Principles of Bioinorganic Chemistry - 2003 Lecture Date Lecture Topic Reading Problems 1 9/4 ( Th) Intro; Choice, Uptake, Assembly of M n+ Ions Ch. 5 Ch. 1 2 9/ 9 ( Tu) Metalloregulation of Gene Expression Ch. 6 Ch. 2 3 9/11 ( Th) Metallochaperones; Metal Folding, X- Ch. 7 Ch. 3 4 9/16 ( Tu) Zinc Fingers; Metal Folding; Cisplat Ch. 8 Ch. 4 5 9/ 18 ( Th) Cisplatin; Electron Transfer; Fundam Ch. 9 Ch. 5 6 9/23 ( Tu) Long-Distance Electron Transfer Ch. 9 Ch. 6 7 9/25 ( Th) Hydrolytic Enzymes, Zinc, Ni, Co Ch. 10 Ch. 7 8 9/30 (MU Model Complexes for Metallohydrolase Ch. 10 9 10/2 (MU Dioxygen Carriers: Hb, Mb, Hc, Hr Ch. 11 10 10/7 ( Tu) O 2 Activation, Hydroxylation: MMO, P- Ch. 11 Ch. 8 11 10/9 ( Th) Model Chemistry for O 2 Carriers/Activator Ch. 11 Ch. 9 12 10/16 ( Th) Complex Systems: cyt. oxidase; nitrogenas Ch. 12 Ch. 10 13 10/21 ( Tu) Metalloneurochemistry/Medicinal Inorg. Chem Ch. 12 Ch. 11 14 10/23 ( Th) Term Examination Ch. 12 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/
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/
.
DNA is the Biological Target of Cisplatin
+H3N
Pt
ClH3N
OH2
H3N
H3NH3N
ClH3N
Cl passivediffusion aquation
DNA adductformation
PtPt
u Cisplatin diffuses into cells, aquates, and attacks cellular targets, DNA, RNA and proteins.
N
N
N
N
N
N
O
N
N
HH
O
H
HH
C G7
1
89
2 34
56
1 23
456 N
N
N
N
NN
NO
O
H3C
H
H
H
AT7
2 34
561
89
1 23
456
u It is generally accepted that DNA is the main target, with platinum coordinating to N7 of the purine nucleobases guanine and adenine.
Transport of Pt in the Body
Transport
Kidney (toxicity)
Injection Pt
Excretion: 50% <48hrs; rest <2months
Oral Drugs
1997
LIVER
Pt enters all cellsSome Pt expelled
CELL NUCLEUSDNA binding
HMG binding
Apoptosis
p53 active
Rescue agent
diuretic
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
Structures of the 1,2-d(GpG) Intrastrand Cisplatin Adduct
Pt
5'-G
3'-G
T5
G6
G7
T8
A20
C19
C18
A17
Pt
d(pGpG) adduct
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 Cross-Link 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
TranscriptionUbiquitinationRepairCell cycleOthers, via hijacking
Cell death or viabilityPt G
GH3NH3N
G GG
GPtH3NH3N
Cellular proteins
Functions affected
123, 4
5 6
7
8
9
10
11
12
0
40
80
120
160
200
240
280
320
0 20 40 60 80 100 120 140
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)Cl3]PØ4
9. [Pt(NH3)3Cl]Cl
10. [Pt(lysine)Cl2]
11. [Pt(arginine)Cl2]
12. [Pt(norleucine)Cl2]
Transcription Inhibition Correlates with Cell Deathin a GFP Reporter Assay
LC50 (µM)
IC50 (
µM
)
•Northern blotting and nuclear run-on assays confirm that control of GFP expression is at the transcriptional level.
OHN
NHOO
-O
Cl
O
NO
S
CO2-
S
O-O O
CO2-
OHN
HNOO
-O
Cl
O
CO2-N
S
CO2-
SH
O-O O
CO2-
OHN
NHOO
BtO
Cl
O
NO
S
CO2AM
S
O
O
O
OAcAcO409 nm 520 nm
+
409 nm 447 nm
FRET
CCF2/AMCCF2
BLUE
GREEN
A Reporter Gene Assay Using -Lactamase and a Fluorescent Substrate
Cytoplasmicesterases
cells stay green
•Enzymatic amplification allows detection of low-level gene expression.•Blue:green ratio quantitates gene expression without correcting for cell plating.
-lactamase
platinum block
Control
Cisplatin Inhibits -Lactamase Gene Expression
40 µM cisplatin37°C, 24 h
1 µM CCF2/AM
Consequences of Cisplatin-DNA Damage
DNA
Pol II Cisplatin damage site
Cisplatin damage siteblocks transcription
Stalled Pol II triggersmultiple cellular processes
Failure to recognize the damage in answer to the distress call is desired in the cancer cell
Ub
Repair team
Consequences of Cisplatin-DNA Damage
Ubiquitinated Pol II is replaced.
Cellular repair machineryis recruited
Recognition and repair of the damage in answer to the distress call is desired in healthy cells.
Restart transcription
OR
Dead End
Cell death
Selective cell death of cancer cells is the goal!
Consequence of Cisplatin Damage
1) Damage recognition2) Complex formation, DNA distortion3) XPG binding, dual nicking4) Excision, dissociation of the nuclease5) Repair synthesis
TFIIH
HSSB
F1 A
TFIIH(XPB, XPD), XPC
ATP ADP+Pi
C
XPG
TFIIH
HSSB
F1
A
CG
ATPPCNA
ADP+Pi
PCNA
HSSB
POLdNTPs, ATPLigase Exinuclease
5' nick
3' nick
HSSBXPA, XPF, ERCC1, HSSB
F1
A
Mammalian Nucleotide Excision Repair
oligo
GTGGG
CAGCTGATTGCAGACTCAGTACGAATTC* TGCGGCCCATCG5' 3'
1 156
CFE
Repair of Cisplatin-DNA Intrastrand Cross-links by HumanNucleotide Excision Repair
Synthesis of Site-Specifically PlatinatedDNA Repair Probes (Wang, 2002)
A F
*P
1. Annealing 2. Ligation
B C D E
T4 Kinase ATP
T4 kinase32P-ATP
T4 Kinase ATP
T4 Kinase ATP
EDCB
*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.
P P P
5’ 5’ 5’ 5’
Top strand oligos Bottom strand oligos
Nucleosome Assembly from DNA Repair Probes
Free DNA
Nucleosomal DNA
Free DNA +Histone Octamer
Stepwise dialysisSucrose gradientcentrifugation
Nucleosomal DNA
Nucleosome Inhibits NER of Cisplatin Adducts
1 2 3 4
Dual Incision
0.3% 1% 1% 10%
Lane 1: NER assay of nucleosomal 199GG-Pt DNALane 2: NER assay of naked 199GG-Pt DNALane 3: NER assay of nucleosomal 199GTG-Pt DNALane 4: NER assay of naked 199GTG-Pt DNA
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.
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 nucleosome
Unmodified nucleosome
Post-translationally modified histone octamer
Assembly
Repair assay
Excision signal
Excision
signalComparison
(Expressed) (Native)
NER from Nucleosomes Reconstituted with Native vs
Expressed HistonesGTG GTG GG GG
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.
The efficiency of nucleotide excision repair of cisplatin adducts from native nucleosomes is at least two-fold higher than from expressed nucleosomes.
0
1
2
3
4
5
6
GG-Pt GTG-Pt
Native
Expressed%
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
TranscriptionUbiquitinationRepairCell cycleOthers, via hijacking
Cell death or viabilityPt G
GH3NH3N
G GG
GPtH3NH3N
Cellular proteins
Functions affected
Other proteins recognize cisplatin-DNA cross-links
•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.
NH3+
COO
•An HMG-domain protein, hSSRP, was pulled out of a cDNA expression library screened for binding to cisplatin-modified DNA.
Helix I
Helix II
Helix III
777
G32 C1
G17
C16
{Pt(NH3)2}
Structure of a Complex of HMGB1 Domain Awith Cisplatin-Modified Duplex DNA
HMG-box proteins bind specifically to cisplatin 1,2-intrastrand cross-links.
These major adducts are shielded from nucleotide excision repair in vitro andin vivo.
Individual A and B domains of HMGB1 are responsible for the recognition of cisplatin-modified DNA.
Protein-DNAcomplex
Free DNA
[DNA] = 5 nM
5’- CCTCTCTGGACCTTCC
3’- GGAGAGACCTGGAAGG
10 nM 200 nM 10 nM 200 nM
DomA F37A DomA
The F37A Mutation in HMGB1 Domain A Abrogates Binding to Cisplatin-Modified DNA
Phe Ala C
H
H
H
HMG-Domain Proteins Inhibit Repair of the Major Cisplatin-DNA Adduct
HMGB1Protein Specific Inhibition (µM)
1-40.5-1HMGB1 domain B
ubiquitous (?) architectural factorExpression Function
Zamble, et al. 1996 Biochemistry 35, 10004.Huang, et a.l 1994 Proc. Natl. Acad. Sci. USA 91, 10394.
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).
HMG-domainprotein
Repair complex
Adduct is repaired
Repair is blocked
Pt GG
H3NH3N
GG
GPt
G
GG
GPtH3NH3N
H3NH3N
Repair Shielding by HMG Domain Proteins
Repaircomplex
Overexpression of HMG1 may sensitize cells to cisplatin
Repair Shielding by HMG-Domain Protein
Overexpression of an HMG-domain protein may sensitize cells to cisplatin.
Steroid Hormones: Estrogen and Progesterone
O
O
HO
OH
Estrogen Progesterone
•stimulates cell proliferation
•HMG1 facilitates binding of the estrogen 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)
•does not cause cell proliferation
•HMG1 facilitates binding of the progesterone receptor to its DNA response element
•currently no data that correlates the levels of HMG1 and progesterone
MCF-7 Cells Treated with Estrogen or ProgesteroneExpress Higher Levels of HMG1
1
10
100
0 5 10
% c
ell
su
rviv
al
Estrogen Sensitizes MCF-7 Cells to Cisplatin
MCF-7 cells treated with estrogen are two-fold more sensitive to cisplatin
IC50 = 2 µM 1 µM
Cell Survival Assay
Untreated MCF-7 cells
Estrogen-treated MCF-7 cells
[cisplatin] (µM)
.
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.
1
10
100
0 20 40 60 80 100 120 140 160
no hormone10-7 M estrogen10-7 M progesterone10-7 M estrogen and10-7 M progesterone
MCF-7ER+/PR+
% V
iab
le c
ell
s
[carboplatin] (µM)
H3N
Pt
H3N O
O
O
O
.
Steroid Hormones Increase Cisplatin Sensitivity of Ovarian BG-1 Cells
0.1
1
10
100no hormone
2 x 10 -7 M estrogen
2 x 10 -7 M progesterone
0 1 2 3 4 5 6 7 8 9 10
•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
BG-1ER+/PR+
% V
iab
le c
ell
s
[cisplatin] (µM)
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)
Full characterization by NMR spectroscopy and ESI-MS
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
Strategy to Address Kinetics Issue:Vary the Length of the Linker to Estrogen
Moiety
Barnes & Lippard (2003) unpublished results.
O
O
OH
NH2
O
PtH3N
H3N Cl
Cl
O
O
OHO
O
O
O
HO
PtH3N
H3N Cl
Cl
O
O
HN
O
O
O
O
NH
O
O
OO
O
O
O
O
H
H
n
DIPC, 4-DMAP, DMF
n
n
BEP2 - BEP5
n = 2, 3, 4, or 5
BEP2
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10
Concentration (uM)
% Survival
BEP2 MCF-7
HCC-1937 Average
50% Survival
BEP3
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10
Concentration (uM)
% Survival
BEP3 MCF-7
HCC-1937 Average
50% Survival
BEP4
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10
Concentration (uM)
% Survival
BEP4 MCF-7
HCC-1937 Average
50% Survival
BEP5
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10
Concentration (uM)
% Survival
BEP5 MCF-7
HCC-1937 Average
50% Survival
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. Post- translational histone modification stimulates NER. Cisplatin treatment of cells stimulates histone acetylation.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-SulfurClusters
Properties of Iron-Sulfur Clusters(A) Rubredoxin Fe–S, 2.25 - 2.30 Å in oxidized (FeIII) and reduced (FeII) states Reduction potentials: - 50 to + 50 mV
