 370C

Substrate: KGLGK-MCA KGLGK and AMC
KGLGK(Ac)-MCA KGLGK(Ac)-MCA KGLGK(Ac)-MCA
Fluorescence intensity of AMC was
measured for the HDAC activity
IC50 = Concentration of 50% inhibition
Enzyme: HDAC1
(100% = no enzyme)
1 x 107 293T cell i) Homogenize ii) Centrifuge
iii) Agarose beads, 4 0C, 1 h.
HDAC1
pcDNA-3-HD1 1 mg
Buffer (modified lysis buffer)
Lipofect Amine 2000 reagent iv) Wash agarose beads
50 mM Tris·HCl (pH 7.5)
120 mM NaCl
p21 Promoter Assay (Cell based assay) 5 mM EDTA
0.5% Nonidet P-40
MFLL-9 cell 85000 unit/well
Inhibitor/DMSO 10 mM

6h 18 h

Merged gene of wild human p21 Luminescent intensity was
promoter and luciferase measured for the luciferace activity

Enzyme inhibition and biological activity
(With 0.1 mM DTT)

.

*column: Chromolith performance RP-18e (100 x 4.6 mm)
eluent: 10-100% CH3CN gradient containing 0.1% TFA over 15 min.

• Replacing of aromatic ring of L-Phe in chlamydocin showed 10 fold less activity in p21
promoter assay.
• Aromatic ring is necessary in cyclic cap group for better binding with surface of HDAC enzyme.
• By these results, further investigations were carried out by focusing on aromatic ring in the
chlamydocin framework.

Naturally occurring cyclic tetrapeptide inhibitors

Containing epoxy ketone moiety Containing ketone moiety

Trapoxin A (n = 2), Trapoxin B (n = 1) FR235222

Tan-1746
Chlamydocin

Apicidin

Cyl 1 (n = 1), Cyl 2 (n = 2)


Aromatic ring shifting in chlamydocin framework

cyclo(-L-Am7(S2Py)-A2in-L-Ala-D-Pro-)
cyclo(-L-Am7(S2Py)-D-A1in-L-Ala-D-Pro-)
cyclo(-L-Am7(S2Py)-L-A1in-L-Ala-D-Pro-)
cyclo(-L-Am7(S2Py)-D-2MePhe-L-Ala-D-Pro-)
cyclo(-L-Am7(S2Py)-L-2MePhe-L-Ala-D-Pro-)
cyclo(-L-Am7(S2Py)-Aib-L-Ala-D-Tic-)

A2in A1in 2MePhe Tic

• Shifting the aromatic ring of L-Phe in chlamydocin may give different orientations for
aromatic ring.
• To know proper orientation of aromatic ring showing better binding affinity in inhibition
of HDAC enzymes.

Cyclic framework Possible orientations of benzene ring
on cyclic framework

His
+2 Asp
S- Zn
Asp

Surface of HDAC enzyme


Synthesis of cyclic tetrapeptides
L-Ab7* AA L-Ala D-Pro/D-Tic

Z OH H OtBu
DCC, HOBt
Z OtBu
H2, Pd-C
Pd-
Z OH H OtBu
DCC, HOBt
Z OtBu
H2, Pd-C
Pd-
Boc OH H OtBu
DCC, HOBt
Boc OtBu
TFA
TFA・
TFA・H OH
HATU, DIEA
cyclo（
cyclo（）

*L-Ab7: L-2-amino-7-bromo heptanoic acid.
AA: A2in, DL-A1in, DL-2MePhe


Scheme 1. Reagents and conditions: (a) AcSK, DMF, r. t.; (b) 2,2’-dipyridyl disulphide, MeNH2/MeOH

cyclo(-L-Am7(S2Py)-A2in-L-Ala-D-Pro-) cyclo(-L-Am7(S2Py)-D-A1in-L-Ala-D-Pro-) cyclo(-L-Am7(S2Py)-L-A1in-L-Ala-D-Pro-)

cyclo(-L-Am7(S2Py)-D-2MePhe-L-Ala-D-Pro-) cyclo(-L-Am7(S2Py)-L-2MePhe-L-Ala-D-Pro-) cyclo(-L-Am7(S2Py)-Aib-L-Ala-D-Tic-)


Variation of spacer length of L-Phe in chlamydocin framework

n = 0, 2, 3

cyclo(-L-Am7(S2Py)-Aib-L-Phg-D-Pro-)
cyclo(-L-Am7(S2Py)-Aib-L-Ph4-D-Pro-)
cyclo(-L-Am7(S2Py)-Aib-L-Ph5-D-Pro-)
cyclo(-L-Am7(S2Py)-Aib-L-Ser(Bzl)-D-Pro-)
cyclo(-L-Am7(S2Py)-Aib-L-Ser-D-Pro-)

Phg Ph4 Ph5 Ser(Bzl) Ser


Variation of spacer of L-Phe in chlamydocin may show
better binding Affinity in inhibition of HDAC enzymes.

Cyclic framework

( )n

S- O

Zn2
＋
＋
＋
＋ His

Asp
Asp
Surface of HDAC enzyme HO


Synthesis of cyclic tetrapeptides

L-Ab7* Aib AA D-Pro
Z OH H OtBu
DCC, HOBt
Z OtBu
H2, Pd-C
Pd-
Z OH H OtBu
DCC, HOBt
Z OtBu
H2, Pd-C
Pd-
Boc OH H OtBu
DCC, HOBt
Boc OtBu
TFA
TFA・
TFA・H OH
HATU, DIEA
cyclo（
cyclo（）

*L-Ab7: L-2-amino-7-bromo heptanoic acid.
AA: L-Phg, L-Ph4, L-Ph5, L-Ser(Bzl) and L-Ser


Scheme 1. Reagents and conditions: (a) AcSK, DMF, r.t.; (b) 2,2’-dipyridyl disulphide, MeNH2/MeOH

cyclo(-L-Am7(S2Py)-Aib-L-Phg-D-Pro-) cyclo(-L-Am7(S2Py)-Aib-L-Ph4-D-Pro-) cyclo(-L-Am7(S2Py)-Aib-L-Ph5-D-Pro-)

cyclo(-L-Am7(S2Py)-Aib-L-Ser-D-Pro-)
cyclo(-L-Am7(S2Py)-Aib-L-Ser(Bzl)-D-Pro-)


(With 0.1 m M DTT)

*column: Chromolith performance RP-18e (100 x 4.6 mm). Eluent: 10-100% CH3CN gradient containing 0.1% TFA over 15 min.
† Selectivity among HDAC1 and HDAC4


Enzyme inhibition and biological activity of diastereomers
(With 0.1 mM DTT)
IC50 (µM) p21 promoter assay.
No. Compounds
HDAC1 HDAC4 HDAC6 EC1000 (µM)

4 cyclo(-L-Am7(S2Py)-D-A1in-L-Ala-D-Pro-) 0.0027 0.0024 0.0120 0.055

5 cyclo(-L-Am7(S2Py)-L-A1in-L-Ala-D-Pro-) 0.0360 0.0250 0.0329 2.0
6 cyclo(-L-Am7(S2Py)-D-2MePhe-L-Ala-D-Pro-) 0.1700 0.0700 0.0710 25.6
7 cyclo(-L-Am7(S2Py)-L-2MePhe-L-Ala-D-Pro-) 0.0037 0.0022 0.0560 0.55

1. Conformational analysis of diastereomers by CD spectra

2. cyclo(-L-Am7(S2Py)-Aib-L-Phe-D-Pro-)
4. cyclo(-L-Am7(S2Py)-D-A1in-L-Ala-D-Pro-)

5. cyclo(-L-Am7(S2Py)-L-A1in-L-Ala-D-Pro-)

6. cyclo(-L-Am7(S2Py)-D-2MePhe-L-Ala-D-Pro-)
7. cyclo(-L-Am7(S2Py)-L-2MePhe-L-Ala-D-Pro-)

• At 220 nm region, compounds 4 and 7 show
-ve ellipticity, but 5 and 6 show +ve ellipticity.
• Compounds with –ve ellipticity show good
biological activity.


2. Conformational analysis of diastereomers by NMR spectrometry

Compound 4 Compound 5


Molecular architecture of HDAC1 and HDAC8 with compound 4
(Study carried out by Dr. Y. Hirashima )

HDAC1 & Comp. 4

• Compound 4 binds in the active site of
HDAC1 and HDAC8.
• The thiol group, attached with its long
aliphatic chain lying in the 11 Å channel,
binds to the Zn+2ion at the bottom of this
channel.
• In HDAC1, the aromatic ring of D-A1in is bent
toward Glu98, Asp99, and Pro29 residues
• In HDAC8 the aromatic ring if D-A1in is bent
Human HDAC8 & Comp.4 toward Pro273 and Tyr306

Summary

• We have synthesized a library of novel chlamydocin analogs by shifting the
aromatic ring of L-Phe and also varying its spacer length

• The cell-free and cell-based HDAC inhibitory activity of the inhibitors was
evaluated and it was found that most of the inhibitors are potent toward HDACs.

• The cyclic tetrapeptides 4 and 12 containing D-A1in and L-Ser(Bzl) were found
potent inhibitors among synthesized compounds..

• Conformational changes were observed in diastereomers 4/5 and 6/7 due to
aromatic ring shifting.

• Docking of compound 4 with D-A1in into HDAC1 and HDAC8 revealed the
interaction of aromatic ring in cap group with hydrophobic residues on surface of
HDAC enzymes.


Non-peptide HDAC inhibitors
1. Non-peptide HDAC inhibitors with aromatic cap group:

Trichostatin A SAHA
Oxamflatin

2. Non-peptide HDAC inhibitors without aromatic cap group:

Depudecin Butyric acid Valproic acid

(Miller, T. A., et al. J. Med. Chem. 2003, 46, 5097-5116)

• Most of non-peptides containing aromatic cap group are found to be potent inhibitors
of HDACs, than those inhibitors which do not have aromatic cap group.
• Aromatic cap group is needed to interact with surface of HDAC enzymes for better
inhibition.

Recently reported potent non-peptide HDAC inhibitors
(Potent non-peptide HDAC inhibitors containing bulkier aromatic cap groups)

(Shinji, C., et al. Bioorg. Med. Chem. 2006, 14, 7625-7651)

(Marson, C., et al. Bioorg. Med. Chem. Lett.
2006, 17, 136-141) (Marson, C., et al. J. Med. Chem. 2006, 49, 800-805)


Design of non-peptide inhibitors with aromatic cap group in the light
of cyclic tetrapeptide thioethers
O
• Chlamydocin thioether analogues have cyclic
O N HN R
S tetrapeptide cap group.
NH HN O • These compounds were found potent in inhibiting

O HDACs.
Hirashima, Y., et al. • But, synthesis of cyclic tetrapeptides is laborious
Proceedings of 29tth European Peptide Symposium
and expensive process.
(J. Pep. Sci.) 2007 (in press).

1. SAHA analogues 2. TSA like non-peptides
O O
O
H OH
N OH N
N H
H N
O

H O
N S
R S
R
O n= 1, 2
R1
O

R = -CH2COCF3, -CH2COCH3, -CH2-4-C5H4N, -CH2C6H5, -CH2COC6H5, and -S-2-C5H4N
R1 = -H, or –CONHCH3


• Introduction
shifting
– Introduction
– Introduction
– SAHA analogues
– Introduction
amino acids
• Conclusion
• References

Synthesis of SAHA analogues

Scheme 1. Reagents and conditions: (a) AcSK, DMF, r.t.; (b) MeNH2/MeOH, R-Br, Et3N

Scheme 2. Reagents and conditions: (a) o-amino thiophenol, Et3N, DMF, r.t.

Scheme 3. Reagents and conditions: (a) 2,2’-dipyridyl disulphide, MeNH2/MeOH, DMF, r.t.



eluent: 10-100% CH3CN gradient containing 0.1% TFA over 15 min., flow = 2 mL/min.

Synthesis of trichostatin A like non-peptides

Scheme 4. Reagents and conditions: (a) AcSK, DMF, r.t.; (b) MeNH2/MeOH, R-Br, Et3N

Scheme 5. Reagents and conditions: (a) MeNH2/MeOH, DMF, Bzl-Br


Scheme 6. Reagents and conditions: (a) Br(CH2)5Br/Br(CH2)4Cl, NaOEt, EtOH.; (b) AcSK, DMF, r.t.;
(c) MeNH2/MeOH, bromomethyl pyridine.HBr, Et3N

Scheme 7. Reagents and conditions: (a) Br(CH2)5COOEt/Br(CH2)6COOEt, NaOEt, EtOH.; (b) HCl.NH2-OH
KOH in MeOH


Summary

• We have synthesized a library of non peptides inhibitors containing aromatic cap
groups similar to SAHA and TSA.

• We replaced the hydroxamic acid group of SAHA and TSA by several thioethers.

• The cell-free and cell-based HDAC inhibitory activity of inhibitors was evaluated
and it was found that most of the inhibitors shown activity in micromolar range

• Among SAHA analogues, the S-S hybrid shown better activity in presence of
DTT.

• Presence of methyl amide in cap group region of TSA like compounds didn't
show any abrupt change in activity.

• These non-peptide thioethers showed less activity as compared to cyclic
tetrapeptide thioethers, due to smaller aromatic cap groups.


Amino suberoyl hydroxamic acid type HDAC inhibitors
1. Fairlie, D. P., et al. J. Med. Chem. 2006, 49, 7611-7622.

2. Miller, T., et al. Bioorg. Med. Chem. Lett. 2007 (in press).

3. Etzkorn, F. A., et al. J. Med. Chem. 2007, 50, 2003 -2006.


Design of diketopiperazine hydroxamic acids

• Diketopiperazines are smallest cyclic dipeptide scaffolds found in many bioactive
molecules.
• The 2-amino-8-hydroxamido octanoic acid was incorporated into cyclic dipeptide
framework.
• Arrangement of aromatic ring on cyclic dipeptide framework, to know the binding
affinity of DKP cap group with surface of HDAC enzyme.


• The diketopiperazine cyclic framework may act as cap group and
• Hydroxamic acid group will chelate with the Zn+2 ion of HDAC enzyme

H
N O His
H Zn+2
O N O
Asp

R N O Asp
H

Surface of Enzyme Active site pocket


Design diketopiperazine hydroxamic acid containing
iminoacids

Design of cyclic dipeptides
cyclo(-L-Asu(NHOH)-L-Pro-)
cyclo(-L-Asu(NHOH)-D-Pro-)
cyclo(-L-Asu(NHOH)-L-Tic)
cyclo(-L-Asu(NHOH)-D-Tic-)
cyclo(-L-Asu(NHOH)-L-MePhg-)
cyclo(-L-Asu(NHOH)-D-MePhg-)
cyclo(-L-Asu(NHOH)-BzlGly-)

HN OH HN OH HN OH HN OH
R=
O O O O

Pro Tic MePhe BzlGly


Synthesis of diketopiperazine hydroxamic acids

R = -CH3, -Bzl
AA = DL-Pro, DL-Tic, DL-MePhe and
BzlGly

O O
R1 R1
NH O NH O b
a
HN HN
O OH
O O

O O
R1 c R1
NH O NH O
HN O HN OH
N N
H H
O O

R1 = DL-Pro, DL-Tic, DL-MePhe and BzlGly
Scheme 1: Reagents and conditions (a) Pd-C, MeOH, H2. (b) HCl.H2N-OBzl, DCC, HOBt.H2O, Et3N
(c) Pd-BaSO4, AcOH, H2

Design diketopiperazine hydroxamic acid containing
α ,α-dialkylated amino acids

Design of cyclic dipeptides

cyclo(-L-Asu(NHOH)-DL-A1in-)
cyclo(-L-Asu(NHOH)-A2in-)
cyclo(-L-Asu(NHOH)-DL-2MePhe)
cyclo(-L-Asu(NHOH)-L-2MePhg-)
cyclo(-L-Asu(NHOH)-D-2MePhg-)

R= NH N N
H
NH
O H O O
O

A1in A2in 2MePhe 2MePhg


Synthesis of diketopiperazine hydroxamic acids

R = -CH3, -C2H5, -Bzl,
AA = DL-A1in, A2in, DL-2MePhe and
DL-2MePhg

O O
R1 a R1
NH O NH O
HN HN OH
O N
H
O O

R1 = DL-A1in, A2in, DL-2MePhe and DL-2MePhg

Scheme : Reagents and conditions (a) HCl.H2N-OH, NaOMe/MeOH, r.t.


Summary

• We have synthesized a library of diketopiperazine hydroxamic acids by
introducing 2-amino-8-hydraxamido octanoic acid into cyclic dipeptide
framework..

• We did the arrangement of aromatic rings on cyclic dipeptide framework by
incorporating several imino acids and α,α-dialkylated amino acids.

• The cell-free and cell-based HDAC inhibitory activity of inhibitors was evaluated
and it was found that most of the inhibitors shown activity in micromolar range

• Compound 5 containing D-Tic showed good activity among synthesized
compounds

• Though hydroxamic acid is potent zinc ligand, but interaction of cap group with
surface of enzyme was not up to satisfactory level.


Conclusions
• We have successfully synthesized a library of novel histone deacetylase inhibitors by arrangement
of aromatic ring on different cap groups like cyclic tetrapeptides, non-peptides and
diketopiperazines.

• The cell-free and cell-based HDAC inhibitory activity of the inhibitors was evaluated and it was
found that some of the compounds showed exciting results and some were disappointing.

• By the aromatic ring shifting in chlamydocin framework conformational changes were observed in
diastereomers. It further revealed an importance of aromatic ring position at cap group region ,
needed for better interaction with surface of HDAC paralogs.

• Lesser activity of non-peptides thioethers (with cap groups similar to SAHA and TSA) as compared
to cyclic tetrapeptide thioethers supports the fact that, macrocyclic cap group with proper
orientation of aromatic ring on its cyclic framework is necessary for better inhibition of HDACs.

• Primitive studies on diketopiperazine hydroxamic acids didn’t show expected activity, may due to
insufficient interaction of aromatic amino acids of diketopiperazine scaffold with the surface of
enzyme, which needs further investigation.

• To conclude, cyclic tetrapeptides with proper orientation of aromatic ring on their macrocyclic cap
group, are showing better interaction with surface of HDACs by inhibiting potently, as compared to
non-peptides and cyclic dipeptides.

• Therefore, cyclic tetrapeptide based HDAC inhibitors can be the challenging antitumor agents


References
1. Grizinger, C. M., Schreiber, S. L. Chem. Biol. 2002, 9, 3-16.

2. Kouzarides, T. Curr. Opin. Genev. Dev. 1999, 9, 48.

3. Hassig, C. A., Schreiber, S. L. Curr. Opin. Chem. Biol. 1997, 1, 300-308.

4. Yoshida, M., Horinouchi, S., Beppu, T., Bioessays 1995, 17, 423-430.

5. Finnin, M. S., Donigian, J. R., Cohen, A., et al. Nature 1999, 401, 188-193.

6. Jose, B., Oniki, Y., Kato, T., Nishino, N., Sumida, Y., Yoshida, M. Bioorg. Med. Chem. Lett. 2004,
14, 5343-5346.

7. Furumai, R., Komatsu, Nishino, N., Khochbin, S., Yoshida, M., Horinouchi, S. Proc. Natl. Acad.
Sci. U.S.A. 2001, 98, 87-92.

8. Komatsu, Y., Tomizaki, K., Tsukamoto, M., Kato, T., Nishino, N., Sato, S., Yamori, T., Tsuruo, T.,
Furumai, R., Yoshida, M. Horinouchi, S., Hayashi, H. Cancer Res. 2001, 61, 4459.

9. Nishino, N., Jose, B., Okamura, S., Ebisusaki, S., Kato, T., Sumida, Y., Yoshida, M., Org. Lett,
2003, 5, 5079-5082.

10. Nishino, N., Jose, B., Shinta, R., Kato, T., Komatsu, Y., Yoshida, M. Bioorg. Med. Chem. Lett.
2004, 12, 5777-5784.


Conference Presentations

1. Poster presentation: “ Design and synthesis of histone deacetylase inhibitors by aromatic ring shifting in

chlamydocin framework”. 29th European Peptide Symposium, Gdansk, Poland (2006).

Gururaj M.Shivashimpi, Satoshi Amagai, Tamaki Kato, Norikazu Nishino, Satoko Maeda,

Tomonori G. Nishino and Minoru Yoshida.

2. Poster Presentation: “Aromatic ring shifting in chlamydocin framework for specific inhibition of histone

deacetylase paralogs.” 43rd Japanese Peptide Symposium, Yokohama. Japan.(2006).

Gururaj M.Shivashimpi. Satoshi Amagai, Tamaki Kato, Norikazu Nishino, Junichi Nakagawa, Satoko

Maeda, Tomonori G. Nishino and Minoru Yoshida.

3. Poster presentation: “Toward potent HDAC inhibitor design: Insights from homology modeling and docking

simulation of HDAC and its inhibitors”. 5th East Asian Biophysics Symposium & 44th Annual Meeting of

the Biophysical Society of Japan, Okinawa, Japan (2006).

Hirashima Y., Shivashimpi G. M., Kato T., Nishino N., Maeda S., Nishino T. G., Yoshida M.


List of publications
1. Design and synthesis of histone deacetylase inhibitors by aromatic ring shifting in chlamydocin framework.
Gururaj M. Shivashimpi, Satoshi Amagai, Tamaki Kato, Norikazu Nishino, Satoko Maeda, Tomonori G.
Nishino and Minoru Yoshida.
Proceedings of 29th European Peptide Symposium (J. Pep. Sci.) 2007 (in press)
2. Aromatic ring shifting in chlamydocin framework for specific inhibition of histone deacetylase paralogs.
Gururaj M. Shivashimpi. Satoshi Amagai, Tamaki Kato, Norikazu Nishino, Junichi Nakagawa, Satoko
Maeda, Tomonori G. Nishino and Minoru Yoshida.
Proceedings of 43rd Japanese Peptide (Peptide Science), 2006, 268-269.
3. Molecular design of histone deacetylase inhibitors by aromatic ring shifting in chlamydocin framework
Gururaj M. Shivashimpi, Satoshi Amagai, Tamaki Kato, Norikazu Nishino, Satoko Maeda, Tomonori G.
Nishino and Minoru Yoshida.
Submitted to Bioorganic and Medicinal Chemistry.
4. Interaction of aliphatic cap group in inhibition of histone deacetylase inhibitors by cyclic tetrapeptides.
Norikazu Nishino, Gururaj M. Shivashimpi, Preeti B. Soni, Mohammed P. I. Bhuiyan, Tamaki Kato,
Satoko Maeda, Tomonori G. Nishino and Minoru Yoshida.
Submitted to Bioorganic and Medicinal Chemistry.
5. Effect of arrangement of aromatic ring on cyclic dipeptides containing 2-amino-8-hydroxamido octanoic
acid.
Gururaj M. Shivashimpi, Tamaki Kato, Norikazu Nishino, Satoko Maeda, Tomonori G. Nishino and
Minoru Yoshida. To be submitted to Bioorganic and Medicinal Chemistry.


Acknowledgements
I am highly grateful to my supervisor Professor Norikazu Nishino (Kyushu Institute of Technology) for
giving me the rare opportunity to engage in this study and for arranging financial support throughout the
work.

My sincere gratitude to Prof. Tamaki Kato (Kyushu Institute of Technology) for his all time cooperation.

I express my thanks to Ms. Satoko Maeda and Prof. Minoru Yoshida (RIKEN) for measurements of
HDAC inhibitory activities

I would like to thank Dr. S. S. Pandey, Dr. Binoy Jose, Dr. M. P. I. Bhuiyan, and Dr. M. Muthukrishnan for
their support and encouragement throughout my research.

Many thanks to my Japanese friends Dr. L. Watanabe, Dr. Y. Hirashima, Mr. S. Okamura, Mr. S.
Ebisusaki and Mr. S. Amagai for their support and help in learning the peptide synthesis.

Many thanks to the present and past members of Nishino Laboratory for their kind support, encouragement
and making me to feel at home.

Finally, I am very much thankful to my father Mahadev C. Shivashimpi (Ret. High school Teacher), mother
Neelambika M. Shivashimpi, brother Veerendra, sisters Veeshalaxi and Shilpa, who were always
encouraging me during this work.

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