1. G1G1 G2G2 S M G0G0 G0G0 “Restriction Point” A, T, G, C + DNA Polymerase Prophase Metaphase Anaphase Telophase

2. G1G1 G2G2 S M Prophase Metaphase Anaphase Telophase Tubulin Inhibitors Block Cells in G2/M-Phase

3. Transcription Translation DNA Synthesis (Replication) G1/G2-PhaseS-Phase Post-Translational Modification “Flow of Genetic Information”

4. Acoelomates/ Pseudocoelomates Protostomes Ectoprocta (“Bryozoa”) Brachiopoda Phoronida Echinodermata Urochordata (“tunicates”) Vertebrata Chordates

5. Bioactive Compounds from Tunicates Phylum Urochordata (Tunicata) a.k.a. “Sea Squirts” Chordate Characteristics: i.e. Notochord, Dorsal Nerve Cord, Pharyngeal Slits

6. Bioactive Compounds from Tunicates: Ecteinascidin-743 Ecteinascidin-743 (ET-743) Ecteinascidia turbinata

7. ET-743 Slows G1 to G2 Transition

8. G1G1 G2G2 S M Prophase Metaphase Anaphase Telophase Cells Treated with ET-743 Accumulate in G1-Phase

9. A:T G:C ET-743 Alkylates Guanine in Minor Groove of DNA

12. Bends DNA into Major Groove

13. Sequence-Specificity of ET-743 Binding/Alkylation 5’-PuGC-3’ 5’-PyGG-3’ e.g.5’-C-G-G-3’ 3’-G-C-C-5’ e.g.5’-A-G-C-3’ 3’-T-C-G-5’ HB1 HB3 HB2 HB1HB3 HB1 HB3 HB2

14. Binding of ET-743 to DNA Impairs DNA Repair and Leads to Apoptosis Apoptosis

15. Natural “Analogues” of ET-743 Bind DNA …but don’t inhibit cancer cells...lack C-ring. ET-743 Saframycin

16. C-Ring of ET-743 Blocks Transcription Factors C-Ring

17. Transcription Factors Recognize Specific Sequences in Promoters/Enhancers Promoter Gene Enhancer RNA Pol TF Promoter Gene Enhancer TF RNA Pol

18. “CCAAT Element” 5’-CTGATTGGPyPyPuPu-3’ 5’-PyPyPuPuCCAATCAG-3’ 3’-GACTAACCPuPuPyPy-5’3’-PuPuPyPyGGTTAGTC-5’ Nuclear Factor Y (NF-Y) Recognizes “CCAAT Element” Present in 25% of Eukaryotic Promoters! Three Subunits (i.e. A, B and C) Only Trimer Binds CCAAT

19. ET-743 Blocks Transcription of P- P-Glycoprotein Multi-Drug Resistance (MDR) Gene MDR1 (a.k.a. Pgp) Gene ATP-Binding Cassette (ABC) Family CCAAT Element in Promoter Region!!

20. Bioactive Compounds from Tunicates: Didemnin-B Didemnin-B (from Trididemnum solidum) Rinehart et al. (1981a) Science, 212: 933-5. Rinehart et al. (1981b) J. Am. Chem. Soc., 103: 1857-9.

21. Cells in G1- and S-Phase Most Sensitive to Didemnins G1G1 G2G2 S M Prophase Metaphase Anaphase Telophase

22. Didemnin-B Inhibits Protein Synthesis Protein Synthesis Cmpd.IC50 (µM) 10.89-2.0 25.4 31.0 43.0 59.0 62.5 74.3 84.3 95.3 104.0 114.5 1224.0 1310.0

23. Protein Synthesis 60S 40S Ribosome (= Ribonucleoprotein Complex) 3 rRNAs (5S, 5.8S and 28S) + 40 Proteins 1 rRNA (18S) + 33 Proteins 80S (4200 Kda)

24. Ribosome Structure

25. Transfer RNA (tRNA) Transports Amino Acid to Ribosome/mRNA

26. Activating Enzyme Binds Amino Acid to tRNA Aminoacyl-tRNA Synthetase (Activating Enzyme) Aminoacyl-tRNA + ATP+ PPi+ AMP + 2 Pi

27. Protein Synthesis 5’ CapAUGGAACGCUAC3’ 7-Methylguanosine UAA/G Start Codon (= Met) Stop Codon

28. Protein Synthesis Met-tRNA i Met Initiation Factors (IFs) 5’ CapAUGGAACGCUAC3’UAA/G

29. Protein Synthesis ATPADP Met-tRNA i Met 5’ CapAUGGAACGCUAC3’UAA/G

30. Protein Synthesis 5’ CapAUGGAACGCUAC3’ Met Peptidyl Site (P-Site) Aminoacyl Site (A-Site) UAA/G

31. Protein Synthesis 5’ CapAUGGAACGCUAC3’ Met Glu Elongation Factors (EFs) EF1  GTP GDP EF1  GTP+ GDP UAA/G GTP Glu

32. Protein Synthesis 5’ CapAUGGAACGCUAC3’ Met Glu Peptidyl Transferase UAA/G Glu GTP GDP EF1  GTP+ GDP Elongation Factors (EFs) EF1  GTP

33. Protein Synthesis 5’ CapAUGGAACGCUAC3’UAA/G Glu GTP GDP EF1  GTP+ GDP Elongation Factors (EFs) EF1  GTP OH Glu-Met

34. Protein Synthesis 5’ CapAUGGAACGCUAC3’ OH Glu-Met Translocation EF2 UAA/G

35. Protein Synthesis 5’ CapAUGGAACGCUAC3’ Glu-Met Translocation EF2 UAA/G

36. Protein Synthesis 5’ CapAUGGAACGCUAC3’ Glu-Met Translocation EF2 UAA/G

37. Protein Synthesis 5’ CapAUGGAACGCUAC3’ Glu-Met Translocation EF2 UAA/G

38. Protein Synthesis 5’ CapAUGGAACGCUAC3’ Translocation UAA/G Met-Glu

39. Protein Synthesis 5’ CapAUGGAACGCUAC3’ Translocation UAA/G Met-Glu Release Factor (RF)

40. Protein Synthesis 5’ CapAUGGAACGCUAC3’ Translocation UAA/G Met-Glu Release Factor (RF) OH H2OH2O

41. Didemnin-B Binds EF-1  Affinity Chromatography Biotinylation

42. Didemnin-B Binds EF-1  agarose Streptavidin-Agarose Affinity Chromatography Streptavidin Biotin-”Tag” Didemnin

43. Didemnin-B Binds EF-1  agarose Affinity Chromatography Didemnin-Binding Protein Elute Didemnin-Binding Proteins

44. Didemnin-B Binds EF-1 

45. Didemnin-B Binds EF-1  /GTP NOT EF-1  /GDP

46. Didemnin-Bound EF-1  Prevents EF-2 Binding 5’ CapAUGGAACGCUAC3’ Met Glu UAA/G EF-1  Didemnin-B EF2

47. Didemnin-B Binds Palmitoyl Protein Thioesterase (PPT1) Meng et al. (1998) Biochemistry, 37: 10488-92. Affinity Chromatography

48. Proteins are Post-Translationally Modified by Acylation N-Myristoylation/Palmitoylation S-Palmitoylation + + + +

49. Palmitoyl Protein Thioesterase

50. Didemnin-B Inhibits Depalmitoylation of Protein Meng et al. (1998) Biochemistry, 37: 10488-92.

51. Didemnin-B Non-Competitively Inhibits PPT1 Meng et al. (1998) Biochemistry, 37: 10488-92.

52. Protein Synthesis Cmpd.IC50 (µM) 10.89-2.0 25.4 31.0 43.0 59.0 62.5 74.3 84.3 95.3 104.0 114.5 1224.0 1310.0 Structure Activity Relationships of Didemnins

53. Aplidine (Dehydrodidemnin-B) Aplidium albicans

54. Clinical Promise of Aplidine