structural studies of chemotherapeutic biomolecules through nmr
DESCRIPTION
Structural Studies of Chemotherapeutic Biomolecules Through NMR. Teresa Lehmann. 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. Glycopeptide antibiotic MW 1500 Da - PowerPoint PPT PresentationTRANSCRIPT
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