G9600 TFF 2003

Apoptosis: Mechanisms (Day 1)

• Historical Perspective• Serine-threonine kinases, survival signaling

downstream of PTEN/PI3K/Akt• Bcl-2 family members, potential usage of

BH3 domains as drug targets• Mitochondrial pathway, apoptogenic factors

G9600 TFF 2003

Apoptosis: Drug targets (Day 2)

• Caspases, apoptosome, inflammasome• TNFR, CD95/Fas, ApoL• TRAIL and decoy receptors• Bcl-2/xL: Antisense, inhibitors of protein-

protein interactions, SAR• Caspases: Why it did not work

G9600 TFF 2003

Historical Observations of Apoptosis in Development

G9600 TFF 2003

Historical Timeline of Apoptosis Research

G9600 TFF 2003

Cell Death(Apoptosis)

Proliferation

Differentiation

Cell Death is a Physiological Response of CellsTo Changes in the Extracellular Environment

G9600 TFF 2003

Cell Death(Apoptosis)

Tumor suppressor genes(e.g., p53, PTEN, Caspases)

Oncogenes(e.g., AKT, Bcl-2)

DrugsIrradiation

Stress

Endogenous Genes Modify Cell Death

G9600 TFF 2003

Mitochondria Conduct Apoptotic Signaling

G9600 TFF 2003

Cellular Receptors Transduce Extracellular Signals

G9600 TFF 2003

Cellular Receptors Initiate Intracellular Cascades

G9600 TFF 2003

PtdIns-Dependent Second Messengers FunctionIn Distinct Intracellular Pathways…

G9600 TFF 2003

… Depending on Distinct Regulatory DomainsOf Target Proteins

G9600 TFF 2003

PIP3-Dependent Cell Responses are Mediated byPTEN/PI3K/AKT

G9600 TFF 2003

Multiple Cell Functions are Mediated byPTEN/PI3K/AKT

G9600 TFF 2003

PTEN/PI3K/AKT Signal Transduction isEvolutionarily Conserved

G9600 TFF 2003

Loss of AKT Signaling Leads to Lifespan Extensionand Reduced Cell Size (=Smaller Organisms)

Yeast (sch9) Fly (Dakt) Mouse (Akt1,2,3)

G9600 TFF 2003

Decreased Akt Gene Function = Mini-Mice

Akt1 KO(~20% smaller)

Akt1 wtLittermate

G9600 TFF 2003

Increased Strength of AKT Signaling = Cancer

G9600 TFF 2003

AKT Regulates Distinct Downstream Substrates

G9600 TFF 2003

AKT: A BAD Kinase Makes Good

G9600 TFF 2003

Mitochondria = Pandora’s Box of Cell Death

G9600 TFF 2003

Bcl-2 Proteins as Conserved GatekeepersOf Mitochondrial Integrity

G9600 TFF 2003

Death Signals Inhibit the Anti-Apoptotic Bcl-2Family Member CED-9 in Lower Eukaryotes

G9600 TFF 2003

Conservation of a Common Death PathwayInvolving Bcl-2-Type Proteins

G9600 TFF 2003

Pro-Apoptotic Bcl-2 Family Members in MammalsComprise Functionally Distinct Sentinels…

G9600 TFF 2003

…That Sense Cellular Stress and are Activated byMultiple Mechanisms…

G9600 TFF 2003

…Including Cleavage by Cellular Proteases

BID (inactive) tBID (active)

G9600 TFF 2003

Bcl-2-Type Proteins are Characterized by the BH3Domain of Protein-Protein Interactions…

G9600 TFF 2003

…but Comprise a Family of Structurally andFunctionally Diverse Proteins

G9600 TFF 2003

Inhibition of Bcl-2/Bcl-xL Results in the Release of Apoptogenic Factors

G9600 TFF 2003

Bcl-2/Bcl-xL Retain Apoptogenic Proteins in theMitochondrial Intermembrane Space…

G9600 TFF 2003

…by Regulating Mitochondrial Channels andInhibiting Pore Formation by Bax/Bak

G9600 TFF 2003

Mitochondrial Breakdown Results from thePermeabilization, Rupture or Leakage of the

Outer Mitochondrial Membrane (OMM)

G9600 TFF 2003

Bcl-2/Bcl-xL as Drug Targets

• Antisense Oligonucleotides [e.g. Genta=G-3139 (5’-d(P-thio)TCT-CCC-AGC-GTG-CCC-CAT-3’)

• Natural Compounds (Antimycin A3 binds to the hydrophobic groove in Bcl-xL and inhibits protein-protein interactions)

• SAR by NMR = structure-activity relationships by nuclear magnetic resonance

G9600 TFF 2003

SAR-by-NMR Method: Screening for Small Moleculesand Improving their Affinities using Linkers and NMR

G9600 TFF 2003

Advantage over Conventional Library Methods:Fewer Compounds have to be Synthesized

G9600 TFF 2003

G9600 TFF 2003

Sequence-Specific Proteases (Caspases) ExecuteApoptotic Disassembly of Cellular Protein

G9600 TFF 2003

Caspases Contain Similar Catalytic SubunitsAnd Distinct N-terminal Regulatory Domains

G9600 TFF 2003

Initiator Caspases (Caspase-8,9) are ActivatedAccording to the Forced-Proximity-Model…

G9600 TFF 2003

…Downstream of Death Signals (Caspase-8)Or Mitochondrial Damage (Caspase-9)

G9600 TFF 2003

Executioner Caspases (Caspase-3,6,7) FormStructural Heterodimers

G9600 TFF 2003

Executioner Caspases Require Processing by UpstreamProteases for Dimer Formation (=Activation)

G9600 TFF 2003

Caspase-9 Differs from Other Caspases byHaving Only a Single Active Site…

G9600 TFF 2003

…that is Formed by the Induced Complex Formation of Caspase-9 Molecules

G9600 TFF 2003

Apaf-1 Mediates the ATP/dATP- and Cytochrome c-Dependent Multimerization of Caspase-9

G9600 TFF 2003

Electron Microscopy Photographs of Apaf-1In Complex with dATP and Cytochrome c

G9600 TFF 2003

Apaf-1 Complexes (=Apoptosomes) Form PropellerStructures with Heptagonal Symmetry (=7-fold)

G9600 TFF 2003

Components of the Apoptosome are LocalizedIn Defined Parts of the Propeller Structure

G9600 TFF 2003

Caspase-9 Molecules are Localized to the HubAnd Form a Dome that is the Basis for the…

G9600 TFF 2003

…Formation of the ‘Evil Oreo’ of Apoptosis

G9600 TFF 2003

Caspase Activity is Determined with FluorogenicTetrapeptides Resembling Protease Cleavage Sites

G9600 TFF 2003

Systematical Permutations of Tetrapeptide SequencesReveal Synthetic Substrate Peptides…

G9600 TFF 2003

…With Increased Specificity for Selected CaspasesAnd Groups of Caspases…

G9600 TFF 2003

…That Form the Rational for the Generation ofPeptoid Caspase Inhibitors

Unfortunately: Limited Practical Use because ofHepatobiliar P1-Asp Transporter System (=Secretion)

G9600 TFF 2003

In Summary: Death Receptor Signals (Extrinsic)Induce Caspase-8 Activation while …

G9600 TFF 2003

…Cellular Stresses (Intrinsic) Cause MitochondrialDamage Leading to Caspase-9 Activation

G9600 TFF 2003

Other Caspases (Caspase-1,5) are Involved inInflammatory Cell Responses…

G9600 TFF 2003

…That Involve Multimolecular Complexes…

G9600 TFF 2003

…And Lead to the Assembly of an Inflammasome

G9600 TFF 2003

Extrinsic Cell Death Pathways Depend onDeath-Receptor Ligands such as TRAIL

G9600 TFF 2003

CD95/Fas/Apo1 Caspase-8 Activation (=Death)

G9600 TFF 2003

Apo2L/TRAIL Caspase-8 Activation (=Death)

Increased Sensitivity in Cancer Cells (Lack of Decoy Receptors)

G9600 TFF 2003

κ

κ

κ

κ

κκ

Apo3L/TNF Caspase-8 Activation (=Death)+ NF-κB Activation (=Survival)

G9600 TFF 2003

Outlook: DNA Damage Responses

G9600 TFF 2003

Outlook: Role of p53