22. The concept of antagonism

Effect of functional antagonists - dose response curve

It causes dose dependent but limited antagonism because receptors (for both agonists) can be saturated - this explains why the potency and effectiveness of adrenaline depends on the extent of ASM (airway smooth muscle) contraction induced by histamine (severity of anaphylactic reaction) adrenaline will induce relaxed smooth muscle of airways and in the presence of increasing concentrations of the broncho-constrictor histamine, the bronchodilatory effect of adrenaline is diminished

3. Irreversible receptor antagonist - example phenoxybenzamine (assessible)

PBZ forms a covalent bond with the agonist binding site on the alpha adrenoceptor (and some other receptors) *S-TM3 refers to a cysteine (sulphur containing) aa residue within transmembrane 3 of the G protein coupled receptor (adrenoceptors are GPCRs) it can bind to particular amino acids within the binding site of the receptor and form this adduct - so there is covalent bond formed between the amino acid (of the binding site of the receptor) and phenoxybenzamine - in the binding site of the receptor we have a molecule (the antagonist) that can produce steric hinderance that prevents agonist binding to the binding site.

5. Inverse receptor agonists

Receptor with high constitutive activity means that many of those receptors in active conformation - effect is seen in absence of agonist The inverse agonist selectively binds to & stabilises inactive conformation of R - equilibrium shifted away from active conformation - causes reduced effect it drives equilibirum to the left so we have less receptors in the active conformation - inhibit constitutive receptor activity because they stabilise the receptor in the inactive conformation

Effects of competitive receptor antagonists shown in the dose response curve

the thing in green is an agonist dose response curve to histamine - get increased amounts of agonist, we will get greater responses What we see in the presence of competitive antagonist, because the antagonist and the agonist bind reversibly to the same site When we increase the concentration of antagonist the dose response curve to the agonist gets shifted to the right - the dose response curve to the agonist gets shifted to the right, you need to use higher and higher concentrations of the agonist to be able to compete for that same receptor (to overcome effects of antagonist)(there can be an unlimited shift to the right due to the fact that it's COMPETITIVE antagonist, you can always increase the dose of agonist and antagonist (not clinically possible though)) a characteristic feature of competitive antagonist is that although they can produce unlimited right shifts, there is no reduction of the maximum response to the receptor

What are negative allosteric modulator (NAM)

they bind to the allosteric binding site and changes the agonist binding site, REDUCING AFFINITY of the agonist for its binding site (there are also ones that decrease efficacy) - because they lower agonist affinity so you need an increased concentration of agonist to bind to and activate receptor - when the NAM leaves the receptor, the agonist binding site returns to normal.

1. Chemical antagonist - mode of action

- bind directly with SOLUBLE AGONISTS to reduce agonist binding and receptor activation -> inhibits agonist-induced effects - include 'biologicals' (drugs that are bigger than 500 Da) - e.g. monoclonal antibodies (mAbs) and soluble receptors which bind to (& neutralise the effects of protein agonists involved in disease processes) e.g. infliximab

4. Allosteric antagonists

- bind to a site on the receptor that is distinct from the binding site for the agonist. - produce a conformational change in the receptor which , in turn, reduces the affinity and or efficacy of the agonist - they DON'T compete with the agonist for the agonist binding site *the interaction takes place through the receptor - they are also called negative allosteric modulators (NAMs)

2. Competitive receptor antagonists

- compete with agonists for the same binding site on the receptor - common approach to block the effects of endogenous agonists (neurotransmitters, hormones, mediators) Neurotransmitters such as acetylcholine and noradrenaline Hormones such as adrenaline, angiotensin, oestradiol, testosterone, etc. Mediators such as histamine, serotonin (5-HT), prostaglandins e.g. histamine is released and antihistamine such as citerizine can binds to the same binding site on the receptor that histamine does.

The effect of irreversible receptor antagonists

- effectively causes a reduction in the number of functional receptors (RT) than can be bound and activated by the agonist - it reduces the max agonist induced effect

3. Irreversble receptor antagonists

- form an irreversible (or very slowly reversible) bond with the agonist binding site, preventing agonist binding to the receptor e.g. phenoxybenzamine

What other drug is often used instead of PBZ for COPD and asthma

- however, some 'slowly reversible' antagonists are used clinically e.g. tiotropium* is used in COPD/asthma, has a long duration of action ‐ allows once daily dosing & improves patient adherence binds not irrervesibly (not a covalent bond to the muscarinic receptor) - gets trapped in the binding site of the receptor - so it is released from the binding site very slowly - as long as it occupies the binding site of the receptor that receptor can't be activated by the agonist such as acetylcholine works well because it binds extensively to receptor - long duration of action

1. Chemical antagonists - example infliximab (definitely assessible)

- it is a monoclonal antibody and is similar to the drug etanercept (soluble receptor that binds the soluble agonists) - both bind to and NEUTRALISE the pro-inflammatory mediator TNF-alpha, preventing its activation of TNF receptors - treatment for RHEUMATOID ARTHRITIS monoclonal antibody that will neutralise Tumour necrosis factor and therefore it won't activate TNF factors - agents such as infliximab are effective because it can mop up the TNF alpha which is playing a role in this disease

Explain constitutive receptor activity

- seems that unbound receptors exist in many (at least 2) states, inactive and active and they're in equilibrium - for most receptors, in the absence of agonist, the equilibrium favours the inactive states - these receptors have low constitutive activity the conformation of the receptor is able to activate downstream signalling pathways to produce an effect - most receptors are only activated in the process of an agonist and there are only few receptors that do this ... so, little or no effect is observed in absence of agonist

Why is there no reduction in max response/effect with competitive antagonists in the dose response curve

- that's because if you have high enough of the agonist you can still fully occupy the amount of receptors and get the full effect. the fraction of receptors occupied by the agonist (y) in the presence of antagonist B is in the picture - this equation is really similar to the equation of the fraction of target bound by drug. [B] is the concentration of antagonist and Kb is the equilibrium dissociation constant of the antagonist for receptor - as [antagonist] goes up, y will go down and effect will also go down.

Explaining inverse agonism

- the inverse agonist selectively binds to and dose dependently stabilises the inactive conformation (Ri) of the receptor R - reduces effect whereas the conventional agonist selectively binds to and dose dependently stabilises the active conformation of the receptor - increases effect In the graph: it shows constitutively activated receptors because you can see there are some activated (100)even when there are no drugs

6. Inhibitors of intracellular signalling

- typically have widespread effects (and side effects) as many receptors and cell types SHARE intracellular signalling pathways - BUT some cells uniquely express signalling molecules that are essential for that cell's function and these can be specifically targeted.

Different types of antagonist

1. chemical antagonist 2. competitive receptor antagonist 3. Irreversible receptor antagonist 4. negative allosteric modulator 5. inverse agonist 6. signalling pathway inhibitor 7. functional (physiological) antagonist **all of them work by binding to a target

The action of competitive receptor antagonists

Agonist binding usually induces a favorable conformational change in the binding site that is transmitted through to other regions of the receptor (activation) But in this case, when the agonist binds, activated receptor binds to & stimulates downstream signaling pathways to induce a DELETERIOUS change in cellular activity. Competitive Antagonist such as antihistamine, e.g. citerizine Antagonist binds to the same binding site, without activating R (no efficacy). This prevents the agonist from binding to, and activating the receptor, & producing the deleterious effect. ... when the antagonist leaves the receptor (binding is reversible), the binding site it is free to be bound by agonist (or antagonist) molecules.

Example of inhibitors of intracellular signalling - Imatinib (assessible)

BCR-Abl (a tyrosine kinase) is uniquely expressed by leukemic cells and is essential for their survival - specifically inhibited by imatinib - inhibiting these tyrosine kinase we can try and reduce the activity of these leukaemic cells. e.g. CML (chronic myeloid leukaemia)

Effects of competitive receptor antagonists

Because the agonist and competitive antagonist bind reversibly to the same binding site, increasing the concentration of the antagonist will produce increasingly larger rightward shifts of the agonist dose-effect curve (theoretically unlimited), with no reduction in max response

Why do we need to inhibit agonist induced effects.

In many clinical conditions, endogenous agonists (neurotransmitters, mediators, hormones) produce unwanted effects*, and inhibiting these receptor‐mediated effects may be beneficial to the patient. there are many ways to inhibit these effects. * e.g. in people with hay fever, histamine released from mast cells activates histamine receptors to cause itch, secretions and nasal congestion. so we can use antihistamines to counteract this

What is a benefit of having this ceiling effect of allosteric antagonism

The fact that there is limited antagonism means there are less side effects and less likely to cause excessive blokade than other antagonists.

The action of irreversible receptor antagonists

The irreversible antagonist covalently binds to the same binding site, without activating R, preventing the agonist from binding to and activating, the receptor - as the antagonist covalently binds to the receptor it does not leave the receptor, and the receptor cannot be activated by the agonist. - it causes a reduction in the number of functional receptors that can be activated by the agonst

7. Functional (physiological) antagonists

These are agonists that activate different receptors to produce opposing effects within the same cell e.g. during an anaphylactic reaction

The agonist curve of allosteric antagonist (dose response curve)

We have to use higher concentration of the agonist in presence of these NAMS - but the effect of the NAM is limited - the NAM only produce a set amount of shift of the agonist dose response curve - that's because the allosteric site can be saturated - and adding further NAM won't produce any further effect on the receptor - so the effect by these NAM is limited - this ceiling effect (the fact it's limited) it's safer and there isn't excessive blockade - because if you use higher agonist you can overcome it

Why is phenoxybenzamine rarely used clinically

because PBZ lacks selectivity for alpha‐ARs it binds to transport systems and other receptors so it's a 'dirty drug' - too many targets - rarely used clinically

Explain why antagonism is limited in allosteric antagonists

because allosteric site is saturated (i.e. fully occupied) at high [NAM] so it can reach a point where giving more dose doesn't do anything - for NAMs that reduce agonist affinity, the maximum rightward shift of the AGONIST curve is limited (unlike the unlimited shift of a competitive antagonist) there is this ceiling effect

Competition between agonist and antagonist

binds to a number of the receptors and prevents it from being bound and activated by the agonist. - if we increase concentration of the antagonist, we get more bound by antagonist. and so now there is a reduction in agonist induced effect. - if we increase the concentration of the antagonist even further - more of receptors bound by antognoist - even further reduction for the agonist induced effect. - this is a competitive interaction - so if there is a high enough concentration of the agonist then the agonist induced effect can be restored because the agonist can outcompete the antagonist for that binding site on the receptor

Allosteric antagonism decreases agonist affinity (diagram)

creates receptor that has low affinity to the agonist - the low affinity leads to less binding of the agonist and so there is less receptor activation and less magnitude of response the effects of antagonist can be overcome if increase the concentration of the agonist - drives the binding of the agonist to the receptor - many receptors can still be activated

Example of a Negative allosteric modulator (allosteric antagonism) - maraviroc (assessed)

decreases affinity of HIV 1 for CCR5 and decreases viral entry - CCR5 is the receptor used by the virus to enter cells in order for HIV 1 to enter CD4 T cells, a part of it need to bind to chemokine receptor CCR5 (G protein coupled receptor) Maraviroc binds to allosteric site and change the receptor and HIV can't bind as well (less affinity) and there is less entry of the virus into the cell - there are NAMS that decrease affinity, but there are NAMs that decrease agonist efficacy (cause conformation change that is more to do with the downstreamm signalling pathways

The term functional antagonist is relative - explain

i.e. adrenaline is a functional antagonist of histamine‐induced contractions, and histamine is a functional antagonist of adrenaline‐induced relaxations (both are agonists!)

What are positive allosteric modulators?

positive allosteric modulators (PAMs) increase affinity at the agonist binding site - PAMs increase the affinity for receptors for its endogenous agonist e.g. benzodiazepines increase affinity at the GABA receptor

What are constitutively active receptors

receptors that are active in the absence of conventional agonist, there are constitutively active receptors (Conventional agonists - receptor activation depends on agonist binding) If the concentration of these constitutively active receptors is sufficiently high, then an effect can be observed.

What is the orthosteric binding site

same as the agonist binding site

7. Functional antagonists - example in an anaphylactic reaction

some people suffer from anaphylactic reactions to certain things that they're really allergic to, exposure to these agents will cause anaphylactic reactions - they will release histamins - airway smooth muscle cell receptors will lead to airway narrowing and reduce breathing. (the smooth muscle contracts) - the administration of adrenaline by epipen and what adrenaline does also activate a receptor on airway smooth muscle but it is a different receptor (B2 adrenoreceptor that does something else), and cause cAMP dependent relaxation and the airways will open and make breathing easier for these people. - these receptors mediate different effects within the same cell. SO ALTHOUGH adrenaline is agonist, it functionally antagonist the effect from the histamine reaction.

Negative allosteric modulators cause dose-dependent antagonism, what does this mean?

the amount of antagonism it causes is dependent on the concentration of the antagonist (the dose of the antagonist) - BUT antagonism is limited