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Principles of Bioinorganic Chemistry - 2003

Principles of Bioinorganic Chemistry - 2003.

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Principles of Bioinorganic Chemistry - 2003

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  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. 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

  3. 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

  4. 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

  5. Pt Pt Pt Pt Pt

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. Cisplatin Inhibits b-Lactamase Gene Expression 40 µM cisplatin 37°C, 24 h 1 µM CCF2/AM Control

  12. 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

  13. 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.

  14. Comedy Restart transcription OR Tragedy Cell death Dead End Selective cell death of cancer cells is the goal! Consequence of Cisplatin Damage

  15. 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.

  16. 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.

  17. Nucleosomal DNA Free DNA Nucleosome Assembly from DNA Repair Probes Sucrose gradient centrifugation Free DNA + Histone Octamer Stepwise dialysis Nucleosomal DNA

  18. 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%

  19. Does Histone Modification Affect the Process? Strahl, B.D.; Allis, C.D.Nature2000, 403,41-5.

  20. 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

  21. 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

  22. 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.

  23. 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)

  24. + 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.

  25. 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.

  26. 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

  27. 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).

  28. Repair Shielding by HMG-Domain Protein Overexpression of an HMG-domain protein may sensitize cells to cisplatin.

  29. 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

  30. MCF-7 Cells Treated with Estrogen or Progesterone Express Higher Levels of HMG1

  31. 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

  32. 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.

  33. 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

  34. 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)

  35. Full characterization by NMR spectroscopy and ESI-MS no hormone BEP, 2h estrogen, 2h Barnes & Lippard (2003) unpublished results.

  36. 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.

  37. 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

  38. Strategy to Address Kinetics Issue:Vary the Length of the Linker to Estrogen Moiety Barnes & Lippard (2003) unpublished results.

  39. Cytotoxicity Studies: BEP2, BEP3, BEP4, BEP5 Optimal kinetics

  40. 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.

  41. 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

  42. The Major Metal Units in ET Proteins (1) Iron-Sulfur Clusters

  43. 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

  44. 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

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