Structural Studies of Chemotherapeutic Biomolecules Through NMR

Teresa Lehmann

Metallo-bleomycins (BLM)Fe(II)BLMCo(II)BLMCu(I)BLM

CS protein peptides (Plasmodium vivax)GDR(A/D)GQPA, [GDR(A/D)GQPA]2

ANGAGNQPG, (ANGAGNQPG)2

Bleomycins

Glycopeptide antibiotic

MW 1500 Da

Streptomyces verticillus

Non-Hodgkin’s lymphomasSquamous cell carcinomaTesticular carcinomasOvarian cancer

Umezawa et al. J. Antibiot. 1996, 19, 200

F e (II)B L M

F e (II I )B L M

O 2 /e -

e -

Activated BLMDNA

Cleaved DNA

Craig et al. Chem. Rev. 1999, 99, 2797

Also interesting: Cu(I)-, Co(III)-, Mn(II)-, Ni(III)-, and Ru(II)BLM

5’----GT----3’5’----GC----3’

Selective

CONH2

HN

CONH2

NH2

N N

CH3 HNNH

NH

N

O

CH3

HO

CH3

O

HN

O

OO

O

OHHO

OH

OH

OH

OH

O

O NH2

HO CH3

NH

O

S

N S

N

H2N

O

NH

R

O

1

234

56

1

23

4

56

1234

5

12 3

456

H5'

H5

NH CH2 CH2 CH2 S+(CH3)2

NH CH2 CH2 CH2 CH2 NHC(NH)(NH2)

BLM-A2: R =

BLM-B2: R =

-Aminoalanine (A)

Pyrimidinylpropionamide (P)

-Hydroxy histidine (H)

Methylvalerate (V)

Threonine (T)

Bithiazole (B)

-L-Gulose (G)

-D-Mannose

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

CONH2

HN

CONH2

NH2

N N

CH3 HN

NH

N

O

O

H2N

O

1234

5

12 3

456

-Aminoalanine (A)

Pyrimidinylpropionamide (P)

-Hydroxy histidine (H)NH

CH3

HO

CH3

O

HN

HO CH3

O

Methylvalerate (V)

Threonine (T)

NH S

N S

N NH

R

O

H5'

H5

Bithiazole (B)

O

OO

O

OHHO

OH

OH

OH

OH

O

O NH2

1

234

56

1

23

4

56

-L-Gulose (G)

-D-Mannose

Craig et al. Chem. Rev. 1999, 99, 2797

N-terminal

Linker

C-terminal

Disaccharide

Blenoxane™(Sterile Bleomycin Sulfate, USP)

WarningPulmonary fibrosis is the most severe toxicity associated withBlenoxane. The most frequent presentation is pneumonitisoccasionally 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, mentalconfusion, fever, chills, and wheezing has been reported in approximately 1% of lymphoma patients treated with Blenoxane.

Metallo-BLM structural studies

Direct studies FeBLM

Indirect studies Other metallo-BLM

Isostructural?

Different studies Different sets of ligands

Iitaka et al. J. Antibiot. 1978, 31, 1070Sugiyama et al. J. Biol. Chem. 2002, 277, 2311

N

CuN

NN

NH2

N

H2NH

H2NOC

H

HH2NOC

O

NH

CO-L-ALANINE

H

H

H

H

N

Oppenheimer et al. J. Biol. Chem. 1982, 257, 1606

NHNH2

Fe

HH2NOC

NH

ValerateNH2

O

O

Sugar

N

O

CH3

O

N

CO

N

H2N

H2NOC

H NH

N

Wu et al. J. Am. Chem. Soc. 1996, 118, 1268

NCo

N

NNH

N

H2N

H2NOC

H

HH2NOC

NH

O

CH3

Sugars

NH2

O

O OH

N

NH

NH2

Fe

HH2NOC

N

NH2

OO

Sugar

N

OCH3

O

N

CO

N

H2N

H2NOC

H

NH

Akkermann et al. J. Am. Chem. Soc. 1990, 112, 7462

N

CONH2

HN

CONH2

NH2

N N

CH3 HNNH

NH

N

O

CH3

HO

CH3

O

HN

O

OO

O

OHHO

OH

OH

OH

OH

O

O NH2

HO CH3

NH

O

S

N S

N

H2N

O

NH

R

O

1

234

56

1

23

4

56

1234

5

12 3

456

H5'

H5

-Aminoalanine (A)

Pyrimidinylpropionamide (P)

-Hydroxy histidine (H)

Methylvalerate (V)

Threonine (T)

Bithiazole (B)

-L-Gulose (G)

-D-Mannose

N

N

N

N

M

N?

O2

N

N

N

N

M

N?

N?

CONH2

HN

CONH2

NH2

N N

CH3 HNNH

NH

N

O

CH3

HO

CH3

O

HN

O

OO

O

OHHO

OH

OH

OH

OH

O

O NH2

HO CH3

NH

O

S

N S

N

H2N

O

NH

R

O

1

234

56

1

23

4

56

1234

5

12 3

456

H5'

H5

-Aminoalanine (A)

Pyrimidinylpropionamide (P)

-Hydroxy histidine (H)

Methylvalerate (V)

Threonine (T)

Bithiazole (B)

-L-Gulose (G)

-D-Mannose

BLM Fe(II)BLM+ Fe(II)

dxz, dyz, dxy

dx2-y2, dz2

H. S. Fe(II)

Zn(II)BLM

Fe(II)BLM

T1: 0.1-1s

T1: 0.6 - 120 ms

Lehmann et al. Biochemistry 1997, 36, 2807

Akkerman et al. Eur. J. Biochem. 1988, 173, 211

-Saturation Transfer (ST)

-T1 measurements-COrrelated SpectrsocopY (COSY)-TOtally Correlated SpectroscopY (TOCSY)-Heteronuclear correlation through Multiple Quantum Coherence (HMQC)

Lehmann et al. Biochemistry 1997, 36, 2807

Lehmann et al. Biochemistry 1997, 36, 2807

CONH2HN

CONH2

NH2

N N

CH3 HNNH

NH

N

O

CH3

HO

CH3

O

HN

O

OO

O

OHHO

OH

OH

OH

OH

O

O NH2

HO CH3

NH

O

S

N S

N

H2N

O

NH

R

O

1

234

56

1

23

4

56

H5'

H5

-Aminoalanine (A)

Pyrimidinylpropionamide (P)

-Hydroxy histidine (H)

Methylvalerate (V)

Threonine (T)

Bithiazole (B)

-L-Gulose (G)

-D-Mannose

HH

NMR data

Molecular Dynamics (MD)

The best structure 3D structure

MDNuclear Overhauser Effect (NOE) (spatial information)

J coupling spectroscopy (angle information)

Fe(II)BLM shows no NOEs and no splitting!!

(T1dip ) 1 (2

15)(g)2 S(S 1)(RM ) 6 f (T1e )

La Mar & de Roop. In NMR of Paramagnetic Molecules; Berliner L.J. &Reuben J. Eds.; Plenum Press: New York, 1993; p 1-73

RMj RMi(T1 j

T1i

)1

6

~ 5Å

NH

N1234

5

HHM

Spek et al. Acta Crystallogr. C 1983, 39, 1212Mandon et al. Inorg. Chem. 1990, 29, 2442Tolman et al. J. Am. Chem. Soc. 1991, 113, 152

H

Fe

Sugars

Bithiazole-Sulfonium

ALA

NHALA NHIS I

NH2

O

NH2

NPYR NHISA

Fe

NHALA

Bithiazole-Sulfonium

Sugars

ALA

Solvent

NPYR NHISA

NH2

INHIS

Fe

Bithiazole-Sulfonium

NHALA

ALA

Solvent

Sugars

NHIS

NH2

I

NPYRA

NHIS

Fe

Bithiazole-Sulfonium

ALA

NHALA

Sugars

NPYRA

I

NH2

NHIS

H2N

NHIS

O

Fe

NHALA

Sugars

Bithiazole-Sulfonium

ALA

Solvent

ANPYR

NH2

I

NHIS

NHIS

Lehmann J. Biol. Inorg. Chem. 2002, 7, 305 Loeb et al. J. Am. Chem. Soc. 1998, 120, 1249

Lehmann J. Biol. Inorg. Chem. 2002, 7, 305

Fe

NHALA

Sugars

Bithiazole-Sulfonium

ALA

Solvent

ANPYR

NH2

I

NHIS

NHISFe

Bithiazole-Sulfonium

NHALA

ALA

Solvent

Sugars

NHIS

NH2

I

NPYRA

NHIS

Fe

Bithiazole-Sulfonium

ALA

NHALA

Sugars

NPYRA

I

NH2

NHIS

H2N

NHIS

O

Wu et al. J. Am. Chem. Soc. 1996, 118, 1268

HOO-Co(III)BLM

Craig et al. Chem. Rev. 1999, 99, 2797

Fe(II)BLM

.O2-Fe(III)BLM

HOO-Fe(III)BLM(activated bleomycin)

O2

e- (from Fe(II)BLM,.O2-Fe(III)BLM

or cellular reductants)

DNA stand scission

Structurally similar toHOO-Co(III)BLM?

Amine displacementWater release

MAN bindingAmine displacement

Smolentsev et al. Inorg. Chem. 2004, 43, 1825XANES-spectroscopy

Co(II)BLMNMRT1 measurementsMD

Fedeles et al. Inorg. Chem. 2001, 40, 1557

Rajani et al. J. Am. Chem. Soc. 2004, 126, 3829

Lehmann et al. Biochemistry 2000, 39, 3886

Cu(II)BLM

in vivocan inhibit the growth of tumorcells in tissue culture and tumor

growth in animals

in vitrounable to cleave DNA

Ishizuka et al. J. Antibiot. 1967, 20, 15Umezawa et al. J. Antibiot. 1968, 21, 592Antholine et al. Inorg. Biochem. 1982, 17, 75

Sausville et al. Biochem. Biophys. Res. Commun.1976, 73, 814Takahashi et al. J. Antibiot. 1977, 30, 861Sugiura et al. J. Biol. Chem. 1982, 257, 10544Antholine et al. Inorg. Biochem. 1982, 17, 75

BLM (administered as metal free)

+ Cu(II)plama Cu(II)BLM(administered as copper complex)

Cu(II)BLMplasma

Cu(II)BLMnucleus

I

activated CuBLM

oxidative DNA damage

O2

Ehrenfeld et al.Biochemistry 1985, 24, 4719

II

Cu(I) + BLM

Fe(II)

Fe(II)BLMO2

Takahashi et al. J. Antibiot. 1977, 30, 861

Antholine et al. J. Inorg. Biochem.1982, 17, 75Freedman et al. Biochemistry 1982, 21, 2203

Cu(II)BLM + reductant

Cu(I)BLM + oxidant

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

N

CuN

NN

NH2

N

H2NH

H2NOC

H

HH2NOC

O

NH

CO-L-ALANINE

H

H

H

H

N

CONH2

HN

CONH2

NH2

N N

CH3 HNNH

NH

N

O

CH3

HO

CH3

O

HN

O

OO

O

OHHO

OH

OH

OH

OH

O

O NH2

HO CH3

NH

O

S

N S

N

H2N

O

NH

R

O

1

234

56

1

23

4

56

1234

5

12 3

456

H5'

H5

-Aminoalanine (A)

Pyrimidinylpropionamide (P)

-Hydroxy histidine (H)

Methylvalerate (V)

Threonine (T)

Bithiazole (B)

-L-Gulose (G)

-D-Mannose

200-300 million cases

1-2 million deaths per year

NH2 COOH

Repeat Region

Plasmodium CS protein repeat units

bergehiPPPPN(P/A)ND

PAPPNAND

brasilianum N(D/A)AG

knowlesi GQPQAQGDGAN(A/V)

malariae N(D/A)AG

reichenowiNVDP

N(A/V)NP

falciparumNANP

NVDP

vivax vk-210 GDRA(D/A)GQPA

vivax vk-247 ANGA(G/D)(N/D)QPG

NMR and Circular Dichroism (CD) spectroscopies to study the solution conformation of peptides

P. falciparum repeatsNANP, (NANP)2, (NANP)3

NPNA, (NPNA)2, (NPNA)3

H3C C

O

N

H

C

H

CH2

C

OH2N

C

O

N C

H

C

O

N

H

C

H

CH2

C

OH2N

C

O

N

H

C

H

CH3

C

O

NH2

Dyson et al. Biochemistry 1990, 29, 7828

P. vivax

ANGAGNQPG, (ANGAGNQPG)2

GDRADGQPA, (GDRADGQPA)2

GDRAAGQPA, (GDRAAGQPA)2

P. falciparumNANP, (NANP)2, (NANP)3

NPNA, (NPNA)2, (NPNA)3

=

turn-like conformations

extended-chain conformations

Lehmann et al. J. Pep. Res. 2003, 61, 252

Cys N P N A N P N A N P N A NH2

Cys N A N P N A N P N A N P NH2

N

P N

A P

N A

N

Satterthwait et al. Phil. Trans. R. Soc. London B 1989, 323, 565Satterthwait et al. Bull. WHO (Suppl.) 1990, 68, 17

Modified Peptides (MP) ABMP

Native Peptides (NP)ABNP

NO YES

Cys (NH CH C

O

NH CH C

CH2

C

OHN

ALAPRO

)3 NH2ALAPRO

CH2

C

O NH

H2C

CH2

O

random coil

bent

P. vivax peptides AB

complex

P. vivax peptides

free

=?

EEEDDDMGFGLFD T. Cruzi EESDDDMGFGLFD HumanEEADDDMGFGLFD L. braziliensis

Soares et al. FEBS Lett. 2004, 560, 134

Fe(II)BLM Dr. Lawrence Que, Jr and Dr. Mark E. RosenUniversity 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)BLMDr. Gonzalo VisbalVenezuelan Institute for Scientific Research (IVIC)VENEZUELA

PeptidesDr. 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 JaneiroBRAZIL

Backup

CoBLMCleaves DNA

Isostructural with FeBLM

(UV light)

dxz, dyz, dxy

dx2-y2, dz2

H. S. Co(II)

TRe Fe(II )

~ 10 9 10 11s

TRe Co(II )

~ 10 12 10 13 s

TRp 1

TRe

Co(II)BLM

Lehmann et al. Biochemistry 2000, 39, 3886

Absalon et al. Biochemistry 1995, 34, 2076

C-4’rad

C-4’ peroxyl rad

C-4’ hydroperoxide

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

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