Structural Studies of Chemotherapeutic Biomolecules Through NMR

1. Structural Studies of Chemotherapeutic Biomolecules Through NMR Teresa Lehmann

2. Metallo-bleomycins (BLM) Fe(II)BLM Co(II)BLM Cu(I)BLM CS protein peptides (Plasmodium vivax) GDR(A/D)GQPA, [GDR(A/D)GQPA]2 ANGAGNQPG, (ANGAGNQPG)2

3. Glycopeptide antibiotic MW 1500 Da Streptomyces verticillus Non-Hodgkin’s lymphomas Squamous cell carcinoma Testicular carcinomas Ovarian cancer Bleomycins Umezawa et al. J. Antibiot. 1996, 19, 200

4. Selective 5’----GT----3’ 5’----GC----3’ - O / e 2 Activated BLM F e ( I I ) B L M DNA F e ( I I I ) B L M Cleaved DNA - e Also interesting: Cu(I)-, Co(III)-, Mn(II)-, Ni(III)-, and Ru(II)BLM Craig et al. Chem. Rev. 1999, 99, 2797

5. Umezawa H. Biomed. 1973, 18, 459 Takita et al. Tetrahedron Lett.1982, 23, 521

6. N-terminal C-terminal Linker Craig et al. Chem. Rev.1999, 99, 2797 Disaccharide

7. Blenoxane™ (Sterile Bleomycin Sulfate, USP) Warning Pulmonary fibrosis is the most severe toxicity associated with Blenoxane. The most frequent presentation is pneumonitis occasionally progressing to pulmonary fibrosis. Its occurrence is higher in elderly patients and in those receiving greater than 400 units total dose, but pulmonary toxicity has been observed in young patients and those treated with low doses. A severe idiosyncratic reaction consisting of hypotension, mental confusion, fever, chills, and wheezing has been reported in approximately 1% of lymphoma patients treated with Blenoxane.

8. Isostructural? Different studies Different sets of ligands Metallo-BLM structural studies Direct studies FeBLM Indirect studies Other metallo-BLM

9. N N Iitaka et al. J. Antibiot.1978, 31, 1070 Sugiyama et al. J. Biol. Chem.2002, 277, 2311 Oppenheimer et al. J. Biol. Chem.1982, 257, 1606 N N Wu et al. J. Am. Chem. Soc.1996, 118, 1268 Akkermann et al. J. Am. Chem. Soc.1990, 112, 7462

10. N ? N N M N N N?

11. 2 2 2 d x - y , d z H . S . F e ( I I ) + BLM F e ( I I ) B L M F e ( I I ) d x z , d y z , d x y

12. T1: 0.1-1s Zn(II)BLM Akkerman et al. Eur. J. Biochem.1988, 173, 211 T1: 0.6 - 120 ms Fe(II)BLM Lehmann et al. Biochemistry1997, 36, 2807

13. -SaturationTransfer (ST) -T1 measurements -COrrelatedSpectrsocopY(COSY) -TOtallyCorrelatedSpectroscopY(TOCSY) -Heteronuclear correlation throughMultipleQuantumCoherence (HMQC) Lehmann et al. Biochemistry1997, 36, 2807

14. b - A m i n o a l a n i n e ( A ) N C O N H H 2 2 H N N H C O N H B i t h i a z o l e ( B ) 2 P y r i m i d i n y l M e t h y l v a l e r a t e ( V ) p r o p i o n a m i d e ( P ) H 5 C H 3 H H O N H 5 ' H N N 2 H N C H H 3 C H N 3 C H H O H 3 b - H y d r o x y h i s t i d i n e ( H ) T h r e o n i n e ( T ) N O H H H O O H O H O H a - L - G u l o s e ( G ) O H N H 2 a - D - M a n n o s e O R a N b b a O N N S N O a b a b a O S g a O b b N O 1 5 H H O 6 O 2 3 1 5 4 O 6 3 2 O 4 O Lehmann et al. Biochemistry 1997, 36, 2807

15. NMR data MolecularDynamics(MD) The best structure 3D structure NuclearOverhauserEffect(NOE)(spatial information) MD J coupling spectroscopy (angle information) Fe(II)BLM shows no NOEs and no splitting!!

16. La Mar & de Roop. In NMR of Paramagnetic Molecules; Berliner L.J. & Reuben J. Eds.; Plenum Press: New York, 1993; p 1-73 Spek et al. Acta Crystallogr. C1983, 39, 1212 Mandon et al. Inorg. Chem.1990, 29, 2442 Tolman et al. J. Am. Chem. Soc.1991, 113, 152 H ~ 5Å

17. S u g a r s S u g a r s S u g a r s N H 2 S o l v e n t O N H 2 A L A N N N N P Y R P Y R H I S H I S A A N N P Y R H I S A F e F e F e N N N H N H H I S H I S A L A A L A I I N N H H I S A L A I N H N H 2 2 A L A A L A S o l v e n t B i t h i a z o l e - S u l f o n i u m B i t h i a z o l e - S u l f o n i u m B i t h i a z o l e - S u l f o n i u m N H 2 N H A L A 2 A L A N N P Y R H I S N A N P Y R H I S A F e F e N N H H I S A L A N I N H H I S A L A I O S o l v e n t S u g a r s H N 2 S u g a r s B i t h i a z o l e - S u l f o n i u m B i t h i a z o l e - S u l f o n i u m Lehmann J. Biol. Inorg. Chem.2002, 7, 305 Loeb et al. J. Am. Chem. Soc.1998, 120, 1249

18. S u g a r s N H 2 A L A N N P Y R H I S A F e N H N H I S A L A I S o l v e n t B i t h i a z o l e - S u l f o n i u m N H 2 N H A L A 2 A L A N N P Y R H I S N A N P Y R H I S A F e F e N N H H I S A L A N I N H H I S A L A I O S o l v e n t S u g a r s H N 2 S u g a r s m B i t h i a z o l e - S u l f o n i u B i t h i a z o l e - S u l f o n i u m Lehmann J. Biol. Inorg. Chem.2002, 7, 305

19. HOO-Co(III)BLM Wu et al. J. Am. Chem. Soc.1996, 118, 1268

20. Structurally similar to HOO-Co(III)BLM? Craig et al. Chem. Rev.1999, 99, 2797

21. Amine displacement Water release MAN binding Amine displacement Smolentsev et al. Inorg. Chem.2004, 43, 1825 XANES-spectroscopy

22. NMR T1 measurements MD Lehmann et al. Biochemistry2000, 39, 3886 Co(II)BLM Fedeles et al. Inorg. Chem.2001, 40, 1557 Rajani et al. J. Am. Chem. Soc.2004, 126, 3829

23. Cu(II)BLM in vivo can inhibit the growth of tumor cells in tissue culture and tumor growth in animals in vitro unable to cleave DNA Sausville et al. Biochem. Biophys. Res. Commun. 1976, 73, 814 Takahashi et al. J. Antibiot.1977, 30, 861 Sugiura et al. J. Biol. Chem.1982, 257, 10544 Antholine et al. Inorg. Biochem.1982, 17, 75 Ishizuka et al. J. Antibiot.1967, 20, 15 Umezawa et al. J. Antibiot.1968, 21, 592 Antholine et al. Inorg. Biochem.1982, 17, 75

24. Antholine et al. J. Inorg. Biochem. 1982, 17, 75 Freedman et al. Biochemistry 1982, 21, 2203 Takahashi et al. J. Antibiot. 1977, 30, 861 Ehrenfeld et al.Biochemistry1985, 24, 4719

25. Cu(I)BLM coordination chemistry and solution structure NMR and MD Lehmann J. Biol. Inorg. Chem.2004, 9, 323 Traditional NMR experiments -NOEs -J-coupling spectroscopy

26. N

28. 200-300 million cases 1-2 million deaths per year

29. Repeat Region NH2 COOH

31. NANP, (NANP)2, (NANP)3 NPNA, (NPNA)2, (NPNA)3 P. falciparum repeats NMR and Circular Dichroism (CD) spectroscopies to study the solution conformation of peptides Dyson et al. Biochemistry1990, 29, 7828

32. P. vivax ANGAGNQPG, (ANGAGNQPG)2 GDRADGQPA, (GDRADGQPA)2 GDRAAGQPA, (GDRAAGQPA)2 P. falciparum NANP, (NANP)2, (NANP)3 NPNA, (NPNA)2, (NPNA)3 = extended-chain conformations turn-like conformations Lehmann et al. J. Pep. Res. 2003, 61, 252

33. Modified Peptides (MP) ABMP Native Peptides (NP) ABNP YES NO O O A L A A L A ) N H C H C N H C y s ( N H C H C 3 2 P R O P R O C H C H 2 2 H 2 C C C H N O O N H C H 2 Satterthwait et al. Phil. Trans. R. Soc. London B1989, 323, 565 Satterthwait et al. Bull. WHO (Suppl.)1990, 68, 17

34. EEEDDDMGFGLFD T. Cruzi EESDDDMGFGLFD Human EEADDDMGFGLFD L. braziliensis bent Soares et al. FEBS Lett.2004, 560, 134 complex free P. vivax peptides AB P. vivax peptides ? = random coil

35. Fe(II)BLM Dr. Lawrence Que, Jr and Dr. Mark E. Rosen University of Minnesota USA Dr. Li-June Ming University of South Florida USA Co(II)BLM Dr. Lawrence Que, Jr University of Minnesota USA Cu(I)BLM Dr. Gonzalo Visbal Venezuelan Institute for Scientific Research (IVIC) VENEZUELA Peptides Dr. H. Jane Dyson and Dr. Gerard Kroon The Scripps Research Institute USA María Lorenzo and Henry Bermúdez Universidad Central de Venezuela VENEZUELA Dr. Fábio Almeida Universidade Federal do Rio de Janeiro BRAZIL

36. Backup

37. Cleaves DNA Isostructural with FeBLM CoBLM (UV light)

38. Co(II)BLM Lehmann et al. Biochemistry2000, 39, 3886

39. C-4’rad C-4’ peroxyl rad C-4’ hydroperoxide Absalon et al. Biochemistry 1995, 34, 2076

40. Wu et al. J. Am. Chem. Soc. 1996, 118, 1281

41. Wu et al. J. Am. Chem. Soc. 1996, 118, 1281