Identification of the target and its structure

Before we can make a drug, it is necessary to know something about the disease to be treated and the target to be targeted. A target is in the vast majority of cases a protein and knowledge about proteins and their composition is of course important.

There are many different proteins, transport proteins, structure proteins, enzymes, receptors and more. The most important are enzymes and receptors, which also constitute the vast majority of targets for the drugs we know today.

Enzymes

A chemical equilibrium can set itself quickly or slowly, depending on how reactive the reactants are. If a reaction is very slow and you want to increase the speed, you need to use a catalyst. Enzymes are catalysts in that they increase the speed of reaction without even being consumed during the process. Enzymes cannot cause reactions that would not normally take place to happen, but they can increase the speed of reactions in both directions for reactions that would normally occur. Therefore, an equilibrium will adjust faster. The way enzymes increase the reaction rate is by lowering the activation energy, but enzymes do not change where the equilibrium lies.

An enzyme is a protein, and proteins consist of amino acids linked together by peptide bonds. There are twenty different amino acids in the human body, each of which has a variable group, called the R group. Since enzymes are proteins, enzymes are also folded into a certain three-dimensional structure. Due to the three-dimensional structure, there will be different clefts/holes in the protein and in some of them, the reactant(s) (which for enzymes are called the substrate(s)) fit perfectly in, and in such a cleft, the reaction is catalyzed. The gap where the substrate fits in is called the active site. The active site is very specific. This means that only a few substrates with a very similar structure will fit in. This is because in the active site there are a number of R-groups from the amino acids that can make certain bonds with the substrate. These can be both intermolecular and intramolecular bonds. The intramolecular bonds can be temporary, but can also be permanent (which is often the case for inhibitors, see later). If these bonds are not created, the substrate will naturally not bind to the enzyme.

  • In a solution where a reaction is to take place between two molecules, the two molecules must bump into each other correctly in order to react. Here, the enzyme helps by collecting the two right molecules and placing them correctly in relation to each other. The reaction process is therefore made easier.
  • When a reaction is to take place, some covalent bonds often have to be broken. The enzyme makes the covalent bonds in the substrate weaker as the enzymes pull on the bonds. This makes splitting the substrate into two easier.
  • A reaction sometimes takes place by converting the reactant/substrate into an intermediate product. For this intermediate, some functional groups must sometimes be used that do not come from the reactant/substrate itself. Enzymes can add temporary functional groups to the substrate. This is done by means of. The R groups in the enzyme that can “transfer” their functional group or parts of them to the substrate. A number of reactions then take place so that the enzyme’s functional groups are restored.

In addition, some enzymes may require a different molecule in the active site to function. This can either be a coenzyme (molecule containing carbon), a cofactor (metal ion e.g. copper, zinc or iron) or a prosthetic group (molecule that is constantly bound to the enzyme).

 

Figure 7. Activation energy for a reaction without the use of enzyme and with the use of enzyme. When using enzymes, a lower activation energy is seen and the reaction therefore takes place more easily.

 

Induced fit

In the past, it was thought that a substrate’s binding to the active site worked in the same way as when a key fits into a lock. The way in which a substrate binds to the enzyme was therefore described using the “lock and key” model. However, there was one problem with this model. It could not explain how several similar substrates could fit into the enzyme. According to the model, there would only be one substrate that would be specific enough to fit in, just like a lock can only be opened by one particular key.

Figure 8. The enzyme adapts to the substrate by a conformational change.

 

A slightly different model has now been proposed, which is based on the fact that a substrate does not have the perfect structure in relation to the active site. Instead, the active site adapts to the substrate, and thus several substrates that are similar to each other can fit into the active site. This is called induced fit. When a substrate approaches an empty active site, bonds will form between enzyme and substrate. This causes a conformational change in the enzyme, so that the enzyme closes around the substrate. This means that the enzyme will be more tightly packed around the substrate, so that more of the necessary bonds can be formed. A conformation change thus simply means that the enzyme changes its three-dimensional structure in an area.

Induced fit also has another very important function, namely the exclusion of water. When the enzyme closes together, water will automatically be squeezed out of the active site. This is convenient, as some reactions are not favorable when water is present. This may be due to the fact that water forms hydrogen bonds with the target molecule, so that the target molecule will not be able to form the necessary bonds with the drug.

Inhibitors

It was mentioned in the case story that there are two types of drugs, agonists and antagonists. Inhibitors can be said to be the enzymes’ antagonists. Inhibitors bind to the enzyme’s active site, just like the substrate does, but instead of activating the enzyme, the enzyme’s activity is inhibited when an inhibitor binds. The reaction rate of the enzyme is slowed down because the inhibitors interfere with the binding of substrate to the active site.

There are different types of inhibitors. An inhibitor can bind covalently to the enzyme, so that the enzyme is constantly inhibited. These substances are called irreversible inhibitors. Many harmful substances are irreversible inhibitors, for example nerve gases are irreversible inhibitors. However, there are also many non-harmful substances that are irreversible inhibitors such as Antabuse. This is a drug that inhibits the enzyme alcohol dehydrogenase, which breaks down the alcohol we consume. An inhibitor can also bind in the active site for a short time with non-covalent bonds. These substances are therefore called reversible inhibitors.

Receptors

Receptors are very similar to enzymes in their structure, but are usually located on a cell membrane. Receptors, like enzymes, also contain a cleft to which a molecule binds. The cleft is called the binding site, where in the enzymes it is called the active site. The reason why it cannot be called an active site in the receptors is that there is usually no conversion of a precursor in this area. When a ligand (which is the same for a receptor as a substrate is for an enzyme) binds in the binding site, a conformational change occurs, i.e. an induced fit. This happens in all receptors. What this change of conformation entails, however, is different.

 

 

Figure 9. Illustration of the general mechanism of a G-protein-coupled receptor. When a ligand binds to the receptor, a conformational change occurs in the receptor, so that an active site appears inside the cell so that a G protein (bound to GDP) can bind. When the G-protein is bound to the active site, the G-protein will be split into three parts, where the part bound to GDP will have GDP replaced with GTP. This part of the G protein can now move to another place in the cell, where it will bind to another enzyme, thereby activating this.

 

G-protein coupled receptors

As can be seen from Figure 9, the conformational change from the binding to a ligand from the outside can cause the part of the receptor inside the cell to also change its structure. This creates a new binding site, which is somewhat similar to an active site, as it is able to function as an enzyme. A new ligand (G-protein, bound to GDP) from the cell’s interior can now bind to the active site, thereby splitting the G-protein inside the cell. A part of the G protein can then send a signal on to a membrane-bound enzyme inside the cell, which is then activated. The functions of this enzyme vary greatly depending on the type of enzyme to which the G protein binds. These receptors are called G-protein coupled receptors.

 

Ion channels

Another kind of receptor is the ion channels. These consist of five protein subunits that go all the way through the cell membrane, thereby forming a channel through the membrane. This allows ions to enter and exit the cell. The five subunits are not exactly the same, as in one of them there is a binding site. When a ligand binds to the binding site, the ion channel is activated so that the channel opens. The conformational change in an ion channel takes place by the five subunits pulling away from the center, so that a channel is formed.

A drug that works on an ion channel can also work in two different ways. Either as a blocker or as an opener of the channel. A blocker will make sure that the channel is constantly closed and is therefore an antagonist. An opener will keep the ion channel open all the time and is an agonist.

 

Figure 10. Figuren illustrerer hvordan en iokanal er placeret i en cellemembran og hvordan ionkanalen åbner sig når en ligand bindes til ionkanalen

 

Transport proteins

Transport proteins are the body’s “smugglers”, as they smuggle molecules across cell membranes, as the molecule itself is too polar to cross. A transport protein is therefore hydrophobic/nonpolar on the outside, so that it can sit inside the membrane, but is hydrophilic/polar on the inside, so that polar molecules can be transported into the protein. The transport protein closes around the molecule to be transported. It is then transported through the cell membrane and released on the other side.

 

Figure 11. Illustrates how a drug can be transported across a cell membrane via a transport protein.

 

A drug that acts on a transporter protein can work in different ways. The medicine can work by being transported itself across the cell membrane by mimicking the molecules that are normally transported across the membrane. The medicine can also work by blocking the transport protein, thereby inhibiting the absorption of the substance that the protein transports. For example, cocaine works in the central nervous system by inhibiting the reuptake of serotonin and dopamine through a transport protein. This inhibition will cause there to be more serotonin and dopamine in the synaptic cleft between the two nerve endings, and there will therefore be a prolonged and increased signal through the nerves.

Table 1

Drug Target Mechanism of Action
Receptors Agonist / Antagonist
Enzymes Reversible / Irreversible
Ion Channel Blocker / Opens

 

To sum up, there are a number of different types of proteins that can act as a drug target. These are specifically receptors, enzymes and transport proteins. The way in which these drug targets work has been described above and can be summarised in Table 1. In addition to proteins, there are a number of other types of drug targets. These can be DNA and RNA, for example.

Tolerance and dependence

If a cell is exposed to an antagonist for a long time, the cell will receive no signal from the receptor. To compensate for this, the cell will upregulate the formation of new receptors (Figure 12b). The cell can now again detect signals from the natural ligand (Figure 12d). Therefore, in order to get the desired medical effect using the antagonist, a higher dose of the drug must be given (Figure 12c). This cycle, in which the cell makes more receptors and a higher dose is given, can keep repeating. The state the cell gets into, and thus also the state the body gets into, is called tolerance, as the body needs more of the drug to achieve the “normal” effect.

When the intake of the drug stops, all the receptors are released. This means that all the new receptors as well as the original ones will be activated by the natural ligand (Figure 12e). This is very uncomfortable, and one will feel an urge to take the drug again because it will feel like a normal response. This is called addiction because you have to take the drug to feel good. Over a longer period of time, the number of receptors will fall back to a normal level (Figure 12f), but until then the patient is in weaning.

Figure 12. The illustration shows how tolerance and dependence on a drug can occur.