IDENTIFICATION OF THE DRUG TARGET:
Medicinal chemistry is the study of how novel drugs can be designed and developed. Identification of drug target helps immeasurably by a detailed understanding of the structure & function of the molecular targets that are present in body. Major drug targets are normally large molecules (macromolecules) like proteins & nucleic acids. Knowing the structures, properties, and functions of these macromolecules is vital if we want to design new drugs. There are various reasons for this. Firstly, it is important to know what functions different macromolecules have in body and whether targeting them is likely to have a useful effect in treating a particular disease. Secondly, a knowledge of macromolecular structure is crucial if one is to design a drug that will bind effectively to the target. Thirdly, a drug must not only bind to the target, it must bind to the correct region of the target. Proteins and nucleic acids are extremely large molecules in comparison to a drug and if the drug binds to the wrong part of macromolecule, it may not have any effect. Finally, an understanding of how a macromolecule operates is essential if one is going to design an effective drug that will interfere with that process. Example include protease inhibitors used in HIV therapy.
Characteristics of Drug Target:
1.The drug target is a biomolecule usually a protein that could exist in isolated or complex modality.
2. Biomolecules have special sites that match other molecules. These molecules could be endogenous or extraneous substances such as chemical molecules.
3. The biomolecular structure might change when the biomolecule binds to small molecules and the changes in structure are usually reversible.
4. Following the change in the biomolecules structure various physiological responses occur and induce regulation of the cell, organ, tissue or body status.
5. The physiological responses triggered by the changes in biomolecule structure play a major role in complex regulation and have a therapeutic effect on pathological conditions.
Traditional Drug Discovery v/s New Strategies in Drug discovery:
The first stage in drug discovery process is to understand the disease mechanism, using cellular and genetic approaches to identify potential drug targets. Target identification and mechanism of action studies play an important role in small molecule discovery. A good target needs to be efficacious, safe, meet clinical and commercial needs and above all be druggable. A druggable target is accessible to the putative drug molecule, be that a small molecule or larger biological and upon binding, elicit a biological response which may be measured both in vitro and in vivo. Good target identification and validation enables increased confidence in the relationship between target and disease and allows us to explore whether target modulation will lead to mechanism-based side effects. Current therapy is based upon less than 500 molecular targets of about 10000 possible targets: 45% of which are G-protein coupled receptors; 28% are enzymes; 11% are hormones and factors; 5% are ion channels; 2% are nuclear receptors.
CHOICE OF BIOASSAY:
Choosing the right bioassay is the crucial step in discovering drug success of drug research program. The test should be easy, fast and relevant, as there are usually a large number of compounds to be analyzed. Human testing is not possible at such an early stage, so the test has to be done in vitro (isolated cells, tissues, enzymes or receptors) or in vivo ( animals). In general, in vitro tests are preferred over in vivo tests because they are inexpensive, easier to carry out, less controversial and they can be automated. In modern medicinal chemistry, a variety of tests are usually carried out both in vitro and in vivo to determine not only whether the candidate drugs are acting at the desired target, but also whether they have activity at other undesired targets.
In vitro tests:
This test does not involve living animals, instead use specific tissues, cells or enzymes. Enzyme inhibitors can be tested on pure enzyme in solution. Nowadays, genetic engineering can be used to incorporate the gene for a particular enzyme into fast-growing cells like yeast or bacteria. These then produce the enzyme in larger quantities, making isolation easier. For instance, HIV protease has been cloned and expressed in bacterium E. coli. Receptor agonists and antagonists can be tested on isolated tissues or cells which express target receptor on their surface. Sometimes these tissues can be used to test drugs for physiological effects. For example, bronchodilator activity can be checked by observing how well compounds inhibit contraction of isolated tracheal smooth muscle.
In vivo tests:
In vivo tests on animals often involve inducing a clinical condition in the animal to produce observable symptoms. The animal is then treated to see whether the drug reduce the problem by eliminating observable symptoms. Transgenic animals are often used in in vivo testing. These are animals whose genetic code has been altered. For instance, it is possible to replace some mouse genes with human genes. The mouse produces human receptor or enzyme and this allows in vivo testing against that target. Alternatively, the mouse gene could be altered such that animal becomes susceptible to a particular disease like breast cancer. Finally, different results may be obtained in different animal species. For example thalidomide , which is teratogenic in rabbits and humans but has no such effect in mice.
Sometimes validity of testing procedures is easy. For instance, an antibacterial agent can be tested in vitro by measuring how effectively it kills bacterial cells. A local anaesthetic can be tested in vitro on how well it blocks action potentials in isolated nerve tissue
High-throughput screening (HTS):
Robotics and the miniaturization of in vitro tests on genetically modified cells has led to a process called HTS, which is particularly effective in identifying potential new lead compounds. This involves the automated testing of large numbers of compounds versus a large number of targets. Typically, several thousand compounds can be tested at once in 3050 biochemical tests. It is important that the test should produce an easily measurable effect which can be detected and measured automatically. This effect could be cell growth, an enzyme-catalysed reaction which produces a color change, or displacement of radioactively labelled ligands from receptors. Receptor antagonists can be studied using modified cells which contain the target receptor in their cell membrane.
Screening by nuclear magnetic resonance (NMR):
NMR spectroscopy is an analytical tool which has been used for many years to determine the molecular structure of compounds. More recently, it has been used to detect whether a compound binds to a protein target. There are several advantages in using NMR as a detection system:
? It is possible to screen 1000 small-molecular-weight compounds a day with one machine.
? Method can detect weak binding which would be missed by conventional screening methods.
? It can identify the binding of small molecules to different regions of the binding site.
? It is complimentary to HTSthe latter may give false- positive results, but these can be checked by NMR to ensure that the compounds concerned are binding in the correct binding site.
? Identification of small molecules which bind weakly to part of the binding site allows the possibility of using them as building blocks for the construction of larger molecules that bind more strongly.
? Screening can be done on a new protein without needing to know its function.
Af?nity screening :
A nice method of screening mixtures of compounds for active ingredients is to take advantage of binding affinity of compounds target. This not only detects the presence of such agents but also picks them out from the mixture. Like, the vancomycin family of antibacterial agents has a strong binding affinity for the dipeptide d-Ala-d-Ala. d-Ala-d-Ala was linked to sepharose resin, and the resin was mixed with extracts from various microbes which were known to have antibacterial activity
Surface plasmon resonance(SPR):
SPR is an optical method of detecting when a ligand binds to its target. The procedure is patented by Pharmacia Biosensor as BIAcore and makes use of a dextran-coated, gold-surfaced glass chip. A ligand that is known to bind to the target is immobilized by linking it covalently to the dextran matrix, which is in a flow of buffer solution.
Scintillation proximity assay(SPA):
SPA is a visual method of detecting whether a ligand binds to a target. It involves immobilization of the target by linking it covalently to beads which are coated with a scintillant. A solution of a known ligand labelled with iodine-125 is then added to the beads. When labelled ligand binds to the immobilized target,125I acts as an energy donor and scintillant-coated beads act as an energy acceptor, resulting in an emission of light that can be detected. To find out whether a novel compound interacts with the target, the compound is added to the solution of labelled ligand and the mixture is added to the beads. Successful binding by the novel compound will mean that less of labelled ligand will bind, resulting in a reduction in emission of light.
Isothermal titration calorimetry (ITC):
ITC is a technique that is used to determine thermodynamic properties of binding between a drug & its protein target. Two identical glass cells are used which are filled with buffer solution. One of the cells acts as the reference cell, while the other acts as the sample cell and contains protein target in solution. The reference cell is heated slightly to a constant temperature. The sample cell is heated to the same temperature through an automatic feedback system, whereby any temperature difference between the two cells is detected and power is applied to the sample cell to equalize the temperature. Once the apparatus has stabilized, a constant level of power is used to maintain the two cells at the same constant temperature. The drug is now added to the sample cell and binds to the protein target. If binding interaction is exothermic, heat energy is generated within the sample cell and so less external power is needed to maintain the cell temperature. If interaction is endothermic, the opposite holds true and more external power has to be applied to maintain the temperature. The external power required to maintain the temperature of the sample cell is measured with respect to time, with power spikes occurring every time the drug is injected into the cell. Measurement of these spikes allows the determination of the thermo- dynamic properties of binding.
Virtual screening :
Virtual screening involves the use of computer programs to assess whether known compounds are likely to be lead compounds for a particular target. There is no guarantee that positive hits from a virtual screening will be active and the compounds still have to be screened experimentally, but the results from a virtual screening can be used to make experimental screening methods more efficient.