IA.Molecular Biology of Pharmacology 040906.ppt
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Transcript of IA.Molecular Biology of Pharmacology 040906.ppt
Molecular Biology of Pharmacology
Indwiani AstutiDept. of Pharmacology & Toxicology Fac. Of Medicine Gadjah Mada University
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• The flow of the expression of genetic information in cells is almost exclusively one way: DNA RNA Protein
• the central dogma of molecular biology.
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A gene codes for a protein
Protein
mRNA
DNA
transcription
translation
CCTGAGCCAACTATTGATGAA
PEPTIDE
CCUGAGCCAACUAUUGAUGAA
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Introduction• Pharmacology: knowledge of how drugs interact
with body constituents to produce therapeutic effects
• The spectrum from the molecular to the whole body
• The elucidation of molecular mechanisms of drug response, the development of new drugs, & the formulation of clinical guidelines for safe & effective use of drugs in therapy or prevention of disease & in relief symptoms.
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• Pharmacological responses by molecular interaction of drugs with cells, tissues, or other body constituents
• The key word is “molecular”• What specific biological molecules must be
present ?• How do drugs & biological molecules interact to
produce changes ?• How are these changes converted into
observable responses ?
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Control of cell growth complicated, considering the alternate pathways now identified
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I. Describing the concept of membrane & receptors
• For most drugs the site of action is at a specific biological molecule: Receptor
• Drug – organ or tissue selectivity for the biological molecule (Cardiac & pulmonary tissue)
• Concept of receptors as sites of drug action
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• A molecular drug-initiated response (biological target) -> together
• The interaction must result in selective binding of the drug molecule to the biological target before the response can take place. (molecular level recognition)
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• Drugs binding: Langley late 1800 - early 1900 & Ehrlich 1920
• 1930 Clark & Gaddum: drug-receptor interactions• 1960 receptor proteins isolated & purified• 1970 amino acid sequence of receptor subunit
determined• 1980 primary amino acid sequence of many
receptor determined from cDNA• 1990 three-dimentional structure• - improved drug design
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• Drugs are receptor modulators and do not confer new properties on cells or tissues
• Receptors must have properties of recognition and tranduction
• Receptors can be upregulated or downregulated
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Peptidoglycan synthesisPeptidoglycan synthesis
CytoplasmCytoplasm Cell wallCell wall
undecaprenolundecaprenol
sugarsugar
aminoaminoacidacid
Cell MembraneCell Membrane
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CycloserineCycloserine
1.1. alanine (ala) analog alanine (ala) analog 2.2. inhibits conversion of L-ala to D-ala inhibits conversion of L-ala to D-ala 3.3. inhibits formation of D-ala-D-alainhibits formation of D-ala-D-ala
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CycloserineCycloserineAnalog of alanine
XX
CytoplasmCytoplasm
sugarsugar
aminoaminoacidacid
XX
XX
XX
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BacitracinBacitracin
CellCell membrane membrane
undecaprenolundecaprenol
PP
PP
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VancomycinVancomycin
• binds to D-ala-D-alabinds to D-ala-D-ala
• inhibits cross-linkinginhibits cross-linking
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Beta lactam antibioticsBeta lactam antibiotics
• penicillins penicillins
• cephalosporins cephalosporins
• monobactamsmonobactams
• inhibit penicillin binding proteinsinhibit penicillin binding proteins
• stop cross-linkingstop cross-linking
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Beta lactamsBeta lactams
CellCell wall wall
Penicillin binding proteinPenicillin binding protein
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C NH CH CH C
O
O C N CH
CH3
CH3
COOH
S
Site of penicillinase action.Breakage of the lactam ring.
STRUCTURE OF PENICILLIN
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II. Explaining the cell signaling • Important of ligand-receptor binding:
transmembrane signal transmission• The mechanisms:
– Direct receptor control of ion channels (ligand gated or voltage gated)
– Receptor-controlled generation of second messengers (G protein/cAMP or G-protein/phosphoinositide systems)
– Receptor internalization & recycling polypeptide redistribution
– Receptor-initiated phosphorylation involving tyrosine kinase activity
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Classical Receptors & Ligand binding
• Receptors = proteins in a cellular or subcellular membrane facilitate communication between the two sides of the membrane
• Membranes of cells consist: phospholipid bilayers• Phospholipid molecules have two distinct region
(amphipathic). One region nonpolar (tails of the fatty acyl chain). The other very polar (phosphate, choline, & ethanolamine)
• Transmembrane regions of receptors formed by alpha helices (19 to 24 sequential amino acids nonpolar side groups)
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G-protein-based second messenger receptors
• A family of membrane-associated proteins that serve a key role in receptor modification of adenylate cyclase activity
• Guanosine groups= G• G-proteins exist in 2 states: inactive
states GDP bound to the protein, & active form GTP bound to protein interact with adenylate cyclase
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Receptor with intrinsic Tyrosine Kinase Activity
• Large group of receptors for growth factors (insulin, EGF, PDGF, hepatocyte GF etc)
• Extracellular domain contains regions bind GF, Intracellular domain contains a kinase activity capable of phosphorylating proteins on tyrosine residues
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Membrane Receptors• G-Protein Coupled-
receptor biogenic amines, peptides, glycoproteins
– Activate adenylate cyclase
– Inhibit adenylate cyclase
– Activate phospholipase C
– Regulate ion channels
• Tyrosine kinases-receptors for peptide GFs
• Guanylate Cyclases-receptors for atrial natriuretic peptide, E.Coli heat-stable enterotoxin
• Serine/Threonine kinases-receptors for activin, inhibin, TGF beta, Mullerian inhibiting substance
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Membrane receptor
• Growth Hormon Prolactin & Cytokine receptors-receptors that assosiate with tyrosine kinases, reseptors for cytokines, growth hormone prolactin
• The steroid receptor superfamily-transcriptional regulators, receptors for steroid, sterols, T3, retinoic acid & vitamin D
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III. Explaining the biology molecular of drugs action
• For examples: – Hormone– GF (Herseptin=Her)– Tyrosine kinase sensitive
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Second messenger involved in Hormone-Receptor Signaling Pathways
Second messenger Pathway
cAMP G-protein-coupled rec.
cAMP dependent protein kinase
cGMP cGMP dependent protein kinase
Diacylglycerol C-C-kinase
Inositol triphosphate Ca++ realease from endoplasmic reticulum & cell entry
Calcium ions Ca++ calmodulin-dependent protein kinase
Nitric oxide Guanylate cyclase
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G-Protein Coupled ReceptorsHormone Action
Glucagon Stimulate Adenyl cyclase (AC)
Somatostatin Inhibit AC, activate K+ channels
Antidiuretic hormone Stimulate AC & Phospholipase C (PLC)
Oxytoxin Stimulate Adenyl cyclase
Adrenocorticotrophic hormone Stimulate Adenyl cyclase
Thyroid Stimulating Hormone Stimulate Adenyl cyclase
Leutenizing hormone Stimulate Adenyl cyclase
Follicle stimulating hormone Stimulate Adenyl cyclase
Growth hormone-releasing Stimulate Adenyl cyclase & PLC
Corticothrophin-releasing hormone Stimulate Adenyl cyclase
Thyrothrophin releasing hormone Stimulate Adenyl cyclase & PLC
Luteinizing releasing hormone Stimulate PLC
Parathormone Stimulate Adenyl cyclase
Calcitonin Stimulate Adenyl cyclase
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Proximal Elements in Tyrosine Kinase Signaling Pathways
• IRS-1: major substrate of insulin & IGF-1 receptor kinase
• grb-2: adapter protein that functions in the activation of ras by insulin, PDGF & EGF
• P13-kinase: activated by insulin & PDGF as a concequence of binding to autophosphorylated PDGF receptor & tyrosine phosphorylated IRS-1
• Phospholipase Cy: activated by EGF & PDGF not insulin
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Herceptin (c-erb2)
• Product oncogen (EGFR)
• Receptor Growth factors family
• Indication: Solid tumor with Her-2 +
• Breast Cancer
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Inhibitionapoptosis
MetastasisAngiogenesis
Invasion
Proliferation
Tirosine kinase
EGFR
GF/Ligand
Membrane
Tirosine kinase
EGFR
Monoclonal Ab
Membrane
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Tyrosine kinase sensitive
• CML expression of gene bcr-abl + (Chr. Ph)
• Imatinib (ST1571)
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Artificial bone, cartilage, & skin with no immune rejection
• Vitoss (Orthovita): nanoparticels bone growth (orthopaed)
• Navavax-estrasorb (cream): nanoparticels Skin burns
• Nucrest-Silcrest : nanocrystaline Skin burns
• Nanodot (Nasa): cells repairs
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Improved, direct chemotherapy and radiotherapy
• Drug delivery– Maximizing bioavailability both over a
period of time and at specific places in the body
– Deliver drug directly to the site without interacting with the rest of the body
Smart drugs
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Nanotechnology for Drug Delivery
• Molecule encapsulated within nanoscale cavities inside polymer : time-released drugs
• Grind solid drugs into fine powders : to increase surface area and reactivity & to increase solubility
• Encapsulate polar drug in a nonpolar coating : easily pass through the cell membrane
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• Coat DNA with cholesterol to easily pass through the oily cell membrane
• Liposome structures to deliver soluble protein (cytokine) such as interferon to cancer cells
• Magnetic nanopoarticles : local bioavailability control by external magnetic field
• Triggered response : inactive drug molecule“wakes up” on encountering a particular signal
• Antacid enclosed in a coating of polymer that dissolves only in highly acidic conditions
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CTLA-4-mediated inhibition may restrict T cell activation during both the initiation & the progression of an anti-tumor response. So blockade to the CTLA-4 inhibitory signals during T cell-APC interaction might result in enhanced anti-tumor responses.
Modulation of T cell responses to tumors with CTLA-4 blockade
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Simultaneous induction of Tr1 cells & of TH1 cells ensures a balance of the inflammatory response so that infection is terminated with minimal collateral
damage to host tissue
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DNA molecule therapy
• A from of gene therapy taking advantages of DNA’s selfbinding property
• Drug DNA combines with the disease-causing DNA to prevent its replicating again and thus be removed as a threat
• DNA molecule engineering is one of the most active area of bionanoscience
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Strategies for killing cancer cells:
Cancer drugs target:•DNA replication•Transcription•Translation
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Signal cascades are useful:1. At each step of the cascade, the signal is amplified 2. The information that arrived at the plasma membrane in the form of a
signal is communicated to the nucleus3. The multitude of steps enables a signal to have different effects in
different cells (because they have different target proteins)
BLOCK EGFR
Tyrosine kinaseinhibitors
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Amino Acid SLC DNA codons
Isoleucine I ATT, ATC, ATA
Leucine L CTT, CTC, CTA, CTG, TTA, TTG
Valine V GTT, GTC, GTA, GTG
Phenylalanine F TTT, TTC
Methionine M ATG
Cysteine c TGT, TGC
Alanine A GCT, GCC, GCA, GCG
Glycine G GGT, GGC, GGA, GGG
Proline P CCT, CCC, CCA, CCG
Threonine T ACT, ACC, ACA, ACG
Serine S TCT, TCC, TCA, TCG, AGT, AGC
Tyrosine Y TAT, TAC
Tryptophan W TGG
Glutamine Q CAA, CAG
Asparagine N AAT, AAC
Histidine H CAT, CAC
Glutamic acid E GAA, GAG
Aspartic acid D GAT, GAC
Lysine K AAA, AAG
Arginine R CGT, CGC, CGA, CGG, AGA, AGG
Stop codons Stop TAA, TAG, TGA