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Molecular Design of Histone Deacetylase Inhibitors with Aromatic Ring Arrangement

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PhD Defense

  1. 1. 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
  2. 2. 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
  3. 3. • 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 • Molecular design of histone deacetylase Inhibitors by aromatic ring arrangement on cyclic dipeptide hydroxamic acids – Introduction – Diketopiperazine hydroxamic acids containing iminoacids – Diketopiperazine hydroxamic acids containing α, α,-dialkylated amino acids • Conclusions • References
  4. 4. 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. Kyushu Institute of Technology
  5. 5. Histone tail acetylation and bromodomain de la Cruz, X., et al. Bioessays 2005, 27, 164-175. Kyushu Institute of Technology
  6. 6. 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. Kyushu Institute of Technology
  7. 7. 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. Kyushu Institute of Technology
  8. 8. HDAC isoforms Zn+2 ion dependent enzymes NAD dependent Sir2 family Kyushu Institute of Technology
  9. 9. 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. Kyushu Institute of Technology
  10. 10. Proposed catalytic mechanism for the deacetylation of Ac-Lys Finnin, M. S., Donigian, J. R., et al. Nature 1999, 401, 188-193. Kyushu Institute of Technology
  11. 11. 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. Kyushu Institute of Technology
  12. 12. 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) Kyushu Institute of Technology
  13. 13. 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. Kyushu Institute of Technology
  14. 14. 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. Kyushu Institute of Technology
  15. 15. 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 Kyushu Institute of Technology
  16. 16. • 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 • Molecular design of histone deacetylase Inhibitors by aromatic ring arrangement on cyclic dipeptide hydroxamic acids – Introduction – Diketopiperazine hydroxamic acids containing iminoacids – Diketopiperazine hydroxamic acids containing α, α,-dialkylated amino acids • Conclusions • References
  17. 17. 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 Kyushu Institute of Technology
  18. 18. 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 Kyushu Institute of Technology
  19. 19. 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 Kyushu Institute of Technology
  20. 20. 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. Kyushu Institute of Technology
  21. 21. 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) Kyushu Institute of Technology
  22. 22. • 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 • Molecular design of histone deacetylase Inhibitors by aromatic ring arrangement on cyclic dipeptide hydroxamic acids – Introduction – Diketopiperazine hydroxamic acids containing iminoacids – Diketopiperazine hydroxamic acids containing α, α,-dialkylated amino acids • Conclusions • References
  23. 23. Aromatic ring shifting in chlamydocin framework Design of cyclic tetrapeptides 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 Kyushu Institute of Technology
  24. 24. • 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 Kyushu Institute of Technology
  25. 25. 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 Kyushu Institute of Technology
  26. 26. 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-) Kyushu Institute of Technology
  27. 27. • 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 • Molecular design of histone deacetylase Inhibitors by aromatic ring arrangement on cyclic dipeptide hydroxamic acids – Introduction – Diketopiperazine hydroxamic acids containing iminoacids – Diketopiperazine hydroxamic acids containing α, α,-dialkylated amino acids • Conclusions • References
  28. 28. Variation of spacer length of L-Phe in chlamydocin framework n = 0, 2, 3 Design of cyclic tetrapeptides 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 Kyushu Institute of Technology
  29. 29. 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 Kyushu Institute of Technology
  30. 30. 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 Kyushu Institute of Technology
  31. 31. 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-) Kyushu Institute of Technology
  32. 32. Enzyme inhibition and biological activity (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 Kyushu Institute of Technology
  33. 33. • 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 • Molecular design of histone deacetylase Inhibitors by aromatic ring arrangement on cyclic dipeptide hydroxamic acids – Introduction – Diketopiperazine hydroxamic acids containing iminoacids – Diketopiperazine hydroxamic acids containing α, α,-dialkylated amino acids • Conclusions • References
  34. 34. 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. Kyushu Institute of Technology
  35. 35. 2. Conformational analysis of diastereomers by NMR spectrometry Compound 4 Compound 5 Kyushu Institute of Technology
  36. 36. 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 Kyushu Institute of Technology
  37. 37. 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. Kyushu Institute of Technology
  38. 38. • 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 • Molecular design of histone deacetylase Inhibitors by aromatic ring arrangement on cyclic dipeptide hydroxamic acids – Introduction – Diketopiperazine hydroxamic acids containing iminoacids – Diketopiperazine hydroxamic acids containing α, α,-dialkylated amino acids • Conclusions • References
  39. 39. 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. Kyushu Institute of Technology
  40. 40. 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) Kyushu Institute of Technology
  41. 41. 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 Kyushu Institute of Technology
  42. 42. • 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 • Molecular design of histone deacetylase Inhibitors by aromatic ring arrangement on cyclic dipeptide hydroxamic acids – Introduction – Diketopiperazine hydroxamic acids containing iminoacids – Diketopiperazine hydroxamic acids containing α, α,-dialkylated amino acids • Conclusion • References
  43. 43. 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. Kyushu Institute of Technology
  44. 44. Enzyme inhibition and biological activity *column: Chromolith performance RP-18e (100 x 4.6 mm) eluent: 10-100% CH3CN gradient containing 0.1% TFA over 15 min., flow = 2 mL/min. Kyushu Institute of Technology
  45. 45. • 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 • Molecular design of histone deacetylase Inhibitors by aromatic ring arrangement on cyclic dipeptide hydroxamic acids – Introduction – Diketopiperazine hydroxamic acids containing iminoacids – Diketopiperazine hydroxamic acids containing α, α,-dialkylated amino acids • Conclusions • References
  46. 46. 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 Kyushu Institute of Technology
  47. 47. Enzyme inhibition and biological activity *column: Chromolith performance RP-18e (100 x 4.6 mm) eluent: 10-100% CH3CN gradient containing 0.1% TFA over 15 min. Kyushu Institute of Technology
  48. 48. 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 Kyushu Institute of Technology
  49. 49. Enzyme inhibition and biological activity *column: Chromolith performance RP-18e (100 x 4.6 mm) eluent: 10-100% CH3CN gradient containing 0.1% TFA over 15 min. Kyushu Institute of Technology
  50. 50. 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. Kyushu Institute of Technology
  51. 51. • 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 • Molecular design of histone deacetylase Inhibitors by aromatic ring arrangement on cyclic dipeptide hydroxamic acids – Introduction – Diketopiperazine hydroxamic acids containing iminoacids – Diketopiperazine hydroxamic acids containing α, α,-dialkylated amino acids • Conclusions • References
  52. 52. 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. Kyushu Institute of Technology
  53. 53. 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. Kyushu Institute of Technology
  54. 54. • 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 Kyushu Institute of Technology
  55. 55. • 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 • Molecular design of histone deacetylase Inhibitors by aromatic ring arrangement on cyclic dipeptide hydroxamic acids – Introduction – Diketopiperazine hydroxamic acids containing iminoacids – Diketopiperazine hydroxamic acids containing α, α,-dialkylated amino acids • Conclusions • References
  56. 56. 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 Kyushu Institute of Technology
  57. 57. 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 Kyushu Institute of Technology
  58. 58. • 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 • Molecular design of histone deacetylase Inhibitors by aromatic ring arrangement on cyclic dipeptide hydroxamic acids – Introduction – Diketopiperazine hydroxamic acids containing iminoacids – Diketopiperazine hydroxamic acids containing α, α,-dialkylated amino acids • Conclusions • References
  59. 59. 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 Kyushu Institute of Technology
  60. 60. 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. Kyushu Institute of Technology
  61. 61. Enzyme inhibition and biological activity *column: Chromolith performance RP-18e (100 x 4.6 mm) eluent: 10-100% CH3CN gradient containing 0.1% TFA over 15 min. Kyushu Institute of Technology
  62. 62. 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. Kyushu Institute of Technology
  63. 63. • 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 • Molecular design of histone deacetylase Inhibitors by aromatic ring arrangement on cyclic dipeptide hydroxamic acids – Introduction – Diketopiperazine hydroxamic acids containing iminoacids – Diketopiperazine hydroxamic acids containing α, α,-dialkylated amino acids • Conclusions • References
  64. 64. 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 Kyushu Institute of Technology
  65. 65. • 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 • Molecular design of histone deacetylase Inhibitors by aromatic ring arrangement on cyclic dipeptide hydroxamic acids – Introduction – Diketopiperazine hydroxamic acids containing iminoacids – Diketopiperazine hydroxamic acids containing α, α,-dialkylated amino acids • Conclusions • References
  66. 66. 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. Kyushu Institute of Technology
  67. 67. 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. Kyushu Institute of Technology
  68. 68. 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. Kyushu Institute of Technology
  69. 69. 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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