Receptors

RECEPTORS

The largest number of drugs do not bind directly to the effectors, viz. enzymes, channels, transporters, structural proteins, template biomolecules, etc. but act through specific regulatory macromolecules which control the above listed effectors. These regulatory macromolecules or the sites on them which bind and interact with the drug are called ‘receptors’.

Receptor:

It is defined as a macromolecule or binding site located on the surface or inside the effector cell that serves to recognize the signal molecule/drug and initiate the response to it, but itself has no other function.

Though, in a broad sense all types of target biomolecules, including the effectors (enzymes, channels, transporters, etc.) with which a drug can bind to produce its action have been denoted as ‘receptors’ by some authors, such designation tends to steal the specific meaning of this important term. If so applied, xanthine oxidase would be the ‘receptor’ for allopurinol, Ltype Ca2+ channel would be the ‘receptor’ for nifedipine, serotonin transporter (SERT) would be the ‘receptor’ for fluoxetine; a connotation not in consonence with the general understanding of the term. It is therefore better to reserve the term ‘receptor’ for purely regulatory macromolecules which combine with and mediate the action of signal molecules including drugs.

The following terms are used in describing drugreceptor interaction:

Agonist

An agent which activates a receptor to produce an effect similar to that of the physiological signal molecule.

Inverse Agonist

An agent which activates a receptor to produce an effect in the opposite direction to that of the agonist.

Antagonist

An agent which prevents the action of an agonist on a receptor or the subsequent response, but does not have any effect of its own.

Partial agonist

An agent which activates a receptor to produce submaximal effect but antagonizes the action of a full agonist.

Ligand

(Latin: ligare—to bind) Any molecule which attaches selectively to particular receptors or sites. The term only indicates affinity or binding without regard to functional change: agonists and competitive antagonists are both ligands of the same receptor.

The overall scheme of drug action through receptors is depicted in Fig. 4.1D.

Basic Evidences For Drug Action Through Receptors

Many drugs exhibit structural specificity of action, i.e. specific chemical configuration is associated with a particular action, e.g. isopropyl substitution on the ethylamine side chain of sympathetic drugs produces compounds with marked cardiac and bronchial activity—most β adrenergic agonists and antagonists have this substitution. A 3 carbon inter nitrogen separation in the side chain of phenothiazines results in anti dopaminergic antipsychotic compounds, whereas 2 carbon separation produces anti-cholinergic antihistaminic compounds. Further, chiral drugs show stereospecificity in action, e.g. levo noradrenaline is 10 times more potent than dextro noradrenaline; d-propranolol is about 100 times less potent in blocking β receptors than the l-isomer, but both are equipotent local anaesthetics.

Thus, the cell must have some mechanism to recognize a particular chemical configuration and three dimensional structure.

Competitive antagonism is seen between specific agonists and antagonists. Langley in 1878 was so impressed by the mutual antagonism among two alkaloids pilocarpine and atropine that he proposed that both reacted with the same ‘receptive substance’ on the cell. Ehrlich (1900) observed quantitative neutralization between toxins and antitoxins and designated ‘receptor’ to be the anchoring group of the protoplasmic molecule for the administered compound.

It was calculated by Clark that adrenaline and acetylcholine produce their maximal effect on frog’s heart by occupying only 1/6000th of the cardiac cell surface— thus, special regions of reactivity to such drugs must be present on the cell.

Receptor Occupation Theory

After studying quantitative aspects of drug action, Clark (1937) propounded a theory of drug action based on occupation of receptors by specific drugs and that the pace of a cellular function can be altered by interaction of these receptors with drugs which, in fact, are small molecular ligands. He perceived the interaction between the two molecular species, viz. drug (D ) and receptor (R) to be governed by the law of mass action, and the effect (E) to be a direct function of the drug receptor complex (DR) formed:

Subsequently, it has been realized that occupation of the receptor is essential but not itself sufficient to elicit a response; the agonist must also be able to activate (induce a conformational change in) the receptor. The ability to bind with the receptor designated as affinity, and the capacity to induce a functional change in the receptor designated as intrinsic activity (IA) or efficacy are independent properties. Competitive antagonists occupy the receptor but do not activate it. Moreover, certain drugs are partial agonists which occupy and sub-maximally activate the receptor. An all or none action is not a must at the receptor. A theoretical quantity(S) denoting strength of stimulus imparted to the cell was interposed in the Clark’s equation:

Depending on the agonist, DR could generate a stronger or weaker S, probably as a function of the conformational change brought about by the agonist in the receptor. Accordingly:

Agonists have both affinity and maximal intrinsic activity (IA = 1), e.g. adrenaline, histamine, morphine.

Competitive Antagonists have affinity but no intrinsic activity (IA = 0), e.g. propranolol, atropine, chlorpheniramine, naloxone.

Partial Agonists have affinity and submaximal intrinsic activity (IA between 0 and 1), e.g. di chloro iso-proterenol (on β adrenergic receptor), pentazocine (on μ opioid receptor).

Inverse Agonists have affinity but intrinsic activity with a minus sign (IA between 0 and –1), e.g. DMCM (on benzodiazepine receptor).

It has also been demonstrated that many full agonists can produce maximal response even while occupying <1% of the available receptors.

A large receptor reserve exists in their case, or a number of spare receptors are present.

The Two-State Receptor Model

A very attractive alternative model for explaining the action of agonists, antagonists, partial agonists and inverse agonists has been proposed.

The receptor is believed to exist in two interchangeable states: Ra (active) and Ri (inactive) which are in equilibrium. In the case of majority of receptors, the Ri state is favoured at equilibrium—no/very weak signal is generated in the absence of the agonist—the receptor exhibits no constitutive activation (Fig. 4.3I). The agonist (A) binds preferentially to the Ra conformation and shifts the equilibrium → Ra predominates and a response is generated (Fig. 4.3II) depending on the concentration of A. The competitive antagonist (B) binds to Ra and Ri with equal affinity → the equilibrium is not altered → no response is generated (Fig. 4.3 III), and when the agonist is applied fewer Ra are available to bind it— response to agonist is decreased. If an agonist has only slightly greater affinity for Ra than for Ri, the equilibrium is only modestly shifted towards Ra (Fig. 4.3 IV) even at saturating concentrations → a submaximal response is produced and the drug is called a partial agonist (C). The inverse agonist (D) has high affinity for the Ri state (Fig. 4.3V), therefore it can produce an opposite response, provided the resting equilibrium was in favour of the Ra state. Certain receptors (mainly Gprotein coupled ones) such as benzodiazepine, histamine H₂, angiotensin AT₁, adrenergic β₁ and cannabinoid receptors exhibit constitutive activation, i.e. an appreciable intensity signal is generated even in the basal state (no agonist present). In their case the inverse agonist stabilizes the receptor in the inactive conformation resulting in an opposite response. Only few inverse agonists are known at present, but as more receptors with constitutive activation are found, more inverse agonists are likely to be discovered.

This model has gained wide acceptance because it provides an explanation for the phenomenon of positive cooperativity often seen with neurotransmitters, and is supported by studies of conformational mutants of the receptor with altered equilibrium.

Nature Of Receptors

Receptors are regulatory macromolecules, mostly proteins, though nucleic acids may also serve as receptors. They are no longer hypothetical. Hundreds of receptor proteins have been isolated, purified, cloned and their primary amino acid sequence has been worked out. Molecular cloning has also helped in obtaining the receptor protein in larger quantity to study its structure and properties, and in subclassifying receptors. The cell surface receptors with their coupling and effector proteins are considered to be floating in a sea of membrane lipids; the folding, orientation and topography of the system being determined by interactions between the lipophilic and hydrophilic domains of the peptide chains with solvent molecules (water on one side and lipids on the other). Nonpolar portions of the AA chain tend to bury within the membrane, while polar groups tend to come out in the aqueous medium. In such a delicately balanced system, it is not difficult to visualize that a small molecular ligand binding to one site in the receptor molecule could be capable of tripping the balance (by altering distribution of charges, etc.) and bringing about conformational changes at distant sites. Each of the four major families of receptors (described later) have a well defined common structural motif, while the individual receptors differ in the details of amino acid sequencing, length of intra/extracellular loops, etc. Majority of receptor molecules are made up of several nonidentical subunits (hetero-polymeric), and agonist binding has been shown to bring about changes in their quaternary structure or relative alignment of the subunits, e.g. on activation the subunits of nicotinic receptor move apart opening a centrally located cation channel.

Radioligand binding studies have helped in characterizing and classifying receptors even when they have been dissociated from the effector system.

Many drugs act upon physiological receptors which mediate responses to transmitters, hormones, autacoids and other endogenous signal molecules; examples are cholinergic, adrenergic, histaminergic, steroid, leukotriene, insulin and other receptors. In addition, now some truly drug receptors have been described for which there are no known physiological ligands, e.g. benzodiazepine receptor, sulfonylurea receptor, cannabinoid receptor.

Receptor Subtypes

The delineation of multiple types and subtypes of receptors for signal molecules has played an important role in the development of a number of targeted and more selective drugs. Even at an early stage of evolution of receptor pharmacology, it was observed that actions of acetylcholine could be grouped into ‘muscarinic’ and ‘nicotinic’ depending upon whether they were mimicked by the then known alkaloids muscarine or nicotine. Accordingly, they were said to be mediated by two types of cholinergic receptors, viz. muscarinic or nicotinic (N); a concept strengthened by the finding that muscarinic actions were blocked by atropine, while nicotinic actions were blocked by curare. In a landmark study, Ahlquist (1948) divided adrenergic receptors into ‘α’ and ‘β’ on the basis of two distinct rank order of potencies of adrenergic agonists. These receptors have now been further subdivided (M₁, M₂ ….M₅), (N_M, N_N) (α₁, α₂) (β₁, β₂, β₃). Multiple subtypes of receptors for practically all transmitters, autacoids, hormones, etc. are now known and have paved the way for introduction of numerous clinically superior drugs. In many cases, receptor classification has provided sound explanation for differences observed in the actions of closely related drugs.

The following criteria have been utilized in classifying receptors:

 a. Pharmacological Criteria

Classification is based on relative potencies of selective agonists and antagonists. This is the classical and oldest approach with direct clinical bearing; was used in delineating M and N cholinergic, α and β adrenergic, H₁ and H₂ histaminergic receptors, etc.

 b. Tissue Distribution

The relative organ/tissue distribution is the basis for designating the subtype, e.g. the cardiac β adrenergic receptors as β₁, while bronchial as β₂. This division was confirmed by selective agonists and antagonists as well as by molecular cloning.

 c. Ligand Binding

Measurement of specific binding of high affinity radio-labelled ligand to cellular fragments (usually membranes) in vitro, and its displacement by various selective agonists/antagonists is used to delineate receptor subtypes. Multiple 5HT receptors were distinguished by this approach. Autoradiography has helped in mapping distribution of receptor subtypes in the brain and other organs.

d. Transducer Pathway

Receptor subtypes may be distinguished by the mechanism through which their activation is linked to the response, e.g. M cholinergic receptor acts through Gproteins, while N cholinergic receptor gates influx of Na+ ions; α adrenergic receptor acts via IP₃DAG pathway and by decreasing cAMP, while β adrenergic receptor increases cAMP; GABA_A receptor is a ligand gated Cl– channel, while GABA_B receptor increases K+ conductance through a Gprotein.

e. Molecular cloning

The receptor protein is cloned and its detailed amino acid sequence as well as three dimentional structure is worked out. Subtypes are designated on the basis of sequence homology. This approach has in the recent years resulted in a flood of receptor subtypes and several isoforms (which do not differ in ligand selectivity) of each subtype. The functional significance of many of these subtypes/ isoforms is dubious. Even receptors without known ligands (orphan receptors) have been described.

Application of so many approaches has thrown up several detailed, confusing and often conflicting classifications of receptors. However, a consensus receptor classification is now decided on a continuing basis by an expert group of the International Union of Pharmacological Sciences (IUPHAR).

f. Silent Receptors

These are sites which bind specific drugs but no pharmacological response is elicited. They are better called drug acceptors or sites of loss, e.g. plasma proteins which have binding sites for many drugs. To avoid confusion, the term receptor should be restricted to those regulatory binding sites which are capable of generating a response.