Receptors

Views:
 
     
 

Presentation Description

No description available.

Comments

Presentation Transcript

This is Michaelis-Menton Curves or Rectangular Hyperbolae (Enzyme Kinetics- Biochemistry):

This is Michaelis-Menton Curves or Rectangular Hyperbolae (Enzyme Kinetics- Biochemistry)

Drug Receptor Interactions:

Receptor occupancy : is often used as convenient shorthand for the fraction of he binding sites occupied by a ligand . Ligand : Literally: entities do not act unless attached. means here a small molecule that binds to a specific site (or sites) on a receptor macromolecule. The term drug is often used in this context, especially in the older literature. Drug Receptor Interactions modeling” (i.e., making particular assumptions about) the two relationships and comparing the predictions of the models with the actual behavior of isolated tissues. These two are: THE RELATIONSHIP BETWEEN LIGAND CONCENTRATION AND RECEPTOR OCCUPANCY THE RELATIONSHIP BETWEEN RECEPTOR OCCUPANCY AND TISSUE RESPONSE

combination of a ligand, A, with its binding site on a receptor, R::

combination of a ligand, A, with its binding site on a receptor, R: Binding is regarded as a bimolecular reaction and k +1 and k –1 are, respectively, are the association rate constant (M –1 s –1 ) and the dissociation rate constant (s –1 ). Some times some authors use K on & K off for above two terms. THE RELATIONSHIP BETWEEN LIGAND CONCENTRATION AND RECEPTOR OCCUPANCY

law of mass action :

law of mass action The rate of a reaction is proportional to the product of the concentrations of the reactants. Assume that equilibrium has been reached so that the rate at which AR is formed from A and R is equal to the rate at which AR dissociates. This gives: Where [R] = concentrations of receptors binding sites for A are free [AR] = receptor binding sites are occupied

PowerPoint Presentation:

P R = proportion of receptor free = [R]/[R] T or N R /N where [R] T =total concentration of receptors, P AR = proportion of receptor occupied by agonist A =[AR]/[R] T or N AR /N

PowerPoint Presentation:

we are concerned only with equilibrium conditions and not with the rate at which equilibrium is reached, combining k +1 and k –1 to form a new constant, K A = k – / k +1 ,which has the unit of concentration. K A is a dissociation equilibrium constant either equilibrium constant or dissociation constant . Replacing k +1 and k –1 gives : Because binding sites are either free or occupied

Association constant:

Association constant The inverse ratio = KA‘ = K +1 /K –1 , gives the association equilibrium constant, which is usually referred to as the affinity constant. Unit is M -1

PowerPoint Presentation:

P R can be also be defined as N R / N , where N R is the number of receptors in which the binding sites are free of A and N is their total number. Similarly, P AR is given by N AR / N , where N AR is the number of receptors in which the binding site is occupied by A. Hill–Langmuir equation

Hill–Langmuir equation:

Hill–Langmuir equation A. V. Hill was the first (in 1909) to apply the law of mass action to the relationship between ligand concentration and receptor occupancy at equilibrium and to the rate at which this equilibrium is approached. The physical chemist I. Langmuir showed a few years later that a similar equation (the Langmuir adsorption isotherm ) applies to the adsorption of gases at a surface (e.g., of a metal or of charcoal).

:

Assumptions: 1.Concentration of A does not change as ligand receptor complexes are formed. In effect, the ligand is considered to be present in such excess . Thus [A] can be regarded as constant 2. The value of K A has been taken to be 1 μ M. that when [A] = K A , P AR = 0.5; that is, half of the receptors are occupied. Relationship between binding-site occupancy and ligand concentration [A]; For bothe curves KA = 1 μ M.

PowerPoint Presentation:

Rearranging the Hill- Langmuir equation Taking log on both sides A plot of log ( pAR /(1 – pAR ) against log [A] should give a straight line with a slope of one. Such a graph is described as a Hill plot.

The slope of the line is not always unity, or even constant. It is referred to as the Hill coefficient (nH); The term Hill slope is also used. :

The slope of the line is not always unity, or even constant. It is referred to as the Hill coefficient ( nH ); The term Hill slope is also used. y and y max are, respectively, the observed response and the maximum response to a large concentration of the agonist, A. [A] 50 is the concentration of A at which y is half maximal. Because it is a constant for a given concentration–response relationship, it is sometimes denoted by K. While this is algebraically neater (and was the symbol used by Hill), it should be remembered that K in this context does not necessarily correspond to an equilibrium constant.

PowerPoint Presentation:

2. THE RELATIONSHIP BETWEEN RECEPTOR OCCUPANCY AND TISSUE RESPONSE V. Hill and A. J. Clark explored assumptions: (1) The law of mass action applies as equations derived. (2) The response of the tissue is linearly related to receptor occupancy. (3) The relationship might be one of direct proportionality. (4) All occupied receptors are activated. Where Y=response of tissue Rearranging this equation Taking logs Putting value from Hill Langmuir equation

PowerPoint Presentation:

y and y max are, respectively, the observed response and the maximum response to a large concentration of the agonist, A. [A] 50 is the concentration of A at which y is half maximal. Because it is a constant for a given concentration–response relationship, it is sometimes denoted by K. While this is algebraically neater (and was the symbol used by Hill), it should be remembered that K in this context does not necessarily correspond to an equilibrium constant. Modifying this equation

PowerPoint Presentation:

THE DISTINCTION BETWEEN AGONIST BINDING AND RECEPTOR ACTIVATION This distinction, it is now appreciated, is crucial to the understanding of the action of agonists and partial agonists. * Here, the occupied receptors can exist in two forms. 1st inactive (AR) and 2nd active (AR*) in the sense that its formation leads to a tissue response. AR and AR* can interconvert (often described as isomerization ), and at equilibrium the receptors will be distributed among the R, AR, and AR* conditions. The position of the equilibrium between AR and AR*, and hence the magnitude of the maximum response of the tissue, will depend on the value of the equilibrium constant E. * Suppose that a very large concentration of the agonist A is applied, so that all the binding sites are occupied (i.e., the receptors are in either the AR or the AR* state). If the position of the equilibrium strongly favors AR, with few active (AR*) receptors, the response will be relatively small. * The reverse would apply for a very effective agonist.

PowerPoint Presentation:

1. The above is del Castillo–Katz scheme, as it was first applied to receptor action by J. del Castillo and B.Katz (University College London) in 1957 . 2. The scheme is readily extended to include the possibility that some of the receptors may be active even in the absence of agonist.

PowerPoint Presentation:

Competitive and Noncompetitive Antagonists Receptor antagonists bind to the receptor but do not activate it. In general the effects of these antagonists result from preventing agonists from binding to and activating receptors. Antagonists may be 1. competitive (reversibly displaced by agonists) or 2. noncompetitive (not reversibly displaced by agonists). Competitive (surmountable or reversible) Antagonists Competitive antagonist C, competes with the agonists A for binding to the receptor, R.

PowerPoint Presentation:

The transduction process between receptor occupancy and drug response is termed coupling. The efficiency of coupling is partly determined by the initial conformational change in the receptor. Thus the effects of full agonists may be more efficiently coupled to receptor occupancy than those of partial agonists. However, coupling efficiency is also determined by the biochemical events that transduce receptor occupancy into cellular response. High efficiency coupling may also result from spare receptors.

PowerPoint Presentation:

Receptors may be considered spare when the maximal response is elicited by an agonist at a concentration that does not produce full occupancy of the available receptors. Spare receptors are not different from “ nonspare ” receptors. They are not hidden. When they are occupied they can be coupled to response. Spare receptors may be demonstrated by using irreversible antagonists to inhibit binding of agonists to a portion of the receptor pool then demonstrating that a high concentration of agonist may still produce an undiminished maximal response. e.g. A maximal inotropic response of heart muscle to catecholamines can be elicited when 90% of the ß-ARs are occupied by a quasi-irreversible antagonist. Thus myocardium is said to contain a large proportion of spare ß-ARs .

Spare Receptors:

Spare Receptors Modifications of classical receptor theory came when its basic assumptions were questioned, at first by R P (‘Steve’) Stephenson from Edinburgh, who showed that the assumption of proportionality between occupancy and effect was incorrect, and postulated that a maximum effect can be produced without total occupancy of receptors (spare receptors). He coined the term ‘efficacy’ as a measure of the ability of a drug to activate receptors and cause a response. Other later developments, too many and complex to detail, included Paton’s rate theory , Changeux’s allosteric theory , and the elucidation of models t o try to explain the difficult concept of efficacy .

Spare Receptors :

Spare Receptors Most maximal biological responses are achieved when only a small percentage of receptors is occupied. Remaining receptors: spare receptors These fully functional spare receptors may serve to increase the sensitivity of target cells to activation by low levels of hormone Maximal stimulation of steroidogenesis by Leydig cells occurs when only 1% of LH receptors are occupied.

Spare receptors:

Spare receptors Full steroid-induced transcriptional response may only require 10% receptors If spare receptors are available then tissue is said to have a receptor reserve for this agonist. This does not, of course, mean that we have two kinds of receptors, spare and used; the receptors do not differ. However, only a few must be activated to cause a large or even maximal response. This can occur when the response of the tissue is limited not by the number of active receptors but by one or more of the events that follow receptor activation.

False belief:

False belief “spare” receptors are nonfunctional. The phrase receptor reserve means essentially the same thing and may help avoid this confusion though it is less frequently used in the literature. Although all receptors may not be needed for a maximal response, all receptors contribute to the measured responses, thus the potency of full agonists (and often the physiological agonists) is enhanced by the presence of the spare receptors . it is essential to understand and account for the spare receptor phenomenon. Many compounds that are partial agonists in normal tissues are full agonists in expression systems due to the high receptor number

TCR as spare receptors:

TCR as spare receptors A key concept of spare receptor model is that a maximal response can be achieved by an agonist occupying only a small proportion of the receptors. However, weak partial agonists must bind to more receptors to produce a maximum response. A receptor reserve allows for responses that are extremely rapid in onset and termination, yet at the same time are sensitive to low, transient ligand concentrations and low-affinity interactions. Several properties of T cells are compatible with this pharmacological theory, including a vast excess of TCRs, variable affinities for ligand , and sensitivity to low concentrations of agonists.

Orphan receptors:

Orphan receptors The molecular function of GPCRs is to recognize ligands and then activate G proteins. G protein-coupled receptors (GPCRs) comprise one of the largest superfamilies of the human genome. The recent achievement of the human genome project : Approximately 700 GPCR genes (excluding pseudo-genes) in the human genome. Most of these genes are identified on the basis of sequence homology to known GPCR genes. Each GPCR gene encodes a protein consisting of an extracellular N-terminal domain, seven transmembrane domains, and intracellular domains responsible for interaction with G proteins or other intracellular signaling molecules .

Orphan receptors:

Orphan receptors However, the ligands of the remaining 120 receptors have not yet been identified, and they are, therefore, referred to as orphan GPCRs. The identification of ligands for orphan GPCRs is expected to lead to the discovery of new regulatory mechanisms of the human body. GPCRs are the most successful targets in the field of drug discovery. Of the approximately 500 drugs currently on the market, more than 30% are GPCR agonists or antagonists, representing approximately 9% of global pharmaceutical sales. Orphan GPCR research is therefore important from the perspectives of both basic and applied science.

Orphan receptors (continue.):

Orphan receptors (continue.) The identification of ligands for orphan GPCRs should yield important clues as to their physiological functions and will help determine whether they are suitable as drug targets. GPCRs for which endogenous ligands were not identified are called orphan GPCRs. It is one of the most important issues in the field of signal transduction to identify endogenous ligands for orphan GPCRs, as identification of endogenous ligands may mean discovery of novel hormones or neurotransmitters. orphan GPCR research started in 1994. hGR3 was isolated, an orphan GPCR, from the human pituitary. This novel orphan GPCR showed low homology to known GPCRs, having at most 30% amino acid identity with the neuropeptide Y receptor.

Orphan receptors:

Orphan receptors Approximately half of GPCR genes are thought to encode sensory receptors for smell, taste, and vision. The other half encode receptors regulating cell functions. To date, natural ligands have been identified for approximately 230 of these receptors. Orphan GPCR research is therefore important from the perspectives of both basic and applied science. The identification of ligands for orphan GPCRs should yield important clues as to their physiological functions and will help determine whether they are suitable as drug targets.

Ligand sources:

Ligand sources Tissue extract Known molecules Database search

Tissue extract:

Tissue extract The most orthodox method of ligand fishing may be to employ a tissue extract as the starting material. In this strategy, the extract of tissue is subjected to a purification procedure, while the particular responses of the cells expressing the target receptor protein are monitored. After the purification steps, which involve a combination of chromatographies , the ligand molecule is finally isolated in homogeneity, and its structure is determined. This was the main approach taken in the early attempts at ligand fishing. e. g nociceptin and orphanin FQ6 to be isolated from rat and porcine brain extracts, respectively, and the discoveries of several novel 6 G Protein-Coupled Receptors: Many novel compounds, all of them peptidic , were discovered using this method.

Known molecules:

Known molecules Another approach to discovering ligands for the orphan receptors involves screening the library of known molecules that includes possible candidate ligands , such as the biogenic amines, peptides, chemokines , bioactive lipids, and metabolic pathway intermediates. In fact, use of this approach led to the discovery of the most ligands , including bioactive peptides, such as melanin-concentrating hormone, urotensin II, motilin , and neuromedin U, and the low molecular weight ligands , such as sphingosylphosphorylcholine , lysophosphatidylcholine , bile acids, and free fatty acids. If the target receptor is expected to possibly pair with a nonpeptidic low molecular weight ligand , this approach can identify an agonistic molecule, because only rarely will a completely novel compound be a specific ligand for such a receptor.

Database search:

Database search Bioactive peptides are usually generated by cleavage at the potential processing sites (cluster of two or three successive basic amino acids) from the precursor proteins equipped with a secretory signal sequence.

authorStream Live Help