adrenergic receptors

Molecule of the Day - Adrenaline/Epinephrine

Adrenaline (C9H13NO3), also known as epinephrine, is a naturally-occurring hormone and neurotransmitter found in our body. Along with noradrenaline, it is produced by the adrenal medulla, which is situated above the kidneys.

As a hormone, adrenaline stimulates the sympathetic nervous system, and is partly responsible for the “fight-or-flight” response.

It binds to adrenergic receptors, which are found in almost all tissues, inducing the breakdown of glycogen into glucose (see below), glycolysis, and also inhibits glycogen synthesis as well as insulin secretion. This results in a surge in glucose availability, providing a burst of energy needed to escape any danger. 

Adrenaline also promotes vasoconstriction, the narrowing of blood vessels, as well as an increase in heart rate to raise the amount of blood being pumped throughout the body. This causes more oxygenated blood to reach the body at a faster rate, enabling cells to carry out respiration to produce more energy as well.

An interesting study revealed that adrenaline is associated with fear. A 1999 study showed that subjects injected with adrenaline experienced greater feelings of fear upon watching horror films. They also expressed greater negative emotions than the control group.

In nature, adrenaline is biosynthesised from phenylalanine through multiple enzyme-catalysed reactions:

On the other hand, adrenaline can be synthesised from resorcinol and 2-chloroethanoyl chloride in the lab:

Adrenergic Receptors (VERRA IMPORTANT, SASSENACHS)

(Sorry, I’ve been re-watching Outlander- but, onwards)

These receptors have their own specific locations and effects

  • alpha-1 (a1)
    -Mostly found in the blood vessel’s endothelium and most sympathetic target organs (NOT THE HEART)
    -Responsible for: peripheral vasoconstriction (to up BP), constrict visceral sphincters and dilate pupils
  • alpha-2 (a2)
    -Located in your pancreas and platelets
    -Inhibits insulin secretion of the pancreas (to up blood glucose) and promotes blood clotting
  • beta-1 (b1)
    -Located in the heart & kidneys
    -This increases HR as well as the contractility of the heart’s muscles (which, in turn, increases SV and BP)
    -Makes the kidney release renin (increases BP also)
  • beta-2 (b2)
    -Located in the lungs and (deep) blood vessels
    -relaxes the smooth muscles in the lungs to increase bronchodilation, which, in turn, increases O2
    -arteries to the liver and skeletal muscles vasodilate to help increase blood glucose
    -Does these 3 things: glycogenolysis, gluconeogenesis and lipolysis to also increase blood glucose 
  • beta-3 (b3)
    -Located in the adipose tissue
    -Stimulates lipolysis (^blood glucose)
just some quick ADHD facts

- Despite being the most commonly studied and diagnosed psychiatric disorder in children and adolescents, the cause is unknown in the majority of cases

- It is classified as neurodevelopmental psychiatric disorder

- The World Health Organization estimated that it affected about 39 million people as of 2013

- Symptoms of hyperactivity tend to go away with age and turn into “inner restlessness” in teens and adults with ADHD

- ADHD is divided into three subtypes: predominantly inattentive, predominantly hyperactive-impulsive, and combined type

- the disorder is often inherited from one’s parents with genetics determining about 75% of cases

- Current models of ADHD suggest that it is associated with functional impairments in some of the brain’s neurotransmitter systems, particularly those involving dopamine and norepinephrine

- The dopamine pathways and norepinephrine pathways which project to the prefrontal cortex and striatum are directly responsible for modulating executive function (cognitive control of behavior), motivation, reward perception, and motor function; these pathways are known to play a central role in the pathophysiology of ADHD

- ADHD psychostimulants possess treatment efficacy because they increase neurotransmitter activity in these systems

- Symptoms of ADHD such as low mood and poor self-image, mood swings, and irritability can be confused with dysthymia, cyclothymia or bipolar disorder as well as with borderline personality disorder

- The management of ADHD typically involves counseling or medications either alone or in combination. While treatment may improve long-term outcomes, it does not get rid of negative outcomes entirely. Medications used include stimulants, atomoxetine, alpha-2 adrenergic receptor agonists, and sometimes antidepressants

- In children ADHD occurs with other disorders about ⅔ of the time. Some commonly associated conditions include: Learning disabilities, Tourette syndrome, Oppositional defiant disorder (ODD) and conduct disorder (CD), Mood disorders (especially bipolar disorder and major depressive disorder), Anxiety disorders, Obsessive-compulsive disorder (OCD), Substance use disorders (this is most commonly seen with alcohol or cannabis), Restless legs syndrome and sleep disorders (problems with sleep initiation are common among individuals with ADHD but often they will be deep sleepers and have significant difficulty getting up in the morning)

- It is estimated that between 2–5% of adults have ADHD. Most adults remain untreated

-  Adults with ADHD may start relationships impulsively, display sensation-seeking behavior, and be short-tempered. Addictive behavior such as substance abuse and gambling are common

- Those affected are likely to develop coping mechanisms as they mature, thus compensating for their previous symptoms

(x)

4

Scenes from the operating room - Part 1

I know a lot of my readers are interested in my profession and have asked me many questions regarding how I got to where I am today. I thought it might be fun to make a post about a few of the things I see everyday and the tools commonly used in anesthesia!

1. Laryngoscope blade and endotracheal tube - if there is any one tool that BEST signifies anesthesia it is the laryngoscope. This tool is ESSENTIAL to an anesthetists’ practice and comes in many different variations. It is basically a metal ‘blade’ attached to a handle with batteries that causes a little light bulb at the end of the blade to illuminate. Once a patient is fully asleep we use the laryngoscope to sweep their tongue out of the way, lift up their epiglottis, and view the vocal cords. If you’re lucky you’ll get a nice clean view of what looks like a white upside down ‘V’. In between the V is where you place the endotracheal tube (ETT). The vocal cords lead to the trachea, and the trachea branches off into our two lungs. The ETT sits inside the trachea and then a little balloon is blown up at the tip to form a seal. This is then how we deliver oxygen, air, nitrous, and anesthetic gases to the patients lungs.

2. Drugs! (Of course) - you learn more about the way drugs work on the human body in anesthesia school than almost anything else. It is MUCH more than, ‘this drug is for blood pressure’. Pictured are only a very MINUTE sample of the many drugs used in anesthesia on a day to day basis. The drugs up top happen to be what we refer to as ‘pressors’ and the red syringe is a drug that is synonymous with anesthesia - succinylcholine. Pressors, or vasopressors, are drugs we use to manipulate (increase) blood pressure. Some examples are Phenylephrine, Ephedrine, and Epinephrine. Phenylephrine causes constriction of our blood vessels by acting on the alpha-adrenergic receptors in the vessel walls. The constriction of the vessels causes an increase in blood pressure. Succinylcholine is a depolarizing muscle relaxant. I would say it is most frequently used when a breathing tube needs to be placed in someone who has a perceived difficult airway. Suxx (common nickname) is used because it is VERY short acting. It lasts about 1-2 minutes and then because of it’s quick metabolism it is gone. It is also used in emergencies/traumas when an endo tracheal tube needs to be placed and the patient has to be paralyzed. This drug you will know IN AND OUT before graduating. ….more to come!
Molecule of the Day: Methylphenidate

Methylphenidate (C14H19NO2), also known as Ritalin, is a white powder that is slightly soluble in water. It is commonly used to treat ADD, ADHD, and narcolepsy.

Methylphenidate inhibits dopamine and norepinephrine transporter proteins, thus preventing dopamine and norepinephrine in the synaptic cleft from being reuptaken into the presynaptic knob. The resultant higher concentration of these substances in the synapse causes the receptors on the postsynaptic knob to be stimulated at a greater frequency, thus achieving greater synaptic transmission. This produces a psychostimulant effect, allowing it to be used in the treatment of ADHD.

In small amounts, methylphenidate has also been shown to enhance memory and control, caused by the activation of dopamine and adrenergic receptors. However, in large doses, it can have the opposite effect.

It has few side effects, which include loss of appetite, nausea, and insomnia. However, like many strong dopamine reuptake inhibitors, it can result in dependence, and is often seen as a gateway drug. 

Methylphenidate is industrially synthesised through a multi-step pathway from 2-bromopyridine and benzyl cyanide.

Requested by @zenbra

The Gland that has got a Secrete Secret

This article will focus on one of the more important glads of the human body; the thyroid. This article will focus on the anatomy and physiology, biochemistry and clinical aspects of the thyroid, hopefully giving our readers a better understanding of this organ.

The Thyroid

Situated on the ventral side of the neck, the thyroid gland is composed of two lobes: right and left that are situated anterolaterally to the trachea. It normally weighs 15 to 20 grams in adults (1), but despite its small size, it is responsible for producing two important bodily hormones.

Follicular cells in the thyroid gland mainly produce the prohormone thyroxine (T4), and a smaller amount of the active hormone, triiodothyronine (T3). Most T4 is converted to T3 in other tissues by thyroxine-specific deiodinase enzymes, activating it when it reaches its target site.


Figure 1. Showing the molecular structure of T3 (left) and T4 (right).

T3 and T4 from thyroid gland to target tissue

Synthesised T3 and T4 diffuse out of follicular cells and enter a blood vessel. Almost all secreted T3 and T4 circulating the bloodstream are bound to proteins; the major binding protein being thyroxine-binding globulin (TBG). A TBG-blood test(2) may be used to diagnose problems with the thyroid such as hypothyroidism, a clinical condition where insufficient production of thyroid hormone occurs.

Free T3 and T4 enter cells by active transport, an energy-dependent transport method. As discussed above, organ tissues with high blood flow (such as liver, skeletal muscles and kidney) possess enzyme deiodinase and catalyses most of the conversion of T3 and T4. Other tissues with low local T3 generation may depend on these tissues to obtain sufficient levels of T3.

At the physiological level

The most important role of thyroid hormones are to control basal metabolic rate (BMR). BMR refers to the basal rate of oxygen consumption and heat production. Normally, mitochondria generate energy by oxidative phosphorylation. During this process, the energy from protons (H+) moving down a proton gradient is used to generate ATP (the energy currency of the cell). This is a similar process to the momentum of water being harnessed by water wheels in old mills.However, a special type of protein, called the uncoupling protein (UCP), is found exclusively in brown adipose tissue (BAT). Mitochondria in these cells can provide an alternative pathway for protons to travel back inside the mitochondria, down their proton gradient. This alternative pathway results in no ATP production with the energy being dissipated as heat.(4)

In the cardiovascular system, thyroid hormones increase the gene expression for β1-adrenergic receptors in cardiac muscle cells and increase the responsiveness of these cells towards β adrenergic activity. The overall effect increases the force of myocardium contraction (positive inotropy) and rate of heart muscle contraction (positive chronotropy), increasing cardiac output and blood vessel dilation in the skin, muscle and heart. The hormone increases tissue sensitivity to beta adrenergic hormones, increasing the heart rate and force of contraction.

Thyroxine hormone also affects the other systems such as the respiratory system, skeletal system, reproduction and nervous system. However, the most important functions of thyroid hormone are the regulation of BMR, maturation and development of nervous system and increase responsiveness of tissue to adrenergic activity.

Mechanism of thyroid hormone

The steps below correspond to the numbers in Figure 2.

1) T3 diffuses into the cytosol and subsequently into the nucleus (8).
2)Thyroid hormone receptor (TR) is located in the nucleus prebound to DNA. TR usually dimerises with a retinoid X receptor (RXR) and this dimer recognises and binds at a specific site on DNA known as the Thyroid Response Element (TRE). TH binds to TR leading to the dissociation of co-repressors (Figure 2).
3)At the same time, recruitment of co-activators (Figure 2) occurs.
4)The TRE mentioned in step 2 is a segment of DNA known as the refulatory sequence, a segment of DNA that increases or decreases the expression of specific genes. In this case, when the T3 binds to the TR-RXR dimer, and the TRE may activate or repress the target genes.


Figure 2. Diagram shows a schematic diagram of the general biochemical action of thyroid hormones on the target DNA

Transcription is followed by RNA translation to form hundreds of new intracellular proteins. T3 changes the rate of expression for hundreds of genes and increases or decreases the production of structural and functional proteins which may be the key molecules in different metabolic processes. T4 also performs such function, but is less potent than its counterpart T3.

With such function, one can imagine just how important the level of thyroid hormone is in the regulation of different physiological processes and how this may impact upon health (9)

Regulation Of Thyroid Hormone Production And Secretion

A hormone with such varied functionality has to be regulated to ensure its adequately supplied to targeted organs. Such intricate control has to be performed by the “endocrine master”; the hypothalamus. The hypothalamus releases thyrotropin hormone (TRH), which stimulates the release of thyroid stimulating hormone (TSH) in the closely linked anterior lobe of pituitary gland. TSH is then transported in the blood where it binds to the TSH receptor on the thyroid gland. TSH speeds up the production and release of thyroid hormones, promoting the growth of the gland with the help of some other growth factors.

When thyroid hormone levels are in excess, circulating molecules act on the hypothalamus and pituitary gland to decrease TRH and TSH secretion respectively. The mechanism involved is a negative-feedback control mechanism. When TRH and TSH secretion decrease, so does the production and the secretion of thyroid hormones. The hormones drop until the optimal physiological level whereby the inhibitions on TRH and TSH secretion are lifted (Figure 3).


Figure 3. Image shows the regulation of thyroid hormone by the hypothalamus and pituitary gland in a negative-feedback loop.

Diseases Related To Thyroid Gland

T3 (Figure 1) contains three iodine atoms. The synthesis of thyroid hormones requires an adequate supply of dietary iodine. The recommended dietary allowance (RDA) for iodine in an adult male is 150µg and slightly higher in pregnant women, 220µg (5). Deficiency of this precursor leads to insufficient production of T3 and T4. The consequences of this are low levels of circulating thyroid hormones which cause an increase of TSH secretion from the pituitary gland interfering with the negative feedback. Increased stimulation of TSH increases the activity of the thyroid gland in an attempt to normalize thyroid hormone level (6). Consequently the gland grows larger than the normal size, producing a condition known as a goitre(Figure 4).

A goitre refers to the enlargement of the thyroid gland (Figure 4), this could be due to hypothyroidism or hyperthyroidism. Goitres are more common in population living in mountainous regions, where access to iodine sources such as seafood are restricted. Such dietary deficiency can be prevented by adding small amounts of iodine to table salt.


Figure 4. Image showing a patient presenting a goitre.

Conclusion

Hopefully after reading this article, you’re a little more in the know about the little gland secretlysecreting hormones to help you stay healthy. Next time you’re calorie counting or checking the nutritional content of your food, make sure that you’re getting enough iodine in your diet as it can ensure that you don’t end up with a large number of problems down the line.

Autonomic Nervous System

Ok, so this little pain is going to be your new bestie.  The Autonomic Nervous System (ANS) is a very important regulator for homeostasis. The ANS has two branches: Sympathetic (fight or flight) and parasympathetic (rest & digest). MOST(not kidney) organs have dual innervation.

Sympathetic
- These postganglionic neurons release norepinephrine (NE), epinephrine (Epi) and dopamine (DA) and MOST of these sympathetic receptors are adrenergic (these receptors will get their own post because they are that important)

Parasympathetic
- These bad boys are mostly responsible for the release of acetylcholine (ACh); these receptors are cholinergic (muscarinic)

My UWorld notes- 5

To those who were waiting yesterday for this post, I’m extremely sorry. For some reason I Thought yesterday was sunday -____- but here it is. 

  • reflex tachycardia caused by nitrate can be prevented by administering beta adrenergic blockers w

  • prazosin- selective alpha 1 adrenergic blocking medication used for HTN and BPH

  • hydrochlorothiazide is a weak diuretic

  • phenylephirine is an alpha agonist which is classified as a vasopressor agent. Its used in cases of shock and severe hypotension

  • hydralazine is a direct acting arteriolar dilator . It causes a reflex tachycardia which can also be prevented with administering beta blocker

  • ataxia telangiectasia an autosomal recessive d/o characterized by DNA hypersensitivity to ionizing radiation. Cerebellar atrophy leads to ataxia that occurs in first years of life. . Patients with ataxia telangiectasia also have sever immunodeficiency with repeated sinopulmonary infections. This risk of cancer in these patients in increased significantly b/c of inefficient DNA repair

  • XP is characterized by DNA hypersensitivity to UV radiation causing premature skin agin and increased risk of skin cancer (malignant melanoma and squamous cell carcinoma)

  • Fanconi anemia is caused by hypersensitivity of DN TO CROSS LINKING AGENTS

  • Bloom syndrome is characterized by generalized chromosome instability. Increased susceptibility to neoplasms is present

  • HNPCC d/t to defect in DNA mismatch repair enzymes leading to increased susceptibility to colon cancer

  • caudate nucleus atrophy- huntington dz

  • Lewy bodies- Parkinsonism

  • loss of neurons in substantia nigra- Parkinson’s dz

  • neurofibrillary tangles in neocortex – Alzheimer’s dz

  • double vision while walking down the stairs or while reading the newspaper- palsy of cranial nerve 4 (trochlear nerve)

  • optic nerve (CN2) transmits visual info to brain damage causes loss of vision

  • CN3 occulomotor nerve innervates superior rectus medial rectus inferior rectus and inferior oblique. Which all collectively perform most ye movement. Palsies of this nerve can cause vertical and horizontal diplopia and an enlarged nonreactive pupil

  • abducens nerve CN6 innervates lateral rectus which is responsible for abdcution of eye. Palsy of this nerve can cause horizontal diplopia and inward deviation ( inward deviation)

  • MLF lesion a/w internuclear ophthalmoplegia, which presents with impaired horizontal eye movement and weak adduction of affected eye with simultaneous abduction nystagmus of contralateral eye

  • at low doses atenolol is a selective beta 1 adrenergic antagonist . Beta 1 receptors are found in cardiac tissue and on renal juxtaglomerular cells but not on vascular smooth muscle. The beta 1 receptor is G protein coupled receptor a/w Gs G protein which increases cAMP levels. Blockage of beta receptor therefore means decreased cAMP levels in cardia and renal tissue without affecting cAMP levels in vascular smooth muscle

  • pure red cell aplasia is a rare form of marrow failure characterized by sever hypoplasia of marrow erythroid elements in setting of normal granulopoiesis and thrombopoiesis. Pure red cell aplasia is a/w thymoma lymphocytic leukemias and parvovirus B19 infections

  • deficiency of 21 hydroxylase is MC type of CAH. . These patients present with cortisol and aldosterone deficiency combined with androgen excess.. genitalia of females infants may be masculinized to some degree; male infants however are normal in appx

  • When asked a question regarding DKA know that ph is decreased H2PO4 is increased (it is titratable acid) and also bicarbonate excretion is decreased. This response is overtime meaning these changes are made because of the acidosis that the patient has . Therefore in order to fix metabolic acidosis d/t DKA bicarbonate excretion is decreased and urinary ph is decreased and titratable acid excretion is increased.

  • Musculocutaneous nerve innervates flexor muscles of upper arm and provides sensory innervation to lateral forearm. Musculocutaneous nerve is derived from upper trunk of brachial plexus and can be injured by forceful injuries that cause separation of neck and shoulder. It is derived from C5-C7 ventral rami

  • posterior arm and forearm are both innervated by the branch of the radial nerve which is posterior cutaneous nerve of the arm and posterior cutaneous nevre of the forearm

  • thenar eminence is innervated by recurrent branch of median nerve

  • g6pd deficiency – cant convert glucose 6 phosphate to 6 phosphogluconate . G6Pd requires NadPH as a cofactor to work

  • urine sample turned black= alkaptonuria which is an autosomal recessive d/o in which lack of homogentisic oxidase blocks the metabolism of phenylalanine and tyrosine at the level of homogentisic acid leading to accumulation of homogentisic acid. Turns black because homogentisic acid excreted in urine undergoes oxidation when exposed to oxygen in air

  • alkaptonuria cause ochronosis a blue black pigment evident in ears nose and cheeks

  • conversion of phenylalanine to tyrosine is defective in PKU and usually occurs d/t defect in phenylalanine hydroxylase

  • small percentage of PKU cases are also d/t dihydrobiopterin reductase deficiency

  • branched chain a.a. Are valine isoleucine and leucine

  • blastomyces dermatidis is a dimorphic fungus that is seen in tissue as round yeasts with doubly refractive walls and broad based budding. Endemic to great lakes and ohio and Mississippi river regions, present in soil and rotten organic matter

  • blastomyces mold form (branching hyphae) predominates in environment with average temperature 25-30 degrees Celsius. In the human body it assumes yeast form (single cells)

    • blastomyces in lungs assumes yeast form and induces granulomatous response

    • aspergillus fumigatus causes lung dz in immunocompromised and only has a mold form . It is seen as septate hyphae that branch at 45 degrees (angle)

    • oppotunistic mold with irregular non septate hyphae that branch at wide angles (>90)= mucor and rhizopus

    • starts with just a sinus infection and then you end up getting a rapid infection to the brain because this organism can penetrate the cribriform plate causing frontal lobe abscess – Mucor and rhizopus

    • cryptococcus neoformans can also cause lung dz but in addition causes meningitis in immunocompromised and in contrast to blastomycosis it forms narrow based buds and has thick polysaccharide capsule which stains with india ink

    • histoplasma capsulatum causes lung dz and is also a dimorphic fungus like blastomyces but the yeast form of histoplasma is found intracellularly within macrophages

    • coccidioides immitis is also a dimorphic fungus but is seen as spherules (round encapsulated structures containing many endospores) in tissue sample, barrel shaped arthroconidia a/w dust storms which causes San Joaquin Valley fever (inhalation of dust particles) 

You’re clearly not thinking. My brain is a device that consists of a combination of organic matter, electricity, and chemical compounds. The epinephrine response is not necessarily under my control, but by masking monoamines that have already bound to my adrenergic receptors, I can to some extent bypass the side effects that prevent my higher faculties from operating at the peak of their potential. Which is very clearly necessary right now.“

“Okay, in actual English, what you just said was you panicked, and then you got high.

—  All the Best and Brightest Creatures by wordstrings

anonymous asked:

I recently got diagnosed with ADHD and my mom says she doesn't want me on medication because she's heard that it's bad and it doesn't help. I really don't know what ADHD medication does and my doctor hasn't said anything about it, can you help?

ADHD medications function in a myriad of different ways. Very, very, very generally speaking ADHD medications are designed to alleviate some of the attention and cognitive deficits in ADHD.

Stimulant medications, like methylphenidate (Ritalin, Concerta, Quillivant), dextroamphetamine (Dexedrine) and combination amphetamine salts (Adderall) boost attention and focus by increasing the amount of dopamine between signalling neurons.

Dopamine is a neurotransmitter, and it has an important role in marking stimuli in the world around us as salient. Basically, what that means is that dopamine tells you brain “This thing right here is very, very important. Pay attention to this thing.”

Which is great, because ADHD brains don’t tend to use dopamine very efficiently, which leads to poor focus and attention.

But there are other, non-stimulant medications for ADHD. Many of the nonstimulant work by either boosting the levels or mimicking the actions of norepinephrine (noradrenaline) in the brain.

Norepinephrine, of course is one of the chemicals behind the fight or flight response, but the effect it has on the brain depends a lot on which receptors it binds to. The receptors to which norepinephrine binds are called adrenergic receptor, and they are divided into two types (alpha and beta) and then further into five subtypes. To make things even more complicated, the alpha subtype receptors are then further divided into more sub-subtypes.

But because we’re talking about ADHD, all you need to care about is the  Alpha-2A adrenergic receptor.  This receptor is important because it helps regulate higher cognitive functions, the sort of functions that are often lacking in ADHD, like attention and organisation.

Clonidine (Kapvay) and Guanfacine (Intuniv) are adrenergic agonists, which work to mimic norepinephrine by binding to the Alpha-2A receptors in the prefrontal cortex, and boosting attention and focus.

Atomoxetine (Strattera), on the other hand, is a reuptake inhibitor, which prevents the ‘recycling’ of norepinephrine. This means there’s more norepinephrine between cells, and therefore more norepinephrine binding to those awesome Alpha-2A receptors.

Where things get sticky, and potentially “bad” is in the side effects. So I’ve told you how these medications help with the symptoms of ADHD, but because each of them is a different chemical, with different structures, our bodies all respond to them differently.

And because our bodies are all different, the way I respond to a medication can be vastly different to the way you respond to that medication. It might work brilliantly, or you might get terrible side effects or it might not work for you at all. Part of your doctor’s job is to help you manage the potential side effects

This sounds scary, and risky, I know but it’s worth stressing that this is a risk that is found with any and all medications. Right down to basic things you can buy in the supermarket like aspirin, paracetamol (acetaminophen), or ibuprofen.

- Prue

A review of the development of structure-specific drugs for Beta-­2 adrenergic receptors.

Its time to recycle a paper I wrote for my Pharmcology course last semester. (P.S. Never write a receptor-based drug discovery paper for a clinical aspect-oriented Pharmacology course.)    

Introduction:

 

Since the discovery of the adrenergic system of receptors, there have been numerous attempts to develop drugs that can modulate their effects. The earlier attempts were mostly synthetic approaches based on the structures of endogenous ligands like epinephrine and norepinephrine. Over the years our understanding of the ligand binding sites and the general structure of the receptor has improved considerably and therefore development of drugs for specific receptors is possible now. The x-ray crystallography structure of b­2 adrenergic receptor was resolved recently and this provides a very good insight into the structure of the receptor and its differences from other GPCRs.

 

G-protein coupled receptors (GPCRs):

These are seven transmembrane receptors associated with heterotrimeric guanine nucleotide-binding proteins (G-proteins) that mediate a number of cellular responses to hormones and neurotransmitters. There are more than 800 different G-protein coupled receptors identified so far and a number of them are potential drug targets for a wide range of diseases.

 

Multiple phylogenetic analyses of GPCRs have revealed that they can be commonly divided into five families on the basis of their sequences and structural similarities. GRAFS classification system - Rhodopsin (Family A), Secretin (Family B), Glutamate (Family C), Adhesion (Family D) and Frizzled/Taste2 (Family E).

 

The general structure of GPCRs includes the characteristic seven membrane-spanning a-helical segments connected by alternating 3 intracellular loops and 3 extracellular loops. The ligand binds to the binding site on the extracellular surface resulting in conformational changes in the final intracellular loop. This conformational change allows for the binding of specific G-protein present on the inner surface of the membrane to the intracellular loop followed by the activation of the G-protein (replacement of GDP for GTP). The subunits of the activated G-protein can then trigger a cascade of intracellular signaling pathways that result in the necessary cellular responses. Many GPCRs can activate multiple signaling pathways by coupling with different G-proteins and different ligands.

 

According to the widely accepted, Ternary Complex Model, the receptor exists in two states – R state and R* state. The receptor stays in the R state in the absence of the agonist and in R* state (the activated state) in the presence of the agonist. However, recent studies have shown that a ‘basal activity’ or ‘constitutive activity’ exists even in the absence of a ligand. It is hypothesized that an equilibrium exists between the active state and the inactive state in the absence of a ligand and this equilibrium is shifted to active state (R*) on binding to an agonist. Studies show that mutations at certain positions in the transmembrane segments result in increased basal activity which shows some proof for this hypothesis, although we do not have a complete understanding of these conformations.  

 

Thus according to this modified model, there are four possible types of ligands –

Full Agonists, Partial agonists, Antagonists and Inverse agonists. Full agonists exhibit maximal receptor stimulation whereas partial agonists are unable to elicit full activity. Neutral antagonists have no effect on receptor activation and Inverse agonists decrease the baseline receptor activity or ‘constitutive activity’.

 

Numerous biophysical studies suggest that a specific ligand stablizes a distinct receptor conformation and has distinct efficacies for different signaling pathways. This implies that a ligand can act as an agonist for one signaling pathway and simultaneously act as an antagonist to a different pathway on binding to the same receptor. 

 

GPCRs are extensively targeted for the treatment of numerous diseases and therefore resolving their structures is vital for the development of novel and subtype-specific drugs. Owing to their structural flexibility and instability, only 6 GPCR structures have been resolved so far - b­1, b­2 adrenergic receptors, Adenosine receptor, Rhodopsin receptor, Dopamine (D3) receptor and Chemokine receptor (CXCR4).

 

b­2 adrenergic receptors:

b­2 Adrenergic receptors (b­2-AR) are a part of the Rhodopsin family and are a good example for ligand-binding GPCRs. b­2-AR are located almost throughout the body and are mainly implicated in cardiovascular and bronchial therapy. They are also clinically important in glaucoma treatment, smooth muscle relaxation (especially uterine relaxation, GIT relaxation and ciliary relaxation).

 

b­2-AR interacts with both the stimulatory G-protein (Gs) and the inhibitory G-protein (Gi) and also MAPK. They can also activate MAPK through the b­-Arrestin pathway (Figure 1). The G­­s pathway activates the Adenylyl cyclase, which produces cAMP. The cAMP release results in smooth muscle relaxation and thus has implications in the treatment of asthma, inhibition of uterine contraction in premature labor and in glaucoma. The Gi pathway is regulated by PKA-mediated receptor phosphorylation and occurs only when the Gs pathway is blocked. b­-arrestins are mainly considered to be regulatory proteins that cause receptor desensitization and internalization. But studies show that when certain inverse agonists bind to the receptor, b­-arrestins can cause MAPK activation. So, it was found that individual ligands favor different pathways due to their varying affinities to each conformation of the seven transmembrane helices.

 

b­2-AR-specific drugs are required in order to prevent unwanted side effects especially cardiovascular effects through b­2-ARs. The most commonly used b­2-AR specific drugs are – salbutamol, salmeterol, terbutaline etc. A number of agonists and antagonists for β-adrenergic receptors have been synthesized and tested for almost 50 years, and about 155 adrenergic drugs have been developed. Of these agents about 32 are considered specific to b­2-AR and have been widely used clinically.

 

Crystal structure of b­2 adrenergic receptor:

X-ray crystallography has been an important tool to understand the structures of cellular proteins and receptors. Bovine rhodopsin was the first GPCR structure to be resolved by X-ray crystallography in the year 2000. But due to the low natural abundance and structural flexibility or instability of most GPCRs, no significant progress was made in solving other structures. Ligand-binding GPCRs such as b­2-AR show large number of conformational changes after ligand binding and therefore are very difficult to crystallize. Therefore, using targeted protein-engineering methods, mutants were developed to increase stability and crystallization of b­2-AR. The following modifications were made –

 

·       Intracellular loop 3 (ICL3) is considered to interact with the G-protein after the ligand binds to the receptor. This loop shows large physical movements thereby causing difficulties during crystallization. Therefore, ICL3 loop was truncated and replaced with the T4 Lysosome sequence that could restrict large movements (Figure A).

·       The b­2-AR-T4L mutants were cloned in Sf9 insect cells to improve yields.

·       This mutant receptor was crystallized in the presence of Carazolol, a b­2-AR-specific partial inverse agonist. This ligand favors the inactive conformation and proved to improve stability of the receptor.

 

But the resulting structure may have been compromised due to the limited movement of the seven transmembrane segments. Therefore, another method was developed using a monoclonal antibody (Mab5) that recognizes that native ICL3 (Figure A). The Fab segment of the antibody binds to the ICL3 segment forming the b­2-AR–Fab complex. This did not alter the agonist-induced conformational changes, and ligand-binding affinities of the wild-type b­2-AR. On comparing the two structures, they were found to be almost similar except for the positions of the extracellular loops and the higher basal activity seen in b2-AR-T4L receptors. Therefore, either structure can be used as a template for In-silico screening studies.

 

In 2008, another b2-AR crystal structure was resolved but this time while binding to Timolol (a partial inverse agonist) along with 2 molecules of cholesterol. This structure provided additional details about the binding sites of cholesterol molecules to the GPCR and also its effect on receptor stability when present in the plasma membrane. It could also provide insights into the trafficking mechanisms of b2-AR through cholesterol sequestration.

 

More recently in Jan 2011, the same lab from Stanford has published the structures of an agonist-bound b2-AR in both high affinity (bound to the G-protein) and low affinity (in the absence of G-protein) states. The b2-AR when bound with the Gs protein shows higher agonist affinity. Therefore, to resolve this high-affinity state structure, a camelid antibody fragment called Nanobody (Nb) was used. The Nb showed Gs-like binding properties and stabilized the high-affinity state during crystallization. The crystal structure of the b2-AR-T4L in the presence of an agonist (BI-167107) was obtained when stabilized with Nb80. For resolving the unstable low-affinity structure, an agonist (Procaterol) was covalently bound using a linker. These structures could reveal vital details about the differences in conformation between the antagonist- and agonist-bound GPCR.

 

In silico screening for drugs:

Virtual screening in general involves the assessment of a large database of compounds and the identification of potential ligands for specific target. There are two main types of methodologies – Ligand-based screening and Receptor-based screening.

 

a)    Ligand-based screening involves the identification of new ligands using the 2D structures of previously known ligands. It produces very few hits as only related compounds from the same or similar classes can be obtained. Since only the 2D structures are being considered, therefore, inaccurate hits maybe obtained. Another ligand-based technique is the use of a 3D structure of the pharmacophore as the template through which some of the problems of 2D structure screening can be avoided.

 

b)    Receptor-based screening or Structure-based drug discovery involves the use of computational docking modules to assess a large database against an X-ray crystal structure of the receptor. Most molecular docking studies include the process of assessing the various conformations of the ligand against the rigid target. Based on the binding force calculations and potential potency values, each ligand is scored and ranked. Recently developed docking software like GLIDE, DOCK, AUTODOCK, etc also take into consideration the flexibility of both the ligand and the receptor (template). Thus using these novel modules, better screening of compound databases can be performed.

 

Requirement of Structure-based drug discovery (SBDD):

Currently, all of the agents that are being used clinically were developed using ligand-based analoging techniques and not using the SBDD studies. Structure-based drug discovery techniques could potentially enable us to optimize selectivity, duration of action as well as to combine β2-agonist activity with desirable ancillary actions (e.g. anti-oxidant activity, calcium channel block, etc.). It also provides us with the possibility to design molecules that interact with multiple subtypes but show subtype selectivity. The most important advantage of SBDD is the potential to find newer classes of ligands whose pharmacophore is different from the regular catecholamine motif. These compounds could not be considered as possibilities during earlier studies.

 

Virtual screening results and potential drugs:

So far, a couple of studies have been published that used the Carazolol-bound b2-AR structure for small molecule screening. One study from Stanford University used the DOCK program to screen about 972,608 molecules from the ZINC database and individual ranks were assigned to each ligand. From the top 500, 25 compounds from 4 classes were selected and studied using radioligand-binding experiments. These were then compared with the known adrenergic ligands (8063 molecules) from the WOMBAT database. Most compounds were found to be similar to known adrenergic agents; although, few were novel chemotypes that have not been explored before. But the most interesting result observed from these studies was that all the compounds from the screen were either antagonists or inverse agonists and none were agonists.

 

Similarly in the second study from Lundbeck research, the GLIDE module was used to screen 400,000 compounds from proprietary database and about 4 million compounds from commercial databases. The physically available ones of the top 150 compounds from each database were selected and studied. These included a number of known antagonists such as carvedilol; thus validating the X-ray structure of the receptor and the in-silico screening program.

 

Recent studies in heart failure have shown that although agonists enhance acute effects, chronic use results in decreased signaling owing to desensitization mechanisms. In case of antagonists, they can potentially increase signaling on chronic use but cause severe side effects in acute conditions. In contrast, inverse agonists inhibit basal signaling initially and with chronic use up-regulate the receptors thus can be used as effective therapeutics.

 

The above studies provide a large list of inverse agonists of different affinities, pharmacokinetics and toxicity profiles which can be further studied for developing into drug candidates. It is clear that structure-based drug discovery using X-ray crystal structures are effective in the discovery of novel subtype specific drugs.

 

Problems faced in X-ray structure-based drug discovery:

The X-ray structures provide considerable advantages relative to the rhodopsin-based homology models using which a number of SBDD was being carried out earlier. But we still face a number of problems for precise determination of active ligands. One of the biggest obstacles with SBDD is the static nature of the X-ray crystal structures. It has been observed that the binding pocket of avian β1-AR bound to cyanopindolol and human β2-AR bound to carazolol are identical owing to a high conservation of binding-site contact residues. However, subtype-specific binding affinities can be observed for both β­1- and β­2-AR. These differences are due to the subtype-specific conformational preferences in distant residues, which in turn influence the amino acid spatial positions at the binding site.

 

Thus the static template of the receptor can only provide the data with respect to that particular conformation which is being used as the template. The above virtual screening studies using the carazolol-bound β2-AR crystal structure as a template identified number of new β2-AR ligands showing high affinities; however, most of the compounds exhibited inverse agonistic or antagonistic activity. Agonists could not be obtained from the screen using an inverse-agonist bound receptor template.

 

Conclusion:

The GPCRs are currently the most widely targeted proteins for therapeutics and the X-ray crystal structures of these complex receptors has given the opportunity to study them in extreme detail and potentially develop novel, more potent drugs for existing and newer disease conditions. The primary intent for resolving the X-ray crystal structure of Beta-2 AR was to study and understand the structural differences and also the conformational mechanisms of signal transduction. But these structures have also been proven to be an excellent resource for drug discovery research.

 

Docking studies have revealed that most of the highly ranked ligands are inverse agonists or antagonists when screened against the inactive state of the receptor. The recently published agonist-bound structures may have to be used for obtaining potential potent agonists. A recent study has shown that by virtually modifying the binding site parameters of the inactive state receptor, the screen can also identify agonists and partial agonists. Such improvements allow us to potentially use any one of these structures to discover a wider range of subtype-specific and potent ligands.

 

Other future directions could include the use of dynamic images from other biophysical methods along with the information from static X-ray structures. To study the conformational changes and the rates of interconversion between these states we need to develop other time-dependent biophysical methods. Developments in fluorescence and NMR spectroscopy may help us to understand GPCR dynamics. Using such novel methods, more effective virtual drug discovery efforts can be undertaken and potentially develop novel, potent drugs for less cost and in less time.

 

References:

 

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