Principles of Bioinorganic Chemistry - 2003
Lectur e
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Date
Lectur e Topic
Reading
n+
9/4 T
( h) Intro; Choice, Uptake, Assembly of MIons
Ch. 5
9/ 9 T
( u) Metalloregulation of Gene Expression
Ch. 6
9/11 T
( h) Metallochaperones; Metal Folding, X-linking
Ch. 7
9/16 T
( u) Zinc Fingers; Metal Folding; C isplatin
Ch. 8
9/ 18 (Th) Cisplatin; Electron Transfer ; Fundamentals Ch. 9
9/23 T
( u) Long-Distance Electron Transfer
Ch. 9
9/25 T
( h) Hydrolytic Enzymes, Zinc, Ni, Co
Ch. 10
9/30 (MU ) Model Com plexes for Metallohydrolases
Ch. 10
10/2 (MU ) Dioxygen Carriers: Hb, Mb, Hc, Hr
Ch. 11
10/7 T
( u) O2 Activation, Hydroxylation: MMO, P-450, R2 Ch. 11
10/9 T
( h) Model Chemistry for O2 Carriers/Activators
Ch. 11
10/16 T
( h) Complex Systems:cyt. oxidase; nitrogenase
Ch. 12
10/21 T
( u) Metalloneurochemistry/MedicinalInorg. Chem. Ch. 12
10/23 T
( h) Term Exam ination
Ch. 12
Pr oblems
Ch. 1
Ch. 2
Ch. 3
Ch. 4
Ch. 5
Ch. 6
Ch. 7
Ch. 8
Ch. 9
Ch. 10
Ch. 11
Ch. 12
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/
Transport of Pt in the Body
Injection Pt
Rescue
agent
Transport
diuretic
Oral Drugs
1997
Pt enters all cells
Some Pt expelled
Apoptosis
Kidney (toxicity)
LIVER
CELL NUCLEUS
DNA binding
HMG binding
p53 active
Excretion: 50% <48hrs; rest <2months
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
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
Cisplatin -DNA Adducts
1,2- Intrastrand
Cross-links
H3N
H3N
Frequency
Pt
G
G
~90%
1,3- Intrastrand
Cross-links
H3N
H3N
Pt
G
T
G
~5%
Interstrand
Cross-links
H3N
H3N
Pt
G
G
~1-5%
Site-Specifically Platinated Duplex DNA
for X-ray and NMR Structure Determinations
Globally Platinated DNA
Many binding sites
Heterogeneous population of molecules
Good representation of in vivo platination
Site-Specifically Platinated DNA
Single binding site
Homogeneous population of molecules
Sequence programmable
Pt
Pt
Pt
Pt
Pt
Pt Pt
Pt
Pt
Pt Pt
Pinto, Naser, Essigmann, Lippard, JACS, 108 (1986) 7405
Manchanda, Dunham, & Lippard, JACS, 118 (1996) 5144 - on automated synthesizer
H 3 N NH 3
Pt
5'-C 1 C 2 T 3 C 4 T 5 G 6 G 7 T 8 C 9 T 10 C 11C 12 -3'
3'-G24G 23A 22G 21A20C 19C 18A 17G16A 15G 14G 13-5'
Structures of the 1,2-d(GpG) Intrastrand Cisplatin
Adduct
5'-G
d(pGpG) adduct
Pt
3'-G
A20
T5
G6
C19
Pt
G7
C18
T8
A17
duplex DNA adduct
Sherman et al. (1985) Science 230, 412
Takahara et al. (1995) Nature 377, 649.
Structure of a {Pt(R,R-DACH)}2+ Intrastrand CrossLink in a Duplex Dodecamer Showing the G*G* Step
A very similar structure occurs for the 3’ orientational isomer of a
{Pt(NH3)(NH2Cy)}2+ G*G* cross-link on the same duplex dodecamer.
Numerous Cellular Proteins Recognize and
Process Platinum-DNA Adducts
Functions affected
H 3N
H 3N
Pt
GG
G
Cellular
proteins
H 3N
H 3N
Pt
G
G
G
Transcription
Ubiquitination
Repair
Cell cycle
Others, via hijacking
Cell death or
viability
Transcription Inhibition Correlates with Cell Death
in a GFP Reporter Assay
320
12
11
280
4. [Pt(en)Cl2]
IC50 (µM)
9
200
10
160
5. cis-[Pt(dach)Cl2]
6. trans-[Pt(NH2CH3)2Cl2]
7. cis-[Pt(NH2-iPr)2Cl2]
8
120
8. [Pt(NH3)Cl3]PØ4
9. [Pt(NH3)3Cl]Cl
7
80
0
2. cis-[Pt(NH3)(NH2C6H11)Cl2]
3. cis-[Pt(NH2CH3)2Cl2]
240
40
1. cisplatin
10. [Pt(lysine)Cl2]
5
6
3, 4
2
1
0
20
11. [Pt(arginine)Cl2]
40
60
80 100 120 140
LC50 (µM)
12. [Pt(norleucine)Cl2]
•Northern blotting and nuclear run-on assays confirm that control of GFP
expression is at the transcriptional level.
A Reporter Gene Assay Using b-Lactamase and a
Fluorescent Substrate
FRET
409 nm
BtO
O
O
Cl
O
AcO
H
N
O
OAc
O
O
NH S
CCF2/AM
O
O
N
Cytoplasmic
esterases
S
-O
O
O
Cl
CCF2
O
-O
O
-O
O
447 nm
O
Cl
O
BLUE
H
N
-O
O
NH
GREEN
S
N
S
CO 2-
platinum
block
+
CO 2-
S
O HN
O
O
CO 2-
O
b-lactamase
O
H
N
CO 2AM
409 nm
520 nm
N
CO 2-
CO 2-
cells stay green
SH
•Enzymatic amplification allows detection of low-level gene expression.
•Blue:green ratio quantitates gene expression without correcting for cell plating.
Cisplatin Inhibits b-Lactamase Gene Expression
Control
40 µM cisplatin
37°C, 24 h
1 µM CCF2/AM
Consequences of Cisplatin-DNA Damage
Cisplatin damage site
blocks transcription
Pol II
Cisplatin damage site
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
Consequences of Cisplatin-DNA Damage
Ubiquitinated Pol II
is replaced.
Ub
Cellular repair machinery
is recruited
Repair team
Recognition and repair of the damage in answer
to the distress call is desired in healthy cells.
Consequence of Cisplatin Damage
Restart transcription
OR
Cell death
Dead End
Selective cell death of cancer cells is the goal!
Mammalian Nucleotide Excision Repair
XPA, XPF, ERCC1, HSSB
ATP
F
1
T FIIH
F
ADP+Pi
A
A
1
H SSB
H SSB
C
TFIIH
(XPB, XPD),
XPC
XPG
5' nick
1)
2)
3)
4)
5)
T FIIH
F
Damage recognition
Complex formation, DNA distortion
XPG binding, dual nicking
Excision, dissociation of the nuclease
Repair synthesis
1
A
H SSB
G
3' nick
C
ATP
PCNA
ADP+Pi
POL
dNTPs, ATP
Ligase
oligo
PCNA
Exinuclease
H SSB
Repair of Cisplatin-DNA Intrastrand Cross-links by Human
Nucleotide Excision Repair
5'
1
CAGCTGATTGCAGACTCAGTACGAATTC* TCTAGGCCTTCT TGCGGCCCATCG
TCTGTGCACTCT
Substrate
CFE
GG
GTG
F
F
H F G G H F G G
H,
HeLa Cell Free Extracts
F,
XPF Cell Free Extracts
G,
XPG Cell Free Extracts
FG, XPF + XPG Cell Free Extracts
30 nt
10% denaturing polyacrylamide gel
Huang, Zamble, Reardon, Lippard, Sancar
(1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10394.
3'
156
Kinetics of Excision of Cisplatin-DNA Adducts
in HeLa Cell Free Extracts
.
Time
(min)
0
GG
15 30 60 90
AG
0 15 30 60 90
120
100
80
60
40
P
er
ce
n
t
R
ep
ai
r
30 nt
GG
AG
20
n
o
rm
al
iz
ed
to
A
G
90
m
in
0
0
20
40
60
Time (min)
80
10% denaturing polyacrylamide gel
Huang, Zamble, Reardon, Lippard, Sancar
(1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10394.
GTG >> AG > GG
100
Structure of Nucleosome Core Particle
Nucleosome
Core Particle
Histone
Octame
r
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.
Synthesis of Site-Specifically Platinated
DNA Repair Probes (Wang, 2002)
5’
Top strand oligos
B
C
5’
5’
T4 kinase
32P-ATP
*P
Bottom strand oligos
D
E
T4 Kinase
ATP
B
P
T4 Kinase
ATP
C
D
A
5’
T4 Kinase
ATP
P
E
P
F
1. Annealing
2. Ligation
199mer
*
A:83-mer; B:20G*G*-Pt or 20G*TG*-Pt; C: 96-mer; D:72-mer; E:40CC or 40CAC; F: 87-mer.
Nucleosome Assembly from DNA Repair Probes
Free DNA
Stepwise dialysis
+
Histone Octamer
Sucrose gradient
centrifugation
Nucleosomal DNA
Nucleosomal DNA
Free DNA
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
Dual
Incision Lane 3: NER assay of nucleosomal 199GTG-Pt DNA
Lane 4: NER assay of naked 199GTG-Pt DNA
0.3% 1% 1% 10%
Does Histone Modification Affect the
Process?
Strahl, B.D.; Allis, C.D. Nature 2000, 403, 41-5.
Nucleosome Assembly from Native
(modified) and Recombinant (E. coli)
Histones
Unmodified
histone octamer
Post-translationally
modified histone octamer
Assembly
Unmodified
nucleosome
Post-translationally
modified nucleosome
(Expressed)
(Native)
Repair assay
Excision
signal
Comparison
Excision
signal
NER from Nucleosomes Reconstituted with
Native vs Expressed Histones
GTG
GTG
GG
GG
6
5
4
%
3
Native
Expressed
2
1
0
GG-Pt
GTGPt
The efficiency of nucleotide excision repair of
cisplatin adducts from native nucleosomes is at least
two-fold higher than from expressed nucleosomes.
Dual
Incision
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.
Western Analysis of Recombinant and
Native Histone Octamers
1
2
3
4
5
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.
Numerous Cellular Proteins Recognize and
Process Platinum-DNA Adducts
Functions affected
H 3N
H 3N
Pt
GG
G
Cellular
proteins
H 3N
H 3N
Pt
G
G
G
Transcription
Ubiquitination
Repair
Cell cycle
Others, via hijacking
Cell death or
viability
Other proteins recognize cisplatin-D NA cross-links
SSRP1; Ixr1; HMGB1; HMGB2; TBP; XPE; RPA; XPC; MutSa; Ku; DNA
photolyase; Histone H1 (Jamieson & Lippard, 1999, Chem. Rev. 99, 24672498)
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
NH 3+
COO
....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.
Structure of a Complex of HMGB1 Domain A
with Cisplatin-Modified Duplex DNA
G32
HMG-box proteins bind
specifically to cisplatin 1,2intrastrand 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 cisplatinmodified DNA.
77
C1
7 {Pt(NH ) }
32
Helix I
Helix III
C16
Helix II
G17
The F37A Mutation in HMGB1 Domain A Abrogates
Binding to Cisplatin-Modified DNA
H
Phe
Ala
C
H
H
DomA
10 nM
5’- CCTCTCTGGACCTTCC
3’- GGAGAGACCTGGAAGG
[DNA] = 5 nM
F37A DomA
200 nM 10 nM
200 nM
Protein-DNA
complex
Free DNA
HMG-Domain Proteins Inhibit Repair of the Major
Cisplatin-DNA Adduct
Protein
Specific Inhibition (µM)
HMGB1
1-4
HMGB1 domain B
0.5-1
Expression
ubiquitous
Function
(?) architectural factor
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 > 4-fold higher than HMGB1 + HMGB2 levels
in most other tissue (Bucci, et al., 1984 J. Biol. Chem., 259, 8840-8846).
Repair Shielding by HMG-Domain Protein
Overexpression of an HMG-domain protein may sensitize cells to cisplatin.
Steroid Hormones: Estrogen and Progesterone
OH
HO
O
O
Estrogen
Progesterone
•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
MCF-7 Cells Treated with Estrogen or Progesterone
Express Higher Levels of HMG1
Estrogen Sensitizes MCF-7 Cells to Cisplatin
% cell survival
100
Cell Survival Assay
Untreated MCF-7 cells
10
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
.
Sensitivity to Carboplatin is also Modulated by Steroid Hormones
100
% Viable cells
MCF-7
ER+/PR+
O
H3N
O
Pt
10
H3N
O
O
no hormone
10-7 M estrogen
10-7 M progesterone
10-7 M estrogen and
10-7 M progesterone
1
0
20
40
60
80
100
120
140
160
[carboplatin] (µM)
•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.
Steroid Hormones Increase Cisplatin Sensitivity of Ovarian BG-1 Cells
.
100
no hormone
-7
2x 10 M estrogen
-7
% Viable cells
2x 10 M progesterone
10
1
BG-1
ER+/PR+
0.1
0
1
2
3
4
5
6
7
8
9
10
[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
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)
Synthesis of BEP, an Estrogen-Tethered Cisplatin Precursor
O
O
H3N
H3N
Pt
Cl
Cl
3% H 2O2
50
o
C, 2h
O
O
OH
H3 N
Cl
4 equiv
Pt
50-60 %
H3 N
Cl
DMSO
O
o
OH
70 C, 12 h
BzO
HO
+
O
H3 N
H3N
4-DMAP
DIPC
DMF
O
O
O
N
H
BzO
H3 N
Pt
O
Cl
Cl
Cl
O
55-65%
O
O
O
NH 2
OH
Pt
H3 N
O
O
O
H
N
O
O
O
Cl
O
Full characterization by NMR
spectroscopy and ESI-MS
no hormone
BEP, 2h
Barnes & Lippard (2003) unpublished results.
estrogen, 2h
OBz
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.
BEP1 Cytotoxicity: Why are ER(+) cells not
sensitized compared to the ER(-) cells?
• Kinetics of HMGB1 upregulation are not optimized for repairshielding 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
Strategy to Address Kinetics Issue:
Vary the Length of the Linker to Estrogen Moiety
O
O
H
O
NH2
n
H 3N
O
O
O
Pt
H 3N
O
HO
O
OH
Cl
O
DIPC, 4-DMAP, DMF
Cl
O
O
O
O
H3N
O
O
H
O
nN
H
H3 N
O
Pt
O
O
O
H
N
Cl
O
nH
Cl
n = 2, 3, 4, or 5
BEP2 - BEP5
Barnes & Lippard (2003) unpublished results.
O
O
O
Cytotoxicity Studies: BEP2, BEP3, BEP4,
BEP5
Optimal kinetics
Summary of Major Findings
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. Posttranslational 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.
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
The Major Metal Units in ET Proteins (1)
Iron-Sulfur
Clusters
Properties of Iron-Sulfur Clusters
(A) Rubredoxin
Fe–S, 2.25 - 2.30 Å in oxidized (FeIII) and reduced (FeII) state
Reduction potentials: - 50 to + 50 mV
(B) 2Fe-2S Ferredoxins (Fd)
FeII FeII
FeII FeIII
reduced
mixed-valent
Reminder:
FeIII FeIII
oxidized
all physiological uses
Reduction potentials: -490 to - 280 mV
eo =
-RT/nF lnQ + pH,
where Q =
[Mn]/[Mn-1]
Thus, at pH 7, the
biological H2/2H+
(C) 3Fe-4S Ferredoxins (cube missing a corner)
standard couple
is - 420 mV.
FeIII 3S4
FeIII 2 FeII S4
Reduction potentials: -700 to - 100 mV
Properties of Iron-Sulfur Clusters, cont’d
(D) 4Fe-4S Ferredoxins and High-potential Iron Proteins
(HiPIPs)
The three state hypothesis:
FeII3 FeIII
FeII2 FeIII2
Ferredoxin
FeII FeIII3
HiPIP
Reduction potentials: -650 to - 280 mV (Fd); + 350 mV (HiPIP)
minimal reorganizational energy