Drug Design Lecture 5 (PhB-3216) PDF

Drug design (PhB-3216) Level 3 Lecture 5 Drug discovery Dr. Asmaa Mohamed Atta [email protected] Office: 223 – Pharmacy Office hours: Monday (11:00 am-1:00 pm)  Drug discovery, de...

Drug design (PhB-3216) Level 3 Lecture 5 Drug discovery Dr. Asmaa Mohamed Atta [email protected] Office: 223 – Pharmacy Office hours: Monday (11:00 am-1:00 pm)  Drug discovery, design, and development: the past  Before the twentieth century, medicines consisted mainly of herbs, and it was not until the mid-nineteenth century that the first serious efforts were made to isolate and purify the active principles of those remedies (i.e. the pure chemicals responsible for the medicinal properties).  The success of these efforts led to the birth of many of the pharmaceutical companies we know today. Since then, many naturally occurring drugs have been obtained and their structures determined (e.g. morphine from opium, cocaine from coca leaves, quinine from the bark of the cinchona tree).  These natural products sparked off a major synthetic effort where chemists made literally thousands of analogues in an attempt to improve on what nature had provided. Opium Cinchona bark Coca leaves  Drug discovery, design, and development: the present  In recent years, medicinal chemistry has undergone a revolutionary change. Rapid advances in the biological sciences have resulted in a much better understanding of how the body functions at the cellular and the molecular level.  As a result, most research projects in the pharmaceutical industry or university sector now begin by identifying a suitable target in the body and designing a drug to interact with that target. An understanding of the structure and function of the target, as well as the mechanism by which it interacts with potential drugs, is crucial to this approach.  Generally, we can identify the following stages in drug discovery, design and development: 1 Choose a drug target Drug 2 Identify a bioassay discovery 3 Find a ‘lead compound’ 4 Isolate and purify the lead compound if necessary  Drug discovery, design, and development  Identify structure–activity relationships (SARs). Drug  Identify the pharmacophore. design  Improve target interactions (pharmacodynamics).  Improve pharmacokinetic properties.  Patent the drug  Carry out preclinical trials (drug metabolism, toxicology, Drug  formulation and stability tests, pharmacology studies, etc.) development  Design a manufacturing process (chemical and process development).  Carry out clinical trials  Register and market the drug  Make money!  Drug discovery, design, and development  Many of these stages run concurrently and are dependent on each other. For example, preclinical trials are usually carried out in parallel with the development of a manufacturing process.  Even so, the discovery, design, and development of a new drug can take 15 years or more, involve the synthesis of over 10,000 compounds, and cost in the region of $800 million (£450 million).  Drug discovery: 1- Choosing a drug target  Once a therapeutic area has been identified, the next stage is to identify a suitable drug target (e.g. receptor, enzyme, or nucleic acid).  An understanding of which biomacromolecules are involved in a particular disease state is clearly important.  This allows the medicinal research team to identify whether agonists or antagonists should be designed for a particular receptor or whether inhibitors should be designed for a particular enzyme.  The caspases are examples of recently discovered enzymes which may prove useful as drug targets. They catalyse the hydrolysis of important cellular proteins, and which have been found to play a role in inflammation and cell death.  By understanding how these enzymes operate, there is the possibility of producing new therapies for a variety of diseases.  For example, agents which promote the activity of caspases and lead to more rapid cell death might be useful in the treatment of diseases such as cancer, autoimmune disease, and viral infections. For example, carboplatin is an anticancer agent that promotes caspase activity.  Alternatively, agents which inhibit caspases and reduce the prevalence of cell death could provide novel treatments for trauma, neurodegenerative disease, and strokes.  Target specificity and selectivity between species  The more selective a drug is for its target, the less chance there is that it will interact with different targets and have undesirable side effects.  For example, penicillin targets an enzyme involved in bacterial cell wall biosynthesis. Mammalian cells do not have a cell wall, so this enzyme is absent in human cells and penicillin has few side effects. Antibiotics mechanism of action on the bacteria cell. (β- Penicillin lactams affects the cell wall synthesis.)  Target specificity and selectivity between species  It is still possible to design drugs against targets which are present both in humans and microbes, as long as the drugs show selectivity against the microbial target.  For example, the antifungal agent fluconazole inhibits a fungal demethylase enzyme involved in steroid biosynthesis. This enzyme is also present in humans, but the structural differences between the two enzymes are significant enough that the antifungal agent is highly selective for the fungal enzyme. Fluconazole Fluconazole interacting with a heme group in the active site of the lanosterol 14 alpha-demethylase  Target specificity and selectivity within the body  Selectivity is also important for drugs acting on targets within the body.  Enzyme inhibitors should only inhibit the target enzyme and not some other enzyme.  Ideally, enzyme inhibitors should show selectivity between the various isozymes of an enzyme (isozymes are the structural variants of an enzyme that result from different amino acid sequences or quaternary structure).  For example, there are three different isoforms of nitric oxide synthase (NOS)—the enzyme responsible for generating the chemical messenger nitric oxide. Selective inhibitors for one of these isoforms (nNOS) could potentially be useful in treating cerebral palsy and other neurodegenerative diseases.  Receptor agonists and antagonists should not only show selectivity for a particular receptor (e.g. an adrenergic receptor) or even a particular receptor type (e.g. the β-adrenergic receptor), but also for a particular receptor subtype (e.g. the β2 -adrenergic receptor). Targeting drugs to specific organs and tissues  Targeting drugs to specific organs and tissues  Targeting drugs against specific receptor subtypes often allows drugs to be targeted to specific organs or to specific areas of the brain.  This is because the various receptor subtypes are not distributed uniformly around the body but are often concentrated in particular tissues.  For example, the β-adrenergic receptors in the heart are predominantly β1, whereas those in the lungs are β2.  This makes it feasible to design drugs that will work on the lungs with a minimal side effect on the heart, and vice versa.  Multi-target drugs  In certain diseases and afflictions, there can be an advantage in ‘hitting’ a number of different targets selectively, as this can be more beneficial than hitting just one.  Combination therapy is normally used to achieve this by administering two or more drugs showing selectivity against the different targets.  The disadvantage of combination therapies is the number of different medications and the associated dose regimens.  Therefore, there are benefits in designing a single drug that can act selectively at different targets in a controlled manner—a multitarget-directed ligand.  Many research projects now set out to discover new drugs with a defined profile of activity against a range of specific targets. Multi-target drugs Rational design of a multitarget-directed ligand  Multi-target drugs Example: Less selective example is olanzapine. This drug binds to more than a dozen receptors for serotonin, dopamine, muscarine, noradrenaline, and histamine. This kind of profile would normally be unacceptable, but olanzapine has been highly effective in the treatment of schizophrenia, Olanzapine probably because it blocks both serotonin and dopamine receptors. Drugs which interact with a large range of targets are called promiscuous ligands or dirty drugs.  Drug discovery: 2-Identifying a bioassay Choice of bioassay  Choosing the right bioassay or test system is crucial to the success of a drug research programme.  The test should be simple, quick, and relevant, as there are usually a large number of compounds to be analysed.  Human testing is not possible at such an early stage, so the test has to be done in vitro (i.e. on isolated cells, tissues, enzymes, or receptors) or in vivo (on animals).  In general, in vitro tests are preferred over in vivo tests because they are cheaper, easier to carry out, less controversial and they can be automated.  However, in vivo tests are often needed to check whether drugs have the desired pharmacological activity and also to monitor their pharmacokinetic properties.  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.  Drug discovery: 2-Identifying a bioassay Invitro test  In vitro tests do not involve live animals. Instead, specific tissues, cells, or enzymes are used. Enzyme inhibitors can be tested on the pure enzyme in solution.  Genetic engineering can be used to incorporate the gene for a particular enzyme into fast- growing cells, such as yeast or bacteria.  These then produce the enzyme in larger quantities, making isolation easier.  For example, HIV protease has been cloned and expressed in the bacterium Escherichia coli. A variety of experiments can be carried out on this enzyme to determine whether an enzyme inhibitor is competitive or non-competitive, and to determine IC50 values.  Drug discovery: 2-Identifying a bioassay 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 alleviates the problem by eliminating the observable symptoms. For example, the development of non-steroidal inflammatory drugs was carried out by inducing inflammation on test animals, then testing drugs to see whether they relieved the inflammation.  Transgenic animals are often used in in vivo testing. These are animals whose genetic code has been altered.  For example, it is possible to replace some mouse genes with human genes. The mouse produces the human receptor or enzyme and this allows in vivo testing against that target.  Alternatively, the mouse’s genes could be altered such that the animal becomes susceptible to a particular disease (e.g. breast cancer). Drugs can then be tested to see how well they prevent that disease.  Drug discovery: 2-Identifying a bioassay In vivo tests  There are several problems associated with in vivo testing. It is slow and expensive, and it also causes animal suffering.  For example, how can one tell whether a negative result is due to the drug failing to bind to its target or not reaching the target in the first place?  Thus, in vitro tests are usually carried out first to determine whether a drug interacts with its target, and in vivo tests are then carried out to test pharmacokinetic properties.  Certain in vivo tests might turn out to be invalid. It is possible that the observed symptoms might be caused by a different physiological mechanism than the one intended. For example, many promising anti-ulcer drugs which proved effective in animal testing were ineffective in clinical trials.  Finally, different results may be obtained in different animal species. For example, penicillin methyl ester prodrugs are hydrolysed in mice or rats to produce active penicillins, but are not hydrolysed in rabbit, dogs, or humans.  Despite these issues, in vivo testing is still crucial in identifying the particular problems that might be associated with using a drug in vivo and which cannot be picked up by in vitro tests.  Drug discovery: 2-Identifying a bioassay High-throughput screening  Robotics and the miniaturization of in vitro tests on genetically modified cells has led to a process called  high-throughput screening (HTS).  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 30–50 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 colour change, or displacement of radioactively labeled ligands from receptors.  Drug discovery: 2-Identifying a bioassay High-throughput screening  High throughput screening (HTS) is the use of automated equipment to rapidly test thousands to millions of samples for biological activity.  In its most common form, HTS is an experimental process in which 103–106 small molecule compounds of known structure are screened in parallel.  Because HTS typically aims to screen 100000 or more samples per day, relatively simple and automation-compatible assay designs, robotic-assisted sample handling, and automated data processing are critical.  The heart of the HTS system is a plate, or tray, which consists of tiny wells in which assay reagents and samples are deposited, and their reactions are monitored.  There are several detection methods in HTS, for example, spectroscopy, mass spectrometry, chromatography, calorimetry, X-ray diffraction, microscopy, and radioactive methods.  The major advantages of an HTS assay are high sensitivity (single molecule detection), high speed (automation), and minimization of sample (microtiter plate assay).  Drug discovery: 2-Identifying a bioassay Screening by nuclear magnetic resonance  Compounds can be tested for their affinity to a macromolecular target by NMR spectroscopy. The relaxation times of ligands bound to a macromolecule are shorter than when they are unbound.  Nuclear magnetic resonance (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. In NMR spectroscopy, a compound is radiated with a short pulse of energy which excites the nuclei of specific atoms, such as hydrogen, carbon, or nitrogen.  Once the pulse of radiation has stopped, the excited nuclei slowly relax back to the ground state, giving off energy as they do so.  The time taken by different nuclei to give off this energy is called the relaxation time , and this varies depending on the environment or position of each atom in the molecule.  Therefore, a different signal will be obtained for each atom in the molecule and a spectrum is obtained which can be used to determine the structure.  First of all, the NMR spectrum of the drug is taken, then the protein is added and the spectrum is re-run, introducing a delay in the measurement such that the protein signals are not detected.  If the drug fails to bind to the protein, then its NMR spectrum will still be detected.  If the drug binds to the protein, it essentially becomes part of the protein. As a result, its nuclei will have a shorter relaxation time and no NMR spectrum will be detected.  Drug discovery: 2-Identifying a bioassay Screening by nuclear magnetic resonance  Drug discovery: 2-Identifying a bioassay Screening by nuclear magnetic resonance  There are several advantages in using NMR as a detection system: 1. it is possible to screen 1000 small-molecular-weight compounds a day with one machine. 2. the method can detect weak binding which would be missed by conventional screening methods. 3. screening can be done on a new protein without needing to know its function.  Disadvantages include the need to purify the protein and to obtain it in a significant quantity (at least 200 mg).  Drug discovery: 2-Identifying a bioassay Virtual screening can be used to identify compounds most likely to be Virtual screening active in experimental 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, in fact, 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.  In other words, if there are several thousand compounds available for testing, virtual screening can be used to identify those compounds which are most likely to be active, and so those are the structures which would be given priority for actual screening.  Virtual screening can involve a search for pharmacophores known to be required for activity, or docking the compounds into target binding sites. Docking and scoring in structure-based virtual screening  Drug discovery: 3- finding a lead Finding a lead  Once a target and a testing system have been chosen, the next stage is to find a lead compound—a compound which shows the desired pharmacological activity.  The level of activity may not be very great and there may be undesirable side effects, but the lead compound provides a start for the drug design and development process.  There are various ways in which a lead compound might be discovered: 1. Screening of natural products. 2. Medical folklore. 3. Screening synthetic compound ‘libraries’. 4. Existing drugs. 5. Starting from the natural ligand or modulator. 6. Combinatorial and parallel synthesis. 7. Computer-aided design of lead compounds. 8. Serendipity and the prepared mind. 9. Computerized searching of structural databases. 10. Fragment-based lead discovery.  Drug discovery: 3- finding a lead Finding a lead 1- Screening of natural products Natural products are a rich source of biologically active compounds. Many of today’s medicines are either obtained directly from a natural source or were developed from a lead compound originally obtained from a natural source. 1-1. The plant kingdom  Plants have always been a rich source of lead compounds (e.g. morphine, cocaine, digitalis, quinine, tubocurarine , nicotine , and muscarine ).  Many of these lead compounds are useful drugs in themselves (e.g. morphine and quinine), and others have been the basis for synthetic drugs (e.g. local anaesthetics developed from cocaine).  Clinically useful drugs which have recently been isolated from plants include the anticancer agent paclitaxel (Taxol) from the yew tree, the antimalarial agent artemisinin from a Chinese plant, and the Alzheimer’s drug galantamine from daffodils.  Drug discovery: 3- finding a lead Finding a lead 1- Screening of natural products Plant natural products as drugs  Drug discovery: 3- finding a lead Finding a lead 1- Screening of natural products Natural products are a rich source of biologically active compounds. Many of today’s medicines are either obtained directly from a natural source or were developed from a lead compound originally obtained from a natural source. 1-2. Microorganisms  Microorganisms such as bacteria and fungi have also provided rich pickings for drugs and lead compounds.  These organisms produce a large variety of antimicrobial agents which have evolved to give their hosts an advantage over their competitors in the microbiological world.  The screening of microorganisms became highly popular after the discovery of penicillin.  Drug discovery: 3- finding a lead Finding a lead 1- Screening of natural products Natural products are a rich source of biologically active compounds. Many of today’s medicines are either obtained directly from a natural source or were developed from a lead compound originally obtained from a natural source. 1-3. Marine sources  In recent years, there has been great interest in finding lead compounds from marine sources. Coral, sponges, fish, and marine microorganisms have a wealth of biologically potent chemicals with interesting inflammatory, antiviral, and anticancer activity.  For example, curacin A is obtained from a marine cyanobacterium, and shows potent antitumour activity.  Drug discovery: 3- finding a lead Finding a lead 1- Screening of natural products Natural products are a rich source of biologically active compounds. Many of today’s medicines are either obtained directly from a natural source or were developed from a lead compound originally obtained from a natural source. 1-3. Marine sources  In recent years, there has been great interest in finding lead compounds from marine sources. Coral, sponges, fish, and marine microorganisms have a wealth of biologically potent chemicals with interesting inflammatory, antiviral, and anticancer activity.  For example, curacin A is obtained from a marine cyanobacterium, and shows potent antitumour activity.  Drug discovery: 3- finding a lead Finding a lead 1- Screening of natural products Natural products are a rich source of biologically active compounds. Many of today’s medicines are either obtained directly from a natural source or were developed from a lead compound originally obtained from a natural source. 1-4. Animal sources  Animals can sometimes be a source of new lead compounds.  Example is a potent analgesic compound called epibatidine, obtained from the skin extracts of the Ecuadorian poison frog.  Drug discovery: 3- finding a lead Finding a lead 1- Screening of natural products Natural products are a rich source of biologically active compounds. Many of today’s medicines are either obtained directly from a natural source or were developed from a lead compound originally obtained from a natural source. 1-5. Venoms and toxins  Venoms and toxins from animals, plants, snakes, spiders, scorpions, insects, and microorganisms are extremely potent because they often have very specific interactions with a macromolecular target in the body.  Venoms and toxins have been used as lead compounds in the development of novel drugs. For example, teprotide, a peptide isolated from the venom of the Brazilian viper, was a lead compound for the development of the antihypertensive agents cilazapril and captopril.  The neurotoxins from Clostridium botulinum are responsible for serious food poisoning ( botulism ), but they have a clinical use as well. They can be injected into specific muscles (such as those controlling the eyelid) to prevent muscle spasm.  Drug discovery: 3- finding a lead Finding a lead 2- Medical folklore  In the past, ancient civilizations depended greatly on local flora and fauna for their survival. They would experiment with various berries, leaves, and roots to find out what effects they had.  As a result, many brews were claimed by the local healer to have some medicinal use.  Some of these extracts may, indeed, have a real and beneficial effect.  Rhubarb root has been used as a purgative for many centuries. The most significant chemicals in rhubarb root are anthraquinones, which were used as the lead compounds in the design of the laxative dantron. Rhubarb Rhubarb root  Drug discovery: 3- finding a lead Finding a lead 3- Screening synthetic compound ‘libraries’  The thousands of compounds which have been synthesized by the pharmaceutical companies over the years are another source of lead compounds.  The vast majority of these compounds have never made the market place, but they have been stored in compound ‘libraries’ and are still available for testing. Pharmaceutical companies often screen their library of compounds whenever they study a new target.  Pharmaceutical companies often try to diversify their range of structures by purchasing novel compounds prepared by research groups.  Drug discovery: 3- finding a lead Finding a lead 4- Existing drugs 4.1. ‘Me too’ and ‘me better’ drugs  Many companies use established drugs from their competitors as lead compounds in order to design a drug that gives them a foothold in the same market area.  The aim is to modify the structure sufficiently such that it avoids patent restrictions, retains activity, and, ideally, has improved therapeutic properties.  For example, the antihypertensive drug captopril was used as a lead compound by various companies to produce their own antihypertensive agents.  For example, modern penicillins are more selective, more potent, and more stable than the original penicillins. Newer statins that lower cholesterol levels also have improved properties over older ones  Drug discovery: 3- finding a lead Finding a lead 4- Existing drugs 4.2. Enhancing a side effect  An existing drug usually has a minor property or an undesirable side effect which could be of use in another area of medicine.  As such, the drug could act as a lead compound on the basis of its side effects. The aim would then be to enhance the desired side effect and to eliminate the major biological activity. This has been described as the SOSA approach— selective optimization of side activities.  Several drugs have been developed by enhancing the side effect of another drug. Chlorpromazine is used as a neuroleptic agent in psychiatry, but was developed from the antihistamine agent promethazine.It is known that promethazine has sedative side effects, and so medicinal chemists modified the structure to enhance the sedative effects at the expense of antihistamine activity.  Drug discovery: 3- finding a lead Finding a lead 4- Existing drugs 4.2. Enhancing a side effect  Similarly, the development of sulphonamide diuretics such as chlorothiazide arose from the observation that sulphanilamide has a diuretic effect in large doses (owing to its action on an enzyme called carbonic anhydrase ).