Role of receptors in drug design

Receptor theories, types, receptor binding studies

1. WELCOME…

2. Prepared by, ROSHNI ANN BABY M.PHARM PART I- PHARM.CHEMISTRY RECEPTORS IN DRUG DESIGN

3. CONTENTS CONCEPT OF RECEPTORS DEFINITION MOLECULAR BIOLOGY RECEPTOR THEORIES RADIOLIGAND BINDING ASSAY

4. Concept of Receptors First postulated by John N Langley (1878 ) -Established after his experiments using nicotine and curare analogues on muscle contraction . -Langley concluded that a protoplasmic "receptive substance" must exist in which the two drugs compete for directly. He further added that the effect of combination of the receptive substance with competing drugs was determined by their comparative chemical affinities for the substance and relative dose.

5. Concept of receptors contd.. Further the term was introduced by Paul Ehrlich (1907) -the compounds do not act unless bound. -Demonstrated that stereoselectivity was imperative in drug-receptor signaling.

6. Definition Receptor-is a protein molecule embedded either within the cell membrane with a part of its structure facing the outside of the cell or inside the cell. Occupation of receptor may result in its activation leading to a cellular response . Receptors have two major properties: Recognition and Transduction

7. Recognition : The receptor protein must exist in a conformational state that allows for recognition and binding of a compound and must satisfy the following criteria : Saturability Reversibility Stereoselectivity Agonist specificity Tissue specificity Transduction : The second property of a receptor is that the binding of an agonist must be transduced into some kind of functional response (biological or physiological).

8. Important Terms Ligand : Any endogenous or exogenous chemical agent that binds to a receptor is known as a ligand Binding domain : The general region on a receptor where a ligand binds is known as binding domain.It is equivalent to enzyme active site but with no catalytic activity. Affinity : The ability of the drug to bind with receptor Intrinsic Activity : The ability of the drug to elicit pharmacological response

9. Agonist : The drug molecule possessing high affinity as well as high intrinsic activity Antagonist :Drugs having high affinity but poor intrinsic activity Partial Agonist : Drug with an affinity equal or less than that of agonist but with less intrinsic activity Inverse agonist : Produces responses opposite to those of the agonist. Signal Transduction : The mechanism by which any message carried by the ligand is translated through the receptor system into tissue response.

10. Receptors-Molecular Biology

11. Structure and function of receptors Globular proteins acting as a cell’s ‘letter boxes’ Located mostly in the cell membrane Receive messages from chemical messengers coming from other cells Transmit a message into the cell leading to a cellular effect Different receptors specific for different chemical messengers Each cell has a range of receptors in the cell membrane making it responsive to different chemical messengers

12. Cell Nerve Messenger Signal Receptor Nerve Nucleus Cell Response Structure and function of receptors

13. Chemical Messengers Neurotransmitters : Chemicals released from nerve endings which travel across a nerve synapse to bind with receptors on target cells, such as muscle cells or another nerve. Usually short lived and responsible for messages between individual cells Hormones : Chemicals released from cells or glands and which travel some distance to bind with receptors on target cells throughout the body Chemical messengers ‘switch on’ receptors without undergoing a reaction Structure and function of receptors

14. Neurotransmitters relay signal between a neuron and another cell

15. Classical Hormones are produced by the glands of the endocrine system, shown below The major endocrine glands: (Male left, female right) 1 Pineal gland 2 Pituitary gland 3 Thyroid gland 4 Thymus 5 Adrenal gland 6 Pancreas 7 Ovary 8 Testes

16. Nerve 1 Nerve 2 Hormone Blood supply Neurotransmitters Structure and function of receptors

17. Mechanism Receptors contain a binding site (hollow or cleft in the receptor surface) that is recognised by the chemical messenger Binding of the messenger involves intermolecular bonds Binding results in an induced fit of the receptor protein Change in receptor shape results in a ‘domino’ effect Domino effect is known as Signal Transduction, leading to a chemical signal being received inside the cell Chemical messenger does not enter the cell. It departs the receptor unchanged and is not permanently bound Structure and function of receptors

18. Chemical messenger do not undergo chemical reaction . It fits into the binding site of receptor protein,passes on it message and then leaves unchanged . The messenger binds to the receptor and induces a change in shape(conformational change) which subsequently affects other components of the cell membrane and leads to a biological effect . The receptor then reforms its original shape. Difference B/W Enzyme and Chemical Messenger

19. Mechanism Cell Membrane Cell Receptor Messenger message Induced fit Cell Receptor Messenger Message Cell Messenger Receptor Structure and function of receptors

20. ENZYME The binding site A hydrophobic hollow or cleft on the receptor surface - equivalent to the active site of an enzyme Accepts and binds a chemical messenger Contains amino acids which bind the messenger No reaction or catalysis takes place Binding site Binding site

21. The binding site

22. Messenger binding Binding site is nearly the correct shape for the messenger Binding alters the shape of the receptor (induced fit) Altered receptor shape leads to further effects - signal transduction 3.1 Introduction Messenger Induced fit M

23. Ionic covalent H-bonding hydrophobic van der Waals 3.2 Bonding forces Example: Receptor Binding site vdw interaction ionic bond H-bond Phe Ser O H Asp CO 2 Messenger binding

24. Substrate binding Induced fit - Binding site alters shape to maximise intermolecular bonding 3.2 Bonding forces Intermolecular bonds not optimum length for maximum binding strength Intermolecular bond lengths optimised Phe Ser O H Asp CO 2 Induced Fit Phe Ser O H Asp CO 2

25. Letting Go

26. Overall process of receptor/messenger interaction M M E R Binding interactions must be: - strong enough to hold the messenger sufficiently long for signal transduction to take place - weak enough to allow the messenger to depart Implies a fine balance Drug design - designing molecules with stronger binding interactions results in drugs that block the binding site - antagonists R M E R Signal transduction

27. Signal transduction Control of ion channels Receptor protein is part of an ion channel protein complex Receptor binds a messenger leading to an induced fit Ion channel is opened or closed Ion channels are specific for specific ions (Na + , Ca 2+ , Cl - , K + ) Ions flow across cell membrane down concentration gradient Polarises or depolarises nerve membranes Activates or deactivates enzyme catalysed reactions within cell

28. Closed or Opened?

29. Signal transduction Hydrophilic tunnel Cell membrane Control of ion channels

30. Opening the Door

31. Cell membrane Five glycoprotein subunits traversing cell membrane Messenger Cell membrane Receptor Induced fit ‘Gating’ (ion channel opens) Cationic ion channels for K + , Na + , Ca 2+ (e.g. nicotinic) = excitatory Anionic ion channels for Cl - (e.g. GABA A ) = inhibitory Binding site Control of ion channels Signal transduction

32. Control of ion channels: Induced fit and opening of ion channel ION CHANNEL (open) Cell Cell membrane MESSENGER Ion channel Ion channel Cell membrane ION CHANNEL (closed) Cell RECEPTOR BINDING SITE Lock Gate Ion channel Ion channel Cell membrane Cell membrane MESSENGER Signal transduction

33. GABA A Receptor

34. P2X 4 Receptor Ion Channel

35. Activation of signal proteins Receptor binds a messenger leading to an induced fit Opens a binding site for a signal protein (G-protein) G-Protein binds, is destabilised then split messenger G-protein split induced fit closed open Signal transduction

36. Activation of signal proteins G-Protein subunit activates membrane bound enzyme Binds to allosteric binding site Induced fit results in opening of active site Intracellular reaction catalysed active site (closed) active site (open) Enzyme Intracellular reaction Enzyme Signal transduction

37. Activation of enzyme active site Protein serves dual role - receptor plus enzyme Receptor binds messenger leading to an induced fit Protein changes shape and opens active site Reaction catalysed within cell closed messenger induced fit active site open intracellular reaction closed messenger Signal transduction

38. Intracellular Receptors

39. Competitive Antagonists

40. Competitive (reversible) antagonists Antagonist binds reversibly to the binding site Intermolecular bonds involved in binding Different induced fit means receptor is not activated No reaction takes place on antagonist Level of antagonism depends on strength of antagonist binding and concentration Messenger is blocked from the binding site Increasing the messenger concentration reverses antagonism An E R M An R

41. Irreversible Antagonists

42. Non competitive (irreversible) antagonists Antagonist binds irreversibly to the binding site Different induced fit means that the receptor is not activated Covalent bond is formed between the drug and the receptor Messenger is blocked from the binding site Increasing messenger concentration does not reverse antagonism X OH OH X O Covalent Bond Irreversible antagonism

43. Non competitive (reversible) allosteric antagonists Antagonist binds reversibly to an allosteric site Intermolecular bonds formed between antagonist and binding site Induced fit alters the shape of the receptor Binding site is distorted and is not recognised by the messenger Increasing messenger concentration does not reverse antagonism ACTIVE SITE (open) ENZYME Receptor Allosteric site Binding site (open) ENZYME Receptor Induced fit Binding site unrecognisable Antagonist

44. The Umbrella Effect

45. Antagonists by umbrella effect Antagonist binds reversibly to a neighbouring binding site Intermolecular bonds formed between antagonist and binding site Antagonist overlaps with the messenger binding site Messenger is blocked from the binding site Antagonist Binding site for antagonist Binding site for messenger messenger Receptor Receptor

46. Agonists

47. Agonists Agonist binds reversibly to the binding site Similar intermolecular bonds formed as to natural messenger Induced fit alters the shape of the receptor in the same way as the normal messenger Receptor is activated Agonists are often similar in structure to the natural messenger E Agonist R E Agonist R Signal transduction Agonist R Induced fit

48. Clonidine Dexmedetomidine

49. RECEPTOR THEORIES

50. Clark’s Occupancy Theory Based on Law of Mass Action [R] + [D] [DR] Response The intensity of the response at any time was proportional to the number of receptors occupied by the drug:the greater the number occupied, the greater the pharmacological effect. drawbacks: Can’t explain inverse agonist Doesnot rationalize partial agonist

51. Derivation Response effect E ∞ [DR] 2 A maximum response would be obtained when all the receptors were occupied Maximum response effect E max ∞ [R T] 3 Where R T is the total number of receptors. Thus for a given dose of a drug the fraction of maximum response is given by Fraction of the maximum response E = [DR] 4 Emax [R T ] The dissociation of the drug receptor complex may be represented as D-R D + R 5

52. Derivation contd.. Applying the law of mass action K D = [D] [R] 6 K D - dissociation constant for [DR] the drug receptor complex. But the total receptor concentration is [R T ] = [R] + [DR ] 7 Substituting 7 in 6 gives K D = [D]([R T ]-[DR]) 8 [DR]

53. Derivation contd … Rearranging eqn K D = [D][R T ] – [D][DR] 9 [DR] [DR] Therefore K D =[D][R T ] – [D] 10 [DR] And K D + [D] = [D][R T ] 11 [DR] K D + [D] = [R T ] 12 [D] [DR] Substituting 12 in 4 E = [DR] = [D] E max [R T K D + [D]

54. Derivation contd … The eqn shows that the relationship between E and molar drug concentration [D] is in the form of a rectangular hyperbola whereas that between E and log [D] is sigmodial . Substituting the value of E\ Emax = ½ in Equation 9 gives the relationship K D = EC 50 ;value of dissociation constant is a measure of affinity of the drug for the receptor

55. Modified occupancy theory By Ariens and Stephenson Pointed out 2 terms: agonist ,antagonist According to this ,drug – receptor interacion involves two stages Complexation -affinity Initiatian of the biological effect-efficacy Drawbacks Does not account for why 2 drug occupy the same receptors can act differently.

56. Major Postulates receptor- ligand complex is reversible. Association is a bimolecular process, while dissociation is a monomolecular process. All receptors of a given class are equivalent and binds to ligand independently of one another. Formation of receptor-ligand doesnot alter free ligand concentration or affinity of receptor for ligand . Response elicited by the number of receptors occupied.

57. Rate theory They proposed that the most important factor determining drug action is the rate at which drug receptor combination takes place. Pharmacological activity is a function of the rate of association and dissociation of the drug with the receptor and not the number of the occupied receptors. Drawback: Does not rationalise why the different types of compounds exhibit the characteristics that they do.

58. Rate theory represented by : E= Veq φ E = Effect produced φ =Proportionality factor e =Efficacy component V =Velocity Rate theory explained is only on the basis of RL formation If the dissociation rate constant is large the ligand will be an agonist If the dissociation rate constant is small, the ligand will be an antagonist

59. The Two state Model Receptors can exist in either an active or inactive state.The active state is known as the relaxed or R state and the inactive state is referred to as the tensed or T state. Receptors in the R state can provide a stimulus but those in the T state are unable to produce a stimulus. The two state model postulates that in the absence of any ligands a population of receptors of the same type will consist of an equilibrium mixture of receptors in the R and T states k 1 k 1 - rate of forward reaction T R k 2 - rate of reverse reaction k 2 k 1 > k 2 Increased R receptors-tissue response k 2 > k 1 Increased T receptors- no response

60. Induced fit theory of Enzymes As the drug approaches the receptor a confirmation change occurs in the receptor to allow for effective binding. The receptor need not necessarily exist in the appropriate conformation.As the drug approaches the receptor a conformational change is induced which orients the essential binding site towards the approaching ligand Proposed by Koshland .

61. Macromolecular perturbation theory Combination of induced fit and rate theories. Two types of conformation changes exist and rate of their existence determines biological response. Agonist produce the specific perturbation required for biological response Antagonist produce a non-specific perturbation which fails to yield a biological response No account on the activity of partial agonist

62. Activation Aggregation Even in the absence of the drug, the receptors are in dynamic equilibrium between the active form (R ) which is responsible for biological response and inactive form(T ). Agonist shift the equilibrium to active form and antagonist shift to the inactive form. Accounts for the activity of inverse agonist.

63. Classification of receptors based on their mechanism Ligand-gated ion channels – nicotinic Ach receptors transmembrane GPCRs – opioid receptors Kinase linked receptors Nuclear receptors

64. Ligand gated ion channels

65. G-PROTEIN COUPLED RECEPTOR

66. Enzyme linked receptor

67. Nuclear receptors Intracellular receptor. Interact with chemical messengers like steroids, thyroid hormones. Hydrophobic in nature eg:oestrogen receptor

68. Nuclear receptors contd..

69. Receptor types and subtypes receptor type subtype egs of agonists egs of antagonists Cholinergic Nicotinic Muscarinic 4 Subtypes M1-M5 GI motility, Glaucoma Nm blockers and muscle relaxants Peptic ulcers Motion sickness Adrenergic α 1, α 2 β α 1 A, α 1B α 1D, α 1A- Α2 c β 1 β 2 β 3 Antiasthmatics β Blockers Dopamine D1-D5 Parkinsons disease Antidepressants Histamine H1-H3 Vasodilation Allergies,ulcers,sedatives Opioid ,ORL-1 Analgesics Antidote to morphine OD. 5 HT 5HT1-5HT7 5HT1A-B 5HT1D-F 5HT2A-C 5HT5A-B Antimigraine GI motility, Antiemetics Ketanserin Oestrogen Contraception Breast Cancer receptor type subtype egs of agonists egs of antagonists Cholinergic Nicotinic Muscarinic 4 Subtypes M1-M5 GI motility, Glaucoma Nm blockers and muscle relaxants Peptic ulcers Motion sickness Adrenergic α 1, α 2 β α 1 A, α 1B α 1D, α 1A- Α2 c β 1 β 2 β 3 Antiasthmatics β Blockers Dopamine D1-D5 Parkinsons disease Antidepressants Histamine H1-H3 Vasodilation Allergies,ulcers,sedatives Opioid Analgesics Antidote to morphine OD. 5 HT 5HT1-5HT7 5HT1A-B 5HT1D-F 5HT2A-C 5HT5A-B Antimigraine GI motility, Antiemetics Ketanserin Oestrogen Contraception Breast Cancer

70. R adioligand binding assay Radiolabelled ligand CELLS OR TISSUES CONTAINING TARGET receptor UNBOUND LIGANDS ARE SEPARATED BY WASHING,FILTRATION OR CENTRIFUGATION EXTEND OF BINDING DETECTED BY MEASURING THE RADIOACTIVITY PRESENT IN THE CELLS/TISSUES AFTER EQUILIBRIUM

71. tissue containing receptor. radiolabelled ligand . technique to separate bound from free ligand . apparatus for determination of samples radioactivity. Essential requirements

72. Equilibrium constant for bound versus unbound radioligand is defined by dissociation binding constant,Kd [L]+[R] [LR](receptor ligand complex) Kd = [L] X [R] eqn -1 [ LR] [L] and [LR] can be found by measuring radioactivity of unbound and bound ligand respectively.

73. total no: of receptors present, R total = no: of receptors occupied by ligand[LR] + those that are unoccupied[R]. No: of receptors unoccupied by a ligand. [R] = R total –[LR] eqn 2

74. Subst:eqn (2) in eqn (1) and rearranging leads to SCATCHARD EQUATION both [LR] and [L] are measurable. [Bound ligand ] = [LR] = R (total )–[LR] [free ligand] [L] Kd

75. Kd and R total can be determined from Schatchard plot. R total Slope-measure of radioligand affinity for receptor ie , -1/ Kd Thus Kd can be determined. SCHATCHARD PLOT

76. Competition binding assays Allows one to determine a rough estimate of an unlabeled ligand’s affinity for a receptor. Competitive or non-competitive. Introduction into the incubation mixture of a non-radioactive drug (e.g. drug B) that also binds to R will result in less of R being available for binding with D*, thus reducing the amount of [D*R] that forms. This second drug essentially competes with D* for occupation of R. Increasing concentrations of B result in decreasing amounts of [D * R] being formed.

77. IC 50- Concentration of unlabelled ligand required to inhibit 50 % of the specific binding of the radio ligand.

78. The inhibitory or affinity constant Ki for the test compound is the same as the IC 50 value if non competitive interactions are involved For compounds that are in competition with the radioligand for the binding site the inhibitory constant depends on the level of radioligand present and is given by Ki = IC 50 1+[L] tot / Kd

79. References Graham. L. Patrick An Introduction to Medicinal Chemistry,3 rd edn , Pg No:43-57 Gareth Thomas, Medicinal Chemistry An Introduction Pg No:287-326 Burgers Medicinal Chemistry and drug discovery; Vol II, VIth edn Pg No:323-345 http://en.wikipedia.org/wiki/receptor http://www.chem.ace.dk/www/weeknotes

80. Thank you