Molecular Biology of Pharmacology

Indwiani AstutiDept. of Pharmacology & Toxicology Fac. Of Medicine Gadjah Mada University

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Structure of DNA

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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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OUT

IN

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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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AntibioticsAntibiotics

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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

• Inhibits dephosphorylationInhibits dephosphorylation

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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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VancomycinVancomycin

Cell wallCell wall

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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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Cross-linking of peptidoglycan

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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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Tirosine kinase

EGFR

GF/Ligand

Membrane

EGFR-TK

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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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Therapeutic use of CTLA-4 chimeras to block T-cell activation

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Blocking of Co-Stimulatory Signals Can Prevent Graft Rejection

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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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DRUG PRODUCTION

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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