GOOD MORNING

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MECHANISM OF DRUG ACTION

DR PRANESH PAWASKARFirst Year ResidentDept. of PharmacologyL.T.M.M.C. Sion, Mumbai 400022Date: 08/10/2016

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OVERVIEW• INTRODUCTION.

• PHARMACODYNAMIC CONCEPTS.

• QUANTITATIVE ACTION OF DRUG INTERACTIONS WITH RECEPTORS.

• PHARMACODYNAMICAL VARIABILITY.

• MECHANISMS OF ACTION.

• RECEPTOR DESENSITISATION AND REGULATION OF RECEPTORS.

• PHARMACODYNAMIC INTERACTIONS IN A MULTICELLULAR CONTEXT.

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INTRODUCTION

PHARMACOLOGY

PHARMACOKINETICS

PHARMACODYNAMICS

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INTRODUCTION

• PHARMACOKINETICS : Understanding the Absorption, Distribution, Metabolism and Elimination.

• PHARMACODYNAMICS : Study of the biochemical and physiological effects of drugs and their mechanism of action.

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WHY TO STUDY PHARMACODYNAMICS?

• Basis for the rational therapeutic use drug.

• Design of new and superior therapeutic agent.

• Effect of drug on body.

• In contrast _ _ _ _

• Many adverse effects and events of drugs and drug toxicities can be anticipated by understanding Drug’s Mechanism Of Action.

• Safety and Success.

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PHARMACODYNAMIC CONCEPTS• Effects of most drugs = macromolecular components ;

Drug Receptor or Drug Target = cellular macromolecule or macromolecular complex.

• Alter the rate or magnitude of an intrinsic cellular response.

• Receptors located on = surface of cells, nucleus.

• Acceptors = interact with acceptors = alter the pharmacokinetics.

• Receptors for hormones, growth factors, transcription factors, and neurotransmitters; the enzymes of crucial metabolic or regulatory pathways = Proteins ; Others like DNA = Cancer chemotherapeutics.

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PHYSIOLOGICAL RECEPTORS• Majority drug receptor = endogenous regulatory ligand

proteins = physiological receptors = great selectivity

• Drugs that bind to physiological receptors and mimic the regulatory effects of the endogenous signalling compounds are termed Agonists.

• If the drug binds to the same recognition site as the endogenous agonist = Orthosteric site = Primary Agonist.

• Allosteric (Allotopic) Agonists bind to a different region on the receptor referred to as an allosteric or Allotopic site.

• Drugs that block or reduce the action of an agonist are termed Antagonists.

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• Competition with an agonist for the same or overlapping site = Syntopic

• Competition with an agonist for the other sites on the receptor = Allosteric

• Competition with an agonist by combining with agonist =

Chemical

• In-Directly inhibiting functions of agonist = Functional

• Agents that are only partly as effective as agonists regardless of the concentration employed are termed Partial Agonists.

• Many receptors show constitutive activity in the absence of a regulatory ligand ;drugs that stabilize such receptors in an inactive conformation are termed Inverse Agonists.

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DRUG SPECIFICITY• Strength of reversible interaction between drug and receptor

= Dissociation constant.

• Chemical structure of drug determines affinity, intrinsic activity and drug’s specificity.

• Drug acting on a single receptor expressed on limited no. of cells exhibit high specificity = Ranitidine (H2). And vice-versa.

• Numerous examples of drugs having discrete action e.g. (Digoxin – NaK ATPase), (methotrexate-dihydrofolate reductase), (lidocaine- peripheral nerves,heart,CNS), (immunosuppressive drug- opportunistic infection), (Furosemide- muscle cramps, arrhythmias)

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• Broad specificity = clinical utility more = adverse effects more

• E.g. Amiodarone (cardiac arrhythmia) (Na,Cl,K Beta) = Thyroid hormone (structural similarity)

• Sterioisomerism (Sotalol- d/k blocker and l/beta agonist) other drug Labetolol

• Chronic administration = Down Regulation/ Desensitization = e.g. Nitroglycerin = also known as Tachyphylaxis.

• Differential tolerance development = Opioid to analgesia but not Respiratory depression

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• No receptor mechanism for = Aluminium hydroxide and Magnesium hydroxide, Mannitol act by colligative property, Cholestyramine resins acts by decreasing dietary cholesterol absorption.• Antibiotics mostly act by inhibiting receptor or enzyme

specific to pathogen not host.• Antibiotics such as Penicillin inhibit a key enzyme

required for synthesis of bacterial cell walls, an enzyme not present in humans or animals.• Mutation of the target receptor, increased expression

of enzymes that degrade or increase efflux of the drug from the infective agent, and development of alternative biochemical pathways.

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STRUCTURE-ACTIVITY RELATIONSHIPS AND DRUG DESIGN• Detail knowledge of a drugs molecular target can inform about

development of new drugs efficacy and toxicity.

• Sequencing entire human genome identified novel receptors for them but ligands for them not known. (Orphan receptors)

• Orphan receptors are still found in G Protein Coupled Receptors and Nuclear Hormone Receptor Families.

• Transgenic animal model helps in predicting Agonism Or Antagonism by genetically altering the receptors mechanism and function

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• Even minor changes in structure can bring major differences in drug pharmacological activities

• This stringent nature of structure to bind to its receptor can be illustrated by capacity of receptors to interact selectively with optical isomers.

• dl-Hyoscyamine and its Atropinic effects.

• Minor modifications in structure have profound effect even on pharmacokinetics e.g. PO4 ester at N3 in Phenytoin makes more soluble

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• Advances in molecular modelling of organic compounds and methods for drug target discovery and biochemical measurements of primary actions of drugs at their receptor have enriched quantitation of structure activity relationship and its use in drug design.

• Drugs binding to selectively mutated receptors improves affinity and selectivity of drug.

• X-ray crystallography helps designing ligands and molecular basis of drug resistance ( BCR-ABL and Imatinib like inhibitors)

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QUANTITATIVE ASPECTS OF DRUG INTERACTIONS WITH RECEPTORS

• Basic currency of receptor pharmacology is dose response or concentration curve.

• Concentration of drug that produces 50% of the maximal response quantifies drug activity and is referred to as the EC50.

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AFFINITY, EFFICACY AND POTENCY

• Drug-receptor interaction is characterized by binding of drug to receptor and generation of a response.

• Drug or ligand is denoted as L and the inactive receptor as R. The first reaction, the reversible formation of the ligand-receptor complex LR, is governed by the chemical property of Affinity.

• Concentration of ligand-receptor complex [LR] is equal to the product of k+1[L][R], the rate of formation of the bi-molecular complex LR, minus the product k–1[LR], the rate dissociation of LR into L and R.

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• The Equilibrium Dissociation Constant (KD) is then described by ratio of the Off And On rate constants (k–1/k+1).• The Affinity Constant or Equilibrium Association

Constant (KA) is the reciprocal of the Equilibrium Dissociation Constant (i.e., KA = 1/KD)• Thus a high-affinity drug has a low KD and will bind a

greater number of a particular receptor at a low concentration than a low-affinity drug.• Note that this relationship describes only receptor

occupancy, not the eventual response that is often amplified by the cell. • Many signaling systems reach a full biological

response with only a fraction of receptors occupied (described later)

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• The relative Potency of two agonists (Drug X, red line; Drug Y, purple line) obtained in the same tissue is a function of their relative affinities and intrinsic efficacies.• The EC50 of Drug X occurs at a concentration that is

one-tenth the EC50 of Drug Y. Thus, Drug X is more potent than Drug Y.

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QUANTIFYING AGONISM AND ANTAGONISM

• Measuring agonist potency by comparison of EC50 values is one method of measuring the capability of different agonists to induce a response in a test system and for predicting comparable activity in another.• Another method of estimating agonist activity is to

compare maximal asymptotes in systems where the agonists do not produce maximal response.

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• Characteristic patterns of antagonism are associated with certain mechanisms of blockade of receptors.• One is straight forward Competitive Antagonism,

whereby a drug with affinity for a receptor but lacking intrinsic efficacy competes with the agonist for the primary binding site on the receptor.

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• If the antagonist binds to the same site as the agonist but does so irreversibly or Pseudo-Irreversibly (slow dissociation but no covalent bond), it causes a shift of the dose-response curve to the right, with further depression of the maximal response.

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• Allosteric effects occur when an Allosteric ligand I or P binds to a different site on the receptor to either inhibit (I) the response (see panel C) or potentiate (P) the response (see panel D). This effect is saturable; inhibition or potentiation reaches a limiting value when the allosteric site is fully occupied.

PHARMACODYNAMICAL VARIABILITY

• Individuals vary in the magnitude of their response to the same concentration of a single drug or to similar drugs, and a given individual may not always respond in the same way to the same drug concentration.• Attempts have been made to define and measure

individual "Sensitivity" (Or "Resistance") to drugs in the clinical setting, and progress has been made in understanding some of the determinants of sensitivity to drugs that act at specific receptors.• Drug responsiveness may change because of disease or

because of previous drug administration.• Receptors are dynamic, and their concentration and

function may be up- or down-regulated by endogenous and exogenous factors.

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FACTORS MODIFYING DRUG ACTION

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PHARMACOGENETICS• Pharmacogenetics refers to the genetic and genomic

variations that give rise to variability in both pharmacokinetic and pharmacodynamic aspects of drug therapy.• Inter-individual variability of responsiveness to many

drugs. e.g. Warfarin.

• Nearly 60% of the variability is due to genetic variation in the primary metabolizing enzyme (CYP2C9) and in the drug's receptor, Vitamin K Epoxide Reductase Complex, subunit 1 (VKORC1). Polymorphisms in CYP2C9 (especially homozygosity in the allele) increase sensitivity towards warfarin, whereas coding region polymorphisms in VKORC1 result in a warfarin-resistant phenotype.• FDA recommended that pharmacogenetics be used to

optimize warfarin dosing, but did not provide specific protocol.

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MECHANISMS OF ACTION

Receptors that Affect Concentrations of Endogenous Ligands:-• Many drugs act on endogenous ligands like

neurotransmitters, hormones and alter their synthesis storage release and transport.• Many examples of drugs that act on neuroeffector

junctions by altering neurotransmitter synthesis, storage of neurotransmitter in vesicles, release of neurotransmitters into the synaptic cleft, and subsequent removal of the neurotransmitter from the synaptic cleft.• E.g. Alpha-Methyltyrosine (inhibits synthesis of

norepinephrine (NE)), Cocaine (blocks NE reuptake), Amphetamine (promotes NE release), and Selegeline (inhibits NE breakdown).

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Receptors that Regulate the Ionic Milieu-

• Some drugs act by affecting the ionic millieu of blood, urine, and the GI tract.• Receptors in this case are ion pumps and transporters.• Drug effects on many of these receptors can have

effects throughout the body due to changes in blood electrolytes and pH.• e.g., Furosemide, Chlorothiazide, Amiloride act by

directly affecting ion pumps and transporters in epithelial cells of the nephron that increase the movement of Na+ into the urine.• Another therapeutically important target is the H+,K+-

ATPase (Proton Pump) of gastric parietal cells like Esmoprazole (90%).

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Cellular Pathways Activated by Physiological Receptors –

1) Signal Transduction Pathways –• Physiological receptors have at least two major

functions, ligand binding and message propagation (i.e., signaling).• So there is existence of at least two functional

domains within the receptor: A Ligand-binding Domain And An Effector Domain.

• Many drugs target the extracelluar ligand-binding domain of physiological receptors. E.g. Beta Blockers.

• However, drugs can affect the receptor by targeting either domain, as in the case of Cetuximab – extra cellular domain, Geftinib Erlotinib on intracellular domain.

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• Regulatory actions of a receptor may be exerted directly on its cellular target called as Transducers.

• The receptor, its cellular target, and any intermediary molecules are referred to as a receptor-effector system or signal transduction pathway.• Frequently ultimate physiological target is an Enzyme,

Ion Channel, Or Transport Protein That Creates, Moves, Or Degrades a small molecule. Termed as Second Messenger.

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2) Signal integration and amplification –

• Receptors and their associated effector and transducer proteins also act as integrators of information as they coordinate signals from multiple ligands with each other and with the differentiated activity of the target cell. • For example, signal transduction systems regulated by

changes in Cyclic AMP (cAMP) and Intracellular Ca2+ are integrated in many excitable tissues. • In cardiac myocytes, an increase in cellular cAMP

caused by activation of adrenergic receptors enhances cardiac contractility by augmenting the rate and amount of Ca2+ delivered to the contractile apparatus; thus, cAMP and Ca2+ are positive contractile signals in cardiac myocytes.

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• Another important property of physiological receptors is their capacity to significantly Amplify a physiological signal.• Neurotransmitters, hormones, and other extracellular ligands

are often present at the ligand-binding domain of a receptor in very low concentrations .(Nano Moles).• Effector domain contains enzymes and enzyme cascades to

catalytically amplify the intended signal.• The ability of virtually all receptors to amplify physiological

signals makes them excellent targets for natural ligands and drugs.• E.g. binding of a single photon to cis-retinal in the

photoreceptor Rhodpsin is eventually amplified ~1 x 106-fold.• A single steroid hormone molecule binding to its receptor

initiates the transcription of many copies of specific mRNAs.

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STRUCTURAL AND FUNCTIONAL FAMILIES OF PHYSIOLOGICAL

RECEPTORS

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G PROTEIN–COUPLED RECEPTORS (GPCRS)

• A bundle of Seven Alfa-helices.• Over 800 GPCRs that make up the third largest family

of genes.• Half of these GPCRs dedicated to sensory perception

(Smell, Taste, And Vision).• Remaining receptors regulate an impressive number

of physiological functions including Nerve Activity, Tension Of Smooth Muscle, Metabolism, Rate And Force Of Cardiac Contraction, And The Secretion of most glands.• ligands for GPCRs are – Ach, NE, Eicosanides,

Peptide Hormones, GABA.• GPCRs are important regulators of CNS and Autonomic

nervous system.

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• Because of their number and physiological importance, GPCRs are the targets for many drugs; perhaps half of all non-antibiotic prescription drugs act at these receptors.

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Receptor Subtypes –

• The Alfa 1, Alfa 2, and Beta adrenergic receptors differ from each other both in ligand selectivity and in coupling to G proteins.• The Beta 1, 2, and 3 adrenergic receptor subtypes

exhibit differences in both tissue distribution and regulation by phosphorylation by G–protein receptor kinases (GRKs) and PKA.• Pharmacological differences among receptor subtypes

are exploited therapeutically through the development and use of receptor-selective drugs.• E.g. Beta 2 Agonist Salbutamol for Broncho-

dilatation. To minimise side effects of Beta 1 effects on heart.

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Receptor Dimerization –

• GPCRs undergo both homo- and Heterodimerization and possibly Oligomerization.• Opioid receptors can exist as Homodimers of mu or

delta receptors, or as mu-delta Heterodimers with distinctly different Pharmacodynamic properties than either Homodimer.• Dimerization also may permit binding of receptors to

other regulatory proteins such as transcription factors.

G PROTEINS

• GPCRs couple to a family of heterotrimeric GTP-binding regulatory proteins termed G proteins.

• G–protein-regulated effectors include enzymes such as Adenylyl Cyclase, Phospholipase C, Cyclic GMP, Phosphodiesterase (PDE6), And Membrane Ion Channels selective for Ca2+ And K+.

• The G protein family is comprised of 23 subunits and 4 families Gs, Gi, Gq, and G12/13.

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G PROTEIN ACTIVATION• When an Agonist binds to a GPCR, there is a conformational

change in the receptor that is transmitted from the ligand-binding pocket to the second and third intracellular loops of the receptor which couple to the G protein.• Conformational change causes the Alfa subunit to

exchange its bound GDP for GTP.• Binding of GTP activates the Alfa subunit and causes it to

release both the Beta-Gamma dimer.• Beta-Gamma heterodimer become active signaling

molecule.• Following activation of one G protein, the receptor is freed

to interact with other G proteins, the active, GTP-bound form binds to and regulates effectors such as adenylyl cyclase (via Gs alfa) or phospholipase C Beta (via Gq Alfa ).

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SECOND MESSENGERSCyclic Amp –

• Cyclic AMP is synthesized by Adenylyl Cyclase under the control of many GPCR.• Stimulation is mediated by the Gs-Alfa subunit,

inhibition by the Gi-Alfa subunit.• Membrane-bound ACs exhibit basal enzymatic activity

that is modulated by binding of GTP-liganded Alfa subunits of the stimulatory and inhibitory G proteins.• Cyclic AMP generated by Adenylyl Cyclases has three

major targets in most cells, the cyclic AMP dependent protein kinase (PKA), cAMP-regulated guanine nucleotide exchange factors termed EPACs (exchange factors directly activated by cAMP), and via PKA phosphorylation.

PKA-

• Target of Cyclic AMP is the PKA Holoenzyme have two catalytic (c) subunits reversibly bound to a Regulatory subunit (R) to form a Heterotetramer (R2C2).• At low concentrations of Cyclic AMP the R subunits

inhibit C subunits thus the holoenzyme is inactive.• When Adenyl Cyclase is activated cAMP

concentration is increased causing C subunit activation.• The active C subunit phosphorylase Serine And

Threonine residues on specific protein substrates. Present in metabolic enzymes, transport proteins and numerous regulatory protein.

PKG –

• Stimulation of receptors that raise Intracellular Cyclic GMP concentrations leads to the activation of the cyclic dependent protein kinase (PKG) that are PKG specific.• The catalytic domain and cyclic nucleotide binding

domains of PKG are expressed as a single polypeptide and forms PKG holoenzyme• Pharmacologically important effects of elevated

cyclic GMP include modulation of platelet activation and relaxation of smooth muscles

PDE –

• Cyclic Nucleotide Phosphodiesterases form another family of important signalling proteins whose activities are regulated via the rate of gene transcription as well as by second messengers.• PDEs Mainly PDE3 are drug targets for treatment of

diseases such as Asthma, Cardio Vascular Diseases Such As Heart Failure, Atherosclerotic Coronary and Peripheral Arterial Disease and neurological disorders.• PDE5 inhibitors are used in treating COPD and

Erectile Dysfunction. Inhibition of PDE5 causes accumulation of cGMP in cells of smooth muscles of corps cavernosum thereby enhancing its relaxation.

OTHER SECOND MESSENGERS

• Ca can enter the cell through Ca channels in the plasma membrane or be released by hormones or growth factors from intracellular stores• The basal Ca level in cell is maintained by membrane

Ca pumps which extrude Ca into extracellular space and sarcoplasmic reticulum.• Hormones and growth factors release Ca from its

intra cellular storage via a signalling pathway that begins with activation of Phospholipase C

• Phospholipase C has two primary forms, PLC beta and PLC gamma.• GPCR activate PLC by activating G protein alfa

subunit.

• PLC isoforms are activated by tyrosine phosphorylation.• Growth factor receptors such as epidermal growth

factor receptor EGFR are receptor tyrosine kinase RTK. • This RTK Auto phosphorylate upon binding their

cognate growth factor.

ION CHANNELS• The lipid bilayer of the plasma membrane is

impermeable to Anions And Cations• To establish and maintain the electrochemical

gradients required to maintain a membrane potential, all cells express ion transporters for Na, K, Ca, Cl.• E.g. Na-K-ATPase pump

• Passive ion fluxes down cellular electrochemical gradients are regulated by a large family of ion channels• Humans express ~232 distinct ion channels to

precisely regulate the flow of Na, K, Ca, Cl across cell membrane• These proteins are important target for drug actions

• This diverse family of ion channels can be divided into sub families depending upon mechanisms of their channels.• They can also be classified as voltage activated,

ligand activated, store activated, stretch activated and temperature activated.

Voltage Gated Channels –• Humans express multiple isoforms of voltage gated

channels for Na, K, Ca, Cl ions.• In nerves and muscle cells, voltage gated Na channels are

responsible for the generation of robust action potentials.• These sodium channels are composed of three subunits, a

pore forming alpha subunit and two regulatory beta subunits.• The alfa subunit is a 260 kDa protein containing four

domains that form a Na ion selective pore.• The voltage activated Na channels in pain neurons are

targets for local anaesthetics such as Lidocaine and Tetracaine which block pore, inhibit depolarisation and thus block the sensation of pain.

• They are also the targets of the naturally occurring Marine Toxins, Tetrodotoxin and Saxitoxin.

• These are also important targets of many drugs used to treat Cardiac Arrhythmias.

Ligand gated channels –

• These channels activated by binding of a ligand to a specific site in the channel protein have a diverse architecture and set of ligands.• Activation of these channels is responsible for the

majority of synaptic transmission by neurons both in the CNS and periphery.• There are a variety of more specialised ion channels

that are activated by intra cellular small molecules and structurally distinct from conventional ligand gated ion channels.• Formally members of Kv family such as

hyperpolarisation and CMP gated (HCN) channel expressed in heart.

• Cyclic Nucleotide-Gated channels (CNG) important for vision.• Ion channels also include the IP3 sensitive Ca

channel responsible for release of Ca from ER and the Sulphonyl urea receptor SUR1 that associates with Kir6.2 channel to regulate the ATP dependent K channel in pancreatic beta cells.• K ATP channel is the target of oral hypoglycaemic

drugs such as Sulphonylurea and Meglitinide.

• Other specialised channels include the 5HT3 regulated channel expressed on vagal nerves that stimulates emesis.• Ondensetron is an important antagonist of the

5HT3 gated channel used to inhibit emesis caused by drugs or disease.

TPR channels –• The Transient Receptor Potential (TRP) channels

comprise a superfamily of ubiquitously expressed ion channels that is remarkable in its diversity and domain structure.• Are not presently targets of approved drugs.• Significant interest in developing drugs that can alter the

function of these ion channels.• Their roles in various sensory phenomena such as Pain,

Temperature, Osmolarity, Touch, Olfaction, Vision, and Hearing.• Most can be activated by multiple mechanisms.• Mutations in TRP channels are known to cause several

disease including Hypomagnesemia And Hypocalcemia, and various Renal Disorders And Neurodegenerative Diseases.

TRANSMEMBRANE RECEPTORS LINKED TO INTRACELLULAR ENZYMES

• Mammalian cells express a diverse group of physiological membrane receptors with extracellular ligand-binding domains and an intrinsic enzymatic activity on the cytoplasmic surface of the cell.• These molecules include the Receptor Tyrosine

Kinases (RTKs) such as the Epidermal Growth Factor (EGF) and Insulin Receptors, which contain intrinsic tyrosine kinases in the cytoplasmic domain.• Tyrosine Kinase-associated receptors without

enzymatic activity, such as the receptors for Gamma-interferon, which recruit the cytoplasmic Janus tyrosine kinases (JAKs).

• Receptor Serine-Threonine Kinases such as the TGF- Receptor.

• Receptors linked to other enzyme activities such as the receptors for Natriuretic Peptides, which have a cytoplasmic guanylate cyclase activity that produces a soluble second messenger, cyclic GMP.

• Receptors responsible for innate immunity, the Toll-like receptors and those for tumor necrosis factors (TNF-Alfa) , have many features in common with the JAK-STAT receptors.

Receptor Tyrosine Kinases-

• The receptor tyrosine kinases include receptors for hormones such as Insulin, for multiple growth factors such EGF, platelet-derived growth factor (PDGF), nerve growth factor (NGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and Ephrins.

• Activation of growth factor receptors leads to cell survival, cell proliferation, and differentiation. Activation of the Ephrin receptors leads to neuronal angiogenesis, axonal migration, and guidance.

JAK-STAT Receptor Pathway -

• Cells express a family of receptors for cytokines such as -Interferon And Hormones like Growth Hormone And Prolactin, which signal to the nucleus by a more direct manner than the receptor tyrosine kinases.• Upon the dimerization induced by ligand binding,

JAKs phosphorylate other proteins termed signal transducers and activators of transcription (STATs), which translocate to the nucleus and regulate transcription.• There are four JAKs and six STATs in mammals.• Prolactin appears to use JAK1, JAK2, and STAT5 to

stimulate milk production.

Receptor Serine-Threonine Kinases -

• Protein ligands such as TGF-Beta activate a family of receptors that are analogous to the Receptor Tyrosine Kinases.• In the basal state, these proteins exist as monomers;

upon binding an agonist ligand, they dimerize, leading to phosphorylation of the kinase domain of the type I monomer, which activates the receptor.• Receptor then phosphorylates a gene regulatory

protein termed a Smad.

• There are multiple Smads in cells.• Regulates genes leading to Morphogenesis And

Transformation.

Toll-like Receptors -

• Signaling related to the innate immune system is carried out by a family of over ten single membrane-spanning receptors termed Toll-like receptors (TLR).

• Highly expressed in Hematopoeitic Cells.• In a single polypeptide chain, these receptors contain

a large extracellular ligand-binding domain, a short membrane-spanning domain.• Ligands for TLR are comprised of a multitude of

pathogen products including Lipids, Peptidoglycans, Lipo-peptides, And Viruses.• Activation of these receptors produces an

Inflammatory Response.

TNF-alfa Receptors -

• The mechanism of action of tumor necrosis factor alfa (TNF-alfa ) signaling to the NF-kappaB transcription factors is very similar to that used by Toll-like receptors.

• Receptor has no enzymatic activity.• TNF receptor is another single membrane-spanning

receptor with an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic domain termed the Death Domain.

RECEPTORS THAT STIMULATE SYNTHESIS OF CYCLIC GMP

• The signalling pathways that regulate the synthesis of cyclic GMP in cells include hormonal regulation of Transmembrane Guanylate Cyclases.

• Such as the atrial natriuretic peptide receptor (ANP)

• Activation of soluble forms of guanylate cyclase by nitric oxide (NO).

• Effects of cyclic GMP are carried out by multiple isoforms of PKG, cyclic GMP-gated ion channels, and cyclic GMP-modulated Phosphodiesterases.

Natriuretic Peptide Receptors -

• Membrane receptors with intrinsic enzymatic activity includes the receptors for three small peptide ligands released from cells in cardiac tissues and the vascular system.• Atrial Natriuretic Peptide (ANP), Brain Natriuretic

Peptide (BNP), C-type Natriuretic Peptide (CNP).

• BNP, CNP is not stored, its synthesis and release are increased by growth factors and sheer stress on vascular endothelial cells.• The major physiological effects of these hormones

are to Decrease Blood Pressure (ANP, BNP), to reduce Cardiac Hypertrophy And Fibrosis (BNP), and to Stimulate Long Bone Growth (CNP).

• The ANP receptor (NPR-A) is the molecule that responds to ANP and BNP.

• ANP and BNP play a role in maintaining the normal state of the cardiovascular system as NPR-A knockout mice have hypertension and cardiac hypertrophy.

• A synthetic BNP Agonist, Nesiritide, is used for treatment of Acute Decompensated Heart Failure.

• The NPR-B receptor widely expressed but prominent in bone, brain, kidney, lung, liver, and cardiac and vascular smooth muscle.

NO Synthase And Soluble Guanylate Cyclase –

• Nitric oxide (NO) is a unique signal.• A very labile gas produced locally in cells by the

enzyme Nitric Oxide Synthase (NOS).

• Resulting NO is able to markedly stimulate the soluble form of guanylate cyclase to produce cyclic GMP.• Three forms of nitric oxide synthase, neuronal NOS

(nNOS or NOS1), endothelial NOS (eNOS or NOS3), and inducible NOS (iNOS or NOS2).• They are found in Myocytes, Vascular Smooth Muscle

Cells, Endothelial Cells, Hematopoietic Cells, And Platelets.

NUCLEAR HORMONE RECEPTORS AND TRANSCRIPTION FACTORS

• In humans, nuclear hormone receptors comprise a superfamily of 48 receptors that respond to a diverse set of ligands.• The receptor proteins are transcription factors able to

regulate the expression of genes controlling numerous physiological processes such as reproduction, development, and metabolism.• Well-known members of the family include receptors

for circulating steroid hormones such as Androgens, Estrogens, Glucocorticoids, Thyroid Hormone, and Vitamin D.

• Examples include Retinoic Acid Receptor (RXR); the Liver X Receptor (LXR —the ligand is 22-OH cholesterol); the Farnesoid X Receptor (FXR—the ligand is chenodeoxycholic acid); and the Peroxisome Proliferator-activated Receptors (PPARs alfa, beta , and gamma ; 15 Deoxy Prostaglandin J2 is one possible ligand for beta PPAR ; the Cholesterol-lowering Fibrates bind to and regulate PPAR gamma ).

APOPTOSIS• The maintenance of many organs requires the

continuous renewal of cells.• Mucosal cells lining the intestine and a variety of cells in

the immune system including T-cells and Neutrophils.• The process by which cells are genetically programmed

for death is termed Apoptosis.• Apoptosis is a highly regulated program of biochemical

reactions that leads to Cell Rounding, Shrinking Of The Cytoplasm, Condensation Of The Nucleus And Nuclear Material, And Changes In The Cell Membrane that eventually lead to presentation of phosphatidylserine on the outer surface of the cell.

• Understanding the pathways regulating apoptosis is important because apoptosis plays an important role in normal cells and because alterations in apoptotic pathways are implicated in a variety of diseases such as Cancer, And Neurodegenerative And Autoimmune Diseases.• Thus, maintaining or restoring normal apoptotic

pathways is the goal of major drug development efforts to treat diseases that involve dysregulated apoptotic pathways and selectively stimulating apoptotic pathways could be useful in removing unwanted cells.

• Two major signaling pathways induce Apoptosis. Apoptosis can be initiated by external signals that have features in common with those used by ligands such as TNF- alfa or by an internal pathway activated by DNA damage, improperly folded proteins, or withdrawal of cell survival factors.• The apoptotic program is carried out by a large

family of Cysteine-proteases termed Caspases.

• The Caspases are highly specific cytoplasmic proteases that are inactive in normal cells but become activated by apoptotic signals.

RECEPTOR DESENSITIZATION AND REGULATION OF RECEPTORS• Receptors are subject to many regulatory and

haemostatic controls.• These controls include regulation of the synthesis

and degradation of the receptor, covalent modification, association with other regulatory proteins, and relocalization within the cell.• Receptors are almost always subject to feedback

regulation by their own signalling.• Continued stimulation of cells with agonists generally

results in a state of Desensitization (also referred to as Adaptation, Refractoriness, or Down-regulation)

• The effect that follows continued or subsequent exposure to the same concentration of drug is diminished. This phenomenon, called Tachyphylaxis.

• Tachyphylaxis, occurs rapidly and is important therapeutically.• An example is attenuated response to the repeated use

of beta adrenergic receptor agonists as bronchodilators for the treatment of asthma.• Desensitization can result from temporary

inaccessibility of the receptor to agonist or from fewer receptors being synthesized and available at the cell surface (e.g., Down-regulation of receptor number).• Phosphorylation of GPCR receptors by specific GPCR

kinases (GRKs) plays a key role in triggering rapid desensitization.

• The - Arrestins recruit proteins, such as PDE4, that limit cyclic AMP signaling, and Clathrin and 2-Adaptin, that promote sequestration of receptor from the membrane (Internalization).• Conversely, Supersensitivity to Agonists also

frequently follows chronic reduction of receptor stimulation. Such situations can result• e.g., following withdrawal from prolonged receptor

blockade.(e.g., the long-term administration of Beta Adrenergic Receptor Antagonists such as Metoprolol)• or in the case where chronic denervation of a

preganglionic fiber induces an increase in neurotransmitter release per pulse, indicating postganglionic neuronal Supersensitivity.

Diseases Resulting From Receptor Malfunction -

• Alteration in receptors and their immediate signaling effectors can be the cause of disease.• Deficiencies in widely employed signaling pathways

have broad effects, as are seen in myasthenia gravis and some forms of insulin-resistant diabetes mellitus, which result from autoimmune depletion of nicotinic cholinergic receptors. Or Insulin receptors.• Among the most significant events is the appearance

of aberrant receptors as products of oncogenes that transform otherwise normal cells into malignant cells. Virtually any type of signaling system may have oncogenic potential.

PHARMACODYNAMIC INTERACTIONS IN A MULTICELLULAR CONTEXT

• It is instructive to examine the pharmacodynamic interactions of physiological ligands and drugs that can occur in the context of a pathophysiological setting.• Consider the vascular wall of an arteriole.• Several cell types interact at this site, including vascular

smooth muscle cells (SMCs), endothelial cells (ECs), platelets, and postganglionic sympathetic neurons.• A variety of physiological receptors and ligands are

represented, including ligands that cause SMCs to contract (Angiotensin II [AngII], Norepinephrine [NE]) and relax (Nitric Oxide [NO], B-type Natriuretic Peptide [BNP], and Epinephrine), as well as ligands that alter SMC gene expression (platelet-derived growth factor [PDGF], AngII, NE, and Eicosanoids).

• Ang-II has both acute and chronic effects on SMC. Interaction of Ang-II with AT1 receptors (AT1-R) causes the formation of the second messenger IP3 causing release of Ca from SR leading to smooth muscle contraction.• NE binds 1 adrenergic receptors that couple to the Gq-

PLC-IP3 pathway, causing an increase in intracellular Ca2+ and, as a result, contraction, an effect that is additive to that of Ang-II.• NO is formed in ECs by the action of two NO synthase

enzymes, eNOS and iNOS. The NO formed in ECs diffuses into SMCs, and activates the soluble form of Guanylate Cyclase (sGC), which catalyzes the formation of cyclic GMP from GTP. The increase in cyclic GMP activates PKG, which phosphorylates protein substrates in SMCs that reduce intracellular concentrations of Ca2+

• Intracellular concentrations of cyclic GMP are also increased by activation of the transmembrane BNP receptor (BNP-R), whose guanylate cyclase activity is increased when BNP binds. BNP is released from cardiac muscle in response to increased filling pressures.• Beta 1 antagonists reduce secretion of renin (the rate-

limiting first step in Ang-II synthesis)• A direct renin inhibitor (Aliskiren) to block the rate-

limiting step in Ang-II production• Angiotensin-converting enzyme (ACE) inhibitors (e.g.,

Enalapril) to reduce the concentrations of circulating Ang-II• AT1 receptor blockers (e.g., Losartan) to block Ang-II

binding to AT1 receptors on SMCs

• Alfa 1 adrenergic blockers to block NE binding to SMCs.• Sodium Nitroprusside to increase the quantities of

NO produced.• Ca2+ channel blocker (e.g., Nifedipine) to block

Ca2+ entry into SMCs.• Thus, the choices and mechanisms are complex, and

the appropriate therapy in a given patient depends on many considerations, including the diagnosed causes of hypertension in the patient, possible side effects of the drug, efficacy in a given patient, and cost.

89

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