Therapies: Apoptosis-based Mechanisms and Applications
Perspective Bcl-2 family members, potential usage of BH3 domains as drug targets Bcl-2/xL inhibitors - Antisense, inhibitors of protein-protein interactions, SAR Caspases, apoptosome, inflammasome Caspase inhibitors and how they work TNFR, CD95/Fas, ApoL TRAIL and decoy receptors
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Cell Death is a Physiological Response of Cells To Changes in the Extracellular Environment
Differentiation Proliferation
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Cell Death (Apoptosis)
Historical Observations of Apoptosis in Development
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Historical Timeline of Apoptosis Research
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Diseases Associated with Defective Apoptosis Regulation
I) Insufficient apoptosis
Cancer (cell accumulation; resistance to chemotherapy, radiation and immunotherapy) Autoimmunity (failure to eliminate autoreactive lymphocytes) Restenosis (accumulation of vascular smooth muscle cells) Persistent infections (failure to eradicate bacteriaor virus-infected cells)
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Diseases Associated with Defective Apoptosis Regulation
II) Excessive apoptosis
Ischemia -- reperfusion injury (stroke, myocardial infarction) Heart failure (myocyte loss) Neurodegeneration (Alzheimer's, Parkinson's, Huntington's and amyothrophic lateral sclerosis=ALS) Inflammation Osteoarthritis (chondrocyte depletion)
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Diseases Associated with Defective Apoptosis Regulation
II) Excessive apoptosis (cont'd)
HIV (depletion of T-lymphocytes) Bacterial infection using apoptosis-inducing virulence proteins Allograft rejection and graft versus host disease Type I diabetes Trauma (spinal cord injury, brain injury)
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Oncology Targets
Drugs Irradiation Stress
Oncogenes (e.g., AKT, Bcl-2)
Tumor suppressor genes (e.g., p53, PTEN, Caspases) Cell Death (Apoptosis)
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The Core Cell Death Machinery
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Mitochondria Conduct Apoptotic Signaling
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Bcl-2 Proteins as Conserved Gatekeepers Of Mitochondrial Integrity
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Death Signals Inhibit the Anti-Apoptotic Bcl-2 Family Member CED-9 in Lower Eukaryotes
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Conservation of a Common Death Pathway Involving Bcl-2-Type Proteins
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Bcl-2-Type Proteins are Characterized by a BH3 Domain of Protein-Protein Interactions...
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...but Comprise a Family of Structurally and Functionally Diverse Proteins
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Pro-Apoptotic Bcl-2 Family Members in Mammals Comprise Functionally Distinct Sentinels...
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...That Sense Cellular Stress and are Activated by Multiple Mechanisms...
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...Including Cleavage by Cellular Proteases
BID (inactive)
tBID (active)
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Inhibition of Bcl-2/Bcl-xL Results in the Release of Apoptogenic Factors
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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 proteinprotein interactions) Small compounds (HA14.1 targeting Bcl-2) SAR by NMR = structure-activity relationships by nuclear magnetic resonance
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Identification of HA14-1 by Computer Screening
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SAR-by-NMR Method: Screening for Small Molecules and Improving their Affinities using Linkers and NMR
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Advantage over Conventional Library Methods: Fewer Compounds have to be Synthesized
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Sequence-Specific Proteases (Caspases) Execute Apoptotic Disassembly of Cellular Proteins
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Caspases Contain Similar Catalytic Subunits And Distinct N-terminal Regulatory Domains
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Initiator Caspases (Caspase-8,9) are Activated According to the Forced-Proximity-Model...
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...Downstream of Death Signals (Caspase-8) Or Mitochondrial Damage (Caspase-9)
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Executioner Caspases (Caspase-3,6,7) Form Structural Heterodimers
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Executioner Caspases Require Processing by Upstream Proteases for Dimer Formation (=Activation)
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Caspase-9 Differs from Other Caspases by Having Only a Single Active Site...
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...that is Formed by the Induced Complex Formation of Caspase-9 Molecules
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Apaf-1 Mediates the ATP/dATP- and Cytochrome cDependent Multimerization of Caspase-9
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Electron TFF Microscopy Photographs of Apaf-1 In Complex with dATP and Cytochrome c
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Apaf-1 Complexes (=Apoptosomes) Form Propeller Structures with Heptagonal Symmetry (=7-fold)
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Components of the Apoptosome are Localized In Defined Parts of the Propeller Structure
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Caspase-9 Molecules are Localized to the Hub And Form a Dome that is the Basis for the...
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...Formation of the `Evil Oreo' of Apoptosis
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Caspase Activity is Determined using Fluorogenic Tetrapeptides Resembling Protease Cleavage Sites
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Systematical Permutations of Tetrapeptide Sequences Reveal Synthetic Substrate Peptides...
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...With Increased Specificity for Selected Caspases And Groups of Caspases...
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...That Form the Rational for the Generation of Peptoid Caspase Inhibitors
Unfortunately: Limited Practical Use because of Hepatobiliar P1-Asp Transporter System (=Secretion)
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In Summary: Death Receptor Signals (Extrinsic) Induce Caspase-8 Activation while ...
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...Cellular Stresses (Intrinsic) Cause Mitochondrial Damage Leading to Caspase-9 Activation
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Other Caspases (Caspase-1,5) are Involved in Inflammatory Cell Responses...
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...That Involve Multimolecular Complexes...
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...And Lead to the Assembly of an Inflammasome
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Extrinsic Cell Death Pathways Depend on Death-Receptor Ligands such as TRAIL
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CD95/Fas/Apo1
Caspase-8 Activation (=Death)
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Apo2L/TRAIL
Caspase-8 Activation (=Death)
Increased Sensitivity in Cancer Cells (Lack of Decoy Receptors)
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Apo3L/TNF Caspase-8 Activation (=Death) + NF-B Activation (=Survival)
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Targeting the PI3K-Akt Pathway: Rationale and Promise
Survival signaling by serine-threonine kinases (PTEN/PI3K/Akt) Akt functioning in metabolism, cell growth and survival Development of Akt inhibitors PI3K/Akt signaling as therapeutic target: ongoing clinical studies (CCI-778, RAD001) Outlook: DNA damage responses, p53
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Cellular Receptors Transduce Extracellular Signals
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Cellular Receptors Initiate Intracellular Cascades
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PtdIns-Dependent Second Messengers Function In Distinct Intracellular Pathways...
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... Depending on Distinct Regulatory Domains Of Target Proteins
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PIP3-Dependent Cell Responses are Mediated by PTEN/PI3K/AKT
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Sequence Alignment of AGC Kinases
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Structur...