Principles of Bioinorganic Chemistry - 2003

1
Principles of Bioinorganic Chemistry - 2003
The grade for this course will be determined by a
term exam (35), a written research paper with
oral presentation (45), problem sets (12) and
classroom participation (8). The oral
presentations will be held in research conference
style at MIT's Endicott House estate in Dedham,
MA, on Saturday, October 18. Please reserve the
date for there are no excused absences. Papers
will be due approximately one week earlier. WEB
SITE web.mit.edu/5.062/www/
2
(No Transcript)
3
Transport of Pt in the Body
Injection Pt
Rescue agent
Transport
Apoptosis
diuretic
Kidney (toxicity)
LIVER
p53 active
Excretion 50 lt48hrs rest lt2months
4
Obstacles for Cisplatin On Route to DNA

• Reagents in blood plasm proteins, protective
  agents
• Receptors at cell wall
• Reagents in cellular membrane
• Reagents inside the cell, such as glutathione,
  S-donor peptides
• Reagents in the nuclear membrane

5
Transport from Outside to Inside Cell

• Cell receptors?
• Active or passive cell-wall transport?
• Relationships with resistance??
• Carrier molecule? YES PS (phos/ser)!
• Inside cell glutathione-like ligands take over
  can some Pt species escape to the nucleus? YES
  transfer proved

6
(No Transcript)
7
(No Transcript)
8
Structures of the 1,2-d(GpG) Intrastrand
Cisplatin Adduct
d(pGpG) adduct
duplex DNA adduct
Sherman et al. (1985) Science 230, 412. Takahara
et al. (1995) Nature 377, 649.
9
Structure of a Pt(R,R-DACH)2 Intrastrand
Cross- Link in a Duplex Dodecamer Showing the
GG Step
A very similar structure occurs for the 3
orientational isomer of a Pt(NH3)(NH2Cy)2 GG
cross-link on the same duplex dodecamer.
10
Numerous Cellular Proteins Recognize and Process
Platinum-DNA Adducts
Functions affected
Transcription Ubiquitination Repair Cell
cycle Others, via hijacking
Cellular proteins
Cell death or viability
11
Transcription Inhibition Correlates with Cell
Death in a GFP Reporter Assay
1. cisplatin 2. cis-Pt(NH3)(NH2C6H11)Cl2 3.
cis-Pt(NH2CH3)2Cl2 4. Pt(en)Cl2 5.
cis-Pt(dach)Cl2 6. trans-Pt(NH2CH3)2Cl2 7.
cis-Pt(NH2-iPr)2Cl2 8. Pt(NH3)Cl3PØ4 9.
Pt(NH3)3ClCl 10. Pt(lysine)Cl2 11.
Pt(arginine)Cl2 12. Pt(norleucine)Cl2
320
12
11
280
240
9
200
10
IC50 (µM)
160
8
120
7
80
5
6
40
3, 4
2
1
0
0
20
40
60
80
100
120
140
LC50 (µM)
Northern blotting and nuclear run-on assays
confirm that control of GFP expression is at
the transcriptional level.
12
FRET
520 nm
409 nm
O
O
O
O
O
O
O
O
O
O
S
O
GREEN
CCF2/AM
O
N
S
O
O
CCF2
S
N
S
O
b-lactamase
platinum block
409 nm
447 nm
O
O
BLUE
O
O

S
cells stay green
O
O
N
Enzymatic amplification allows detection of
low-level gene expression. Bluegreen ratio
quantitates gene expression without correcting
for cell plating.
13
40 µM cisplatin 37C, 24 h 1 µM CCF2/AM
Control
14
Consequences of Cisplatin-DNA Damage
Cisplatin damage site blocks transcription
DNA
Stalled Pol II triggers multiple cellular
processes
Failure to recognize the damage in answer to the
distress call is desired in the cancer cell
15
Consequences of Cisplatin-DNA Damage
Ubiquitinated Pol II is replaced.
Cellular repair machinery is recruited
Recognition and repair of the damage in answer to
the distress call is desired in healthy cells.
16
Consequence of Cisplatin Damage
17
(No Transcript)
18
(No Transcript)
19
(No Transcript)
20
Structure of Nucleosome Core Particle
Nucleosome Core Particle
Histone Octamer
DNA 146 bp
Two H2A/H2B Heterodimer
H3/H4 Tetramer
H2A pink H2B yellow H3 blue H4 bright
green.
Luger, et al., 1997, Nature 389, 251-260.
21
Synthesis of Site-Specifically PlatinatedDNA
Repair Probes (Wang, 2002)
Top strand oligos Bottom strand oligos
B
C
D
E
5
5
5
5
T4 Kinase ATP
T4 kinase ?32P-ATP
T4 Kinase ATP
T4 Kinase ATP
E
D
C
B
P
P
P
P
A
F
1. Annealing 2. Ligation

199mer
A83-mer B20GG-Pt or 20GTG-Pt C 96-mer
D72-mer E40CC or 40CAC F 87-mer.
22
Nucleosome Assembly from DNA Repair Probes
Sucrose gradient centrifugation
Free DNA Histone Octamer
Stepwise dialysis
Nucleosomal DNA
23
Nucleosome Inhibits NER of Cisplatin Adducts
1 2 3 4
1. The nucleosome structure inhibits nucleotide
excision repair of cisplatin cross-links. 2.The
efficiency of dual incision of nucleosomal DNA
GG-Pt is about 30 of naked DNA GG-Pt, whereas
the efficiency of dual incision of nucleosomal
DNA GTG-Pt is about 10 of naked DNA GTG-Pt.
Lane 1 NER assay of nucleosomal 199GG-Pt
DNA Lane 2 NER assay of naked 199GG-Pt DNA Lane
3 NER assay of nucleosomal 199GTG-Pt DNA Lane 4
NER assay of naked 199GTG-Pt DNA
Dual Incision
0.3 1 1 10
24
Does Histone Modification Affect the Process?
Strahl, B.D. Allis, C.D. Nature 2000, 403, 41-5.
25
Nucleosome Assembly from Native (modified) and
Recombinant (E. coli) Histones
Unmodified histone octamer
Post-translationally modified histone octamer
Assembly
Post-translationally modified nucleosome
Unmodified nucleosome
(Expressed)
(Native)
Repair assay
Excision signal
Excision signal
Comparison
26
NER from Nucleosomes Reconstituted with Native vs
Expressed Histones
GTG GTG GG GG

The efficiency of nucleotide excision repair of
cisplatin adducts from native nucleosomes is at
least two-fold higher than from expressed
nucleosomes.
Lanes 1 and 2 NER results for nucleosomes
reconstituted from expressed histones and
199GTG-Pt DNA. Lanes 3 and 4 NER results for
nucleosomes reconstituted from native,
modified histones and 199GTG-Pt DNA.
Dual Incision
27
Western Analysis of Recombinant and Native
Histone Octamers
Western blotting with anti-acetyl-lysine. 1
Native histone octamer. 2 Recombinant histone
octamer. 3 HeLa nuclear extract. 4 HeLa
nuclear extract treated with 4mM sodium
butyrate, a histone deacetylase inhibitor. 5
HeLa nuclear extract treated with 1mM cisplatin.
28
Numerous Cellular Proteins Recognize and Process
Platinum-DNA Adducts
Functions affected
Transcription Ubiquitination Repair Cell
cycle Others, via hijacking
Cellular proteins
Cell death or viability
Other proteins recognize cisplatin-DNA
cross-links
SSRP1 Ixr1 HMGB1 HMGB2 TBP XPE RPA XPC
MutSa Ku DNA photolyase Histone H1 (Jamieson
Lippard, 1999, Chem. Rev. 99, 2467-2498)
29
HMG-Domain Proteins
80 amino-acid DNA-binding motif
nonhistone components of chromatin
regulators of transcription and cellular
differentiation
recognizes DNA structural elements
bends DNA
LEF-1, SRY, hUBF, HMG1/2, mtTFA, tsHMG, Ixr
....and Cisplatin
An HMG-domain protein, hSSRP, was pulled out of
a cDNA expression library screened for binding
to cisplatin-modified DNA.
Almost all of the HMG-domain proteins
investigated specifically bind
cisplatin-modified DNA.
HMG-domain proteins recognize the major
1,2-intrastrand cisplatin-DNA adducts but not
the 1,3-intrastrand cross-link or trans-DDP
adducts.
Exposure to cisplatin, but not trans-DDP,
influences the intracellular distribution of
several HMG-domain proteins in human cell lines.
30
HMG-box proteins bind specifically to cisplatin
1,2-intrastrand cross-links.
These major adducts are shielded from nucleotide
excision repair in vitro and in vivo.
Individual A and B domains of HMGB1 are
responsible for the recognition of
cisplatin-modified DNA.
31
The F37A Mutation in HMGB1 Domain A Abrogates
Binding to Cisplatin-Modified DNA
5
-

C
C
T
C
T
C
T
G
G
A
C
C
T
T
C
C

Phe
Ala
3
-

G
G
A
G
A
G
A
C
C
T
G
G
A
A
G
G



DNA 5 nM
DomA
F37A DomA





10 nM
200 nM 10 nM
200 nM
Protein-DNA complex
Free DNA

32
HMG-Domain Proteins Inhibit Repair of the Major
Cisplatin-DNA Adduct
Protein
Specific Inhibition (µM)
Expression
Function
HMGB1
1-4
ubiquitous
(?) architectural factor
0.5-1
HMGB1 domain B
Huang, et a.l 1994 Proc. Natl. Acad. Sci. USA 91,
10394.
Zamble, et al. 1996 Biochemistry 35, 10004.
HMGB2 levels in rat testis are gt 4-fold higher
than HMGB1 HMGB2 levels in most other tissue
(Bucci, et al., 1984 J. Biol. Chem., 259,
8840-8846).
33
Repair Shielding by HMG-Domain Protein
Overexpression of an HMG-domain protein may
sensitize cells to cisplatin.
34
Steroid Hormones Estrogen and Progesterone
O
H
O
H
O
O
Progesterone
Estrogen
stimulates cell proliferation
does not cause cell proliferation
HMG1 facilitates binding of the estrogen
receptor to its DNA response element
HMG1 facilitates binding of the progesterone
receptor to its DNA response element
treatment of MCF-7 cells with estrogen causes
a 2.5 fold increase in HMG1 mRNA levels (Chau
et al, 1998)
currently no data that correlates the levels
of HMG1 and progesterone
35
MCF-7 Cells Treated with Estrogen or
Progesterone Express Higher Levels of HMG1
36
Estrogen Sensitizes MCF-7 Cells to Cisplatin
100
Cell Survival Assay
Untreated MCF-7 cells
10
cell survival
Estrogen-treated MCF-7 cells
1
0
5
10
cisplatin (µM)
MCF-7 cells treated with estrogen are two-fold
more sensitive to cisplatin IC50 2 µM
1 µM
37
.
Sensitivity to Carboplatin is also Modulated by
Steroid Hormones
Carboplatin is less toxic than cisplatin and
more widely used in the clinic.
Carboplatin-DNA adducts are also recognized by
HMG-domain proteins.
20 h pretreatment of MCF-7 cells with
carboplatin followed by 4 h cotreatment with
hormones yield the maximum cisplatin sensitivity.
Timing of hormone and carboplatin treatment is
important in determining the degree of
sensitization.
38
Steroid Hormones Increase Cisplatin Sensitivity
of Ovarian BG-1 Cells
.
Viable cells
BG-1


ER
/PR
cisplatin (µM)
Steroid hormone treatment increases cisplatin
sensitivity of BG-1 cells two-fold
A pilot study has begun at Dana Farber Cancer
Institute and Mass General
Hospital to determine whether treatment of
ovarian cancer patients with
cisplatin/carboplatin treatment in combination
with steroid hormones will
improve the potency of platinum drugs against
ovarian cancer
39
Why Use Pt(IV)?

• Pt(IV) complexes are kinetically inert
• Facilitates synthetic manipulations
• Allows for oral administration
• Different pharmacological and pharmaco-kinetic
  properties
• Spectrum of activity
• Reduced side effects
• Drug resistance
• Reduction in vivo to reactive Pt(II)

40
Full characterization by NMR spectroscopy and
ESI-MS
no hormone
BEP, 2h
estrogen, 2h
Barnes Lippard (2003) unpublished results.
41
Cytotoxicity Studies BEP1
IC50 3.7 ?M (MCF-7), 3.8 ?M (HCC-1937) Thus
HMGB1 overexpression does not sensitize the ER()
cells.
Barnes Lippard (2003) unpublished results.
42
BEP1 Cytotoxicity Why are ER() cells not
sensitized compared to the ER(-) cells?

• Kinetics of HMGB1 upregulation are not optimized
  for repair-shielding of cisplatin adducts
• Concentration of estrogen delivered to the cell
  is not suitable for desired HMGB1 upregulation
• Estrogen-induced cell proliferation
• Estrogen-compounds derivatized at the 17-position
  are not recognized by the estrogen-receptor with
  strong affinity

43
Strategy to Address Kinetics IssueVary the
Length of the Linker to Estrogen Moiety
Barnes Lippard (2003) unpublished results.
44
Cytotoxicity Studies BEP2, BEP3, BEP4, BEP5
Optimal kinetics
45
Structures of cisplatin-DNA 1,2-intrastrand
cross-link, and in complex with HMG-domain A,
reveal hydrophobic notch and Phe
intercalation. Adduct blocks transcription and
leads to ubiquitination of RNA Pol II large
subunit.
Nucleotide excision repair removes the major
1,2-intrastrand cross-links repair is less
efficient from nucleosomes. Post- translational
histone modification stimulates NER. Cisplatin
treatment of cells stimulates histone acetylation.
HMG-domain proteins shield cisplatin intrastrand
d(GpG) cross-links from nucleotide excision
repair.
Steroid hormones stimulate HMGB1 expression and
sensitize cells to cisplatin and carboplatin.
Phase I clinical trial has commenced at DFCI
and MGH. Novel linked Pt(IV) estradiol complex
strategy for new drug candidates.
46
Electron Transfer (ET) in Living Systems
PRINCIPLES

• M-binding sites tailored to minimize structural
  changes upon ET
• One-electron transfer processes preferred
• Coupling of H with electron transfer controls
  redox potential
• ET can occur over long distances 11-13 Å is
  most common
• Parameters distance, driving force,
  reorganizational energy

TOPICS

• Three major bioinorganic ET units FenSn
  clusters Cu hemes
• Long-distance electron transfer dependence on
  distance, driving force, reorganization
  energy
• Electron supply in the methane monooxygenase
  system

47
The Major Metal Units in ET Proteins (1)
Iron-Sulfur Clusters
48
Properties of Iron-Sulfur Clusters
(A) Rubredoxin FeS, 2.25 - 2.30 Å in
oxidized (FeIII) and reduced (FeII) states
Reduction potentials - 50 to 50 mV (B)
2Fe-2S Ferredoxins (Fd)
Reminder eo -RT/nF lnQ pH, where Q
Mn/Mn-1 Thus, at pH 7, the biological
H2/2H standard couple is - 420 mV.
FeII FeII
FeII FeIII
FeIII FeIII
oxidized
reduced
mixed-valent
all physiological uses
Reduction potentials -490 to - 280 mV
(C) 3Fe-4S Ferredoxins (cube missing a corner)
FeIII 3S4
FeIII 2 FeII S4
Reduction potentials -700 to - 100 mV
49
Properties of Iron-Sulfur Clusters, contd
(D) 4Fe-4S Ferredoxins and High-potential Iron
Proteins (HiPIPs)
The three state hypothesis
FeII3 FeIII
FeII2 FeIII2
FeII FeIII3
HiPIP
Ferredoxin
Reduction potentials -650 to - 280 mV (Fd)
350 mV (HiPIP)
minimal reorganizational energy