Atropine: Structure Activity Relationship

Today we will explore the structure activity relationship of atropine. Definitely more chemistry here,  please revise the previous two articles before continuing. 

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Previously, we learnt that atropine is a tropane alkaloid which is an ester of tropanol alcohol and tropic acid. It acts on the muscarinic acetylcholine receptors (mAChRs) found in the central nervous system, as well as parasympathetic division of the peripheral nervous system. The mAChR is a G-protein coupled receptor which relies on secondary messengers to elicit its action. To be precise, atropine is a reversible competitive antagonist of mAChR. This means that atropine readily competes with the natural (endogenous) neurotransmitter acetylcholine and prevents it from activating mAChRs. In analogy, atropine is a naughty kid who sticks the wrong key into your door every time you wish to unlock it. Given enough naughty kids, you may not be able to unlock your door anymore!

On a molecular level, receptor antagonist binds to the same receptor as endogenous neurotransmitter, but it does not elicit any response. Given atropine and acetylcholine together, atropine of increasing concentration will diminish and eventually abolish the innate activity of acetylcholine. However, if we increase the concentration of acetylcholine, it will  displace atropine from mAChR, which resumes action. This is called reversible competitive antagonism. On a biological level, the antagonism of mAChR will diminish parasympathetic control. This causes the sympathetic division to go into overdrive, giving the impression that atropine does the opposite of acetylcholine. Think about it this way, atropine deactivates the decelerate paddle of a car, this disables the car to slow down. It gradually accelerates out of control, but atropine never affects the accelerate paddle.

That said, atropine must bind to mAChRs, but unlike ACh, atropine induces a different structural change such that mAChR does not become activated (no downstream signaling, no second messenger). That means the two chemical molecules are very similar, but not entirely so. Atropine has to do whatever acetylcholine does in receptor binding, but it has extra chemical substituents. Recall that drug-receptor binding isn’t caused by chemical reaction, instead attraction of certain atoms to one another. These are inter-molecular attractions and they may or may not be permanent. The latter applies to ACh and atropine, hence their reversible effects. If drug binding is not reversible, the outcome can be disastrous.

There are a few types of inter-molecular attractions which are relevant to us. First, there is electrostatic attraction of  positively charged atom to negatively charged atom (+ --- -). Secondly, hydrogen-bonding like those in water (H₂O---H₂O) bonds H to O, N and F atoms. Thirdly, atoms or molecules can attract each other between regions of poor and rich electron density (dipole), a force called Van Der Waals attraction. Finally, there is pi-stacking attraction, where aromatic rings tend to attract each other.
 
Pharmacologists study the structure activity relationship (SAR) of a drug by removing or adding different chemical substituents, then comparing the biological effect with endogenous neurotransmitter or drug standards. Chemical structures which are crucial to receptor binding and activity can therefore be deduced. The advent of physics allows scientists to crystalise proteins including mAChRs together with a drug, and then solve the entire structure using X-ray (X-ray crystallography). This enables visualisation the inter-molecular attraction within drug-receptor complex. Super computers are also employed to calculate and model different shapes of drugs-receptor complexes. Understanding of SAR is vital to the development of more effective and safer drugs.

Figure 1: Structure activity relationship of atropine in relation to MAChR.

Examine Figure 1 carefully, it shows the SAR of ACh and atropine. Notice that both of ACh and atropine are esters (blue). They also have a nitrogen atom (red), and two or three carbon atoms that bridge the nitrogen to ester group (red bolded bonds). In human body, the nitrogen atom of atropine behaves as an alkali, it will react with acids to produce a positively charged ion, called ammonium (NH+). ACh on the other hand carries a permanent positive charge on its nitrogen atom called trimethylammonium (NMe3+). Atropine also has additional aromatic ring and a branched alcohol (OH group) in its tropic acid component. Knowing these, we can deduce the binding requirements of mAChR. First, there is electrostatic attraction between the positive ammonium to negatively charged protein (NH+ ------ -OOC). Two or three carbon bonds away, there is hydrogen bonding between ester (carbonyl oxygen) of atropine and ACh with amino-hydrogen atoms of mAChR (NH₂ ---- O-C=O---- H₂N). The two to three carbon distance is crucial because any shorter or longer would not allow inter-molecular attraction. Similarly, if there is no positive charge on nitrogen atom, or a lack of an ester (carbonyl) group, mAChR binding would fail. In fact, the binding pockets of mAChR is a 'tight-fit' because ACh itself is a small molecule.

Unlike ACh, atropine has an aromatic ring and branched alcohol in its tropic acid fragment. These confer huge antagonistic activity, hence inferring extra inter-molecular interactions with mAChR. First, there is pi-stacking interaction between the aromatic ring of atropine with aromatic amino acids located below the ACh binding site of mAChR. Secondly, hydrogen bonding occurs between branched OH group with carbonyl groups of amino acids. The extra intermolecular attractions induce a different conformational change such that mAChR is not activated. Remember that intermolecular interactions are not permanent? Given enough time, both atropine and ACh will dissociate (unbound) from mAChR all on their own. That’s why they can compete with each other. Nonetheless, atropine and scopolamine have high affinity to mAChR, they are very potent. The two still remain as gold standard anti-muscarinic agents.

Knowing the SAR of atropine allows us to do more. We can alter the structure of atropine, even removing the tropane ring all together, but still develop analogs which are anti-muscarinic. We can make analogs with permanently positive nitrogen atom, which do not cross the blood-brain barrier. This reduces central nervous system side effects like sedation and delirium. Many anti-muscarinic drugs are used in modern medicine to treat motion sickness, reduce secretions and urinary incontinence, dilate pupils for examination (mydriatic), alleviate asthma by dilating airway, inhibit spasm, and control the symptoms of Parkinson’s disease. Atropine is used to speed the heart in an event of abnormally low heart rate (bradycardia, medical emergency). It is also used to counteract the cholinergic effects of neostigmine, a paralytic agent commonly used in general anaesthesia. Similarly, atropine is an antidote to pesticides or even nerve gas poisoning, which causes sympathetic crisis. The toxic effects of an agonist (nerve gas) and antagonist (atropine) cancel out each other!

The infamous scopolamine, AKA the ‘Devil’s breath’ is reportedly used as truth serum to extract information out of people and then have their memories ‘erased’. I remain skeptical of these accounts, but there is a grain of truth. Scopolamine is an oily liquid, its salts are hygroscopic solids (readily absorbs moisture from air). The pure drug cannot be blown into people's face as a power, it probably has to be adsorbed onto some inert material to achieve devious goals. On a brighter side, scopolamine is used as transdermal patch placed behind the ear to treat motion sickness. Figure 2 lists some common anti-muscarinic drugs used in modern medicine, you can examine their structures to determine the SAR of atropine. Remarkably, there is an ultra-potent, but less toxic analog of atropine called 3-quinuclidinyl benzilate, which was developed by the US military (designated as agent BZ) as a psychosomatic chemical weapon! It is aimed to incapacitate a lot of people via anticholinergic toxidrome. Luckily, BZ is outlawed by the chemical weapons convention, let’s hope it stays that way.

Figure 2: Examples of antimuscarinic drugs.

Finally, here's the answer to last week's homework. The M of muscarinic acetylcholine receptor stands for muscarine. Muscarine is an alkaloid toxin which is isolated from hallucinogenic mushrooms such as Amanita muscaria. It activates mAChR so effectively (agonist), scientists named the receptor after muscarine (similar to nicotine and nAChR)! Examine the structure of muscarine in Figure 1 and compare it to the SAR of ACh, you’ll get better answer.

Here's something interesting, perhaps enforced by urban legends and movies. When a patient is about to die,  doctors can give shots which ‘strengthen’ the heart and prevent death-rattle, enabling final words and farewell. That’s likely atropine! What an epiphany, atropine…Atropos… Cut the thread of life? In our journey to understand this most canonical poison, we have seen how humans have transformed poison into medicine, herbs into pills and superstition into science.