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slide1

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/

slide3

Oral Drugs

1997

Pt enters all cells

Some Pt expelled

CELL NUCLEUS

DNA binding

HMG binding

Transport of Pt in the Body

Injection Pt

Rescue agent

Transport

diuretic

Apoptosis

Kidney (toxicity)

LIVER

p53 active

Excretion: 50% <48hrs; rest <2months

slide4

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
slide5

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
slide7

Pt

Pt

Pt

Pt

Pt

slide8

Structures of the 1,2-d(GpG) Intrastrand Cisplatin Adduct

d(pGpG) adduct

duplex DNA adduct

Sherman et al. (1985) Science230, 412.

Takahara et al. (1995) Nature377, 649.

slide9

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.

slide10

G

H

N

G

H

N

G

3

Pt

G

3

Pt

G

H

N

G

H

N

3

3

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

slide11

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)Cl3]PØ4

9. [Pt(NH3)3Cl]Cl

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.

slide12

A Reporter Gene Assay Using b-Lactamase and a

Fluorescent Substrate

B

t

O

A

c

O

O

A

c

H

N

-

O

C

l

-

O

Cytoplasmic

H

N

H

esterases

N

C

l

-

C

O

2

N

H

C

O

A

M

2

-

C

O

2

-

O

-

O

H

-

C

O

N

2

C

l

H

N

-

C

O

2

S

H

-

C

O

2

FRET

520 nm

409 nm

O

O

O

O

O

O

O

O

O

O

S

O

GREEN

CCF2/AM

O

N

S

O

CCF2

O

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.

•Blue:green ratio quantitates gene expression without correcting for cell plating.

slide13

Cisplatin Inhibits b-Lactamase Gene Expression

40 µM cisplatin

37°C, 24 h

1 µM CCF2/AM

Control

slide14

911...AAA

Cisplatin damage site

Pol II

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

slide15

Ub

Repair team

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.

slide16

Comedy

Restart transcription

OR

Tragedy

Cell death

Dead End

Selective cell death of cancer cells is the goal!

Consequence of Cisplatin Damage

slide20

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, Nature389, 251-260.

slide21

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

32P-ATP

T4 Kinase

ATP

T4 Kinase

ATP

T4 Kinase

ATP

B

C

D

E

*P

P

P

P

A

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.

slide22

Nucleosomal DNA

Free DNA

Nucleosome Assembly from DNA Repair Probes

Sucrose gradient

centrifugation

Free DNA

+

Histone Octamer

Stepwise dialysis

Nucleosomal DNA

slide23

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%

does histone modification affect the process
Does Histone Modification Affect the Process?

Strahl, B.D.; Allis, C.D.Nature2000, 403,41-5.

slide25

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

Excision

signal

Comparison

ner from nucleosomes reconstituted with native vs expressed histones
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

slide27

1 2 3 4 5

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.

slide28

G

H

N

G

H

N

G

3

Pt

G

3

Pt

G

H

N

G

H

N

3

3

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)

slide29

+

N

H

3

C

O

O

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.

slide30

Structure of a Complex of HMGB1 Domain A

with 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 and

in vivo.

Individual A and B domains of HMGB1 are responsible for the recognition of cisplatin-modified DNA.

slide31

H

C

H

H

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

slide32

HMG-Domain Proteins Inhibit Repair of the Major Cisplatin-DNA Adduct

Protein

Specific Inhibition (µM)

Expression

Function

HMGB1

1-4

ubiquitous

(?) architectural factor

HMGB1 domain B

0.5-1

Huang, et a.l 1994Proc. Natl. Acad. Sci. USA91, 10394.

Zamble, et al. 1996Biochemistry 35, 10004.

HMGB2 levels in rat testis are > 4-fold higher than HMGB1 + HMGB2 levels in most other tissue (Bucci, et al., 1984J. Biol. Chem., 259, 8840-8846).

slide33

Repair Shielding by HMG-Domain Protein

Overexpression of an HMG-domain protein may sensitize cells to cisplatin.

slide34

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

slide36

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

slide37

100

MCF-7

+

+

ER

/PR

% Viable cells

10

n

o

h

o

r

m

o

n

e

-

7

1

0

M

e

s

t

r

o

g

e

n

-

7

1

0

M

p

r

o

g

e

s

t

e

r

o

n

e

-

7

1

0

M

e

s

t

r

o

g

e

n

a

n

d

-

7

1

0

M

p

r

o

g

e

s

t

e

r

o

n

e

1

0

20

40

60

80

100

120

140

160

[carboplatin] (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.

slide38

100

n

o

h

o

r

m

o

n

e

-

7

2

x

1

0

M

e

s

t

r

o

g

e

n

-

7

2

x

1

0

M

p

r

o

g

e

s

t

e

r

o

n

e

10

1

0.1

0

1

2

3

4

5

6

7

8

9

10

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

why use pt iv
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)
slide40

Full characterization by NMR spectroscopy and ESI-MS

no hormone

BEP, 2h

estrogen, 2h

Barnes & Lippard (2003) unpublished results.

cytotoxicity studies bep1
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
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
Strategy to Address Kinetics Issue:Vary the Length of the Linker to Estrogen Moiety

Barnes & Lippard (2003) unpublished results.

slide45

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.

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.

slide46

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
slide48

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

(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

slide49

Properties of Iron-Sulfur Clusters, cont’d

(D) 4Fe-4S Ferredoxins and High-potential Iron Proteins (HiPIPs)

The three state hypothesis:

FeII3FeIII

FeII2FeIII2

FeII FeIII3

Ferredoxin

HiPIP

Reduction potentials: -650 to - 280 mV (Fd); + 350 mV (HiPIP)

minimal reorganizational energy