Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Unit 9: Medicinal Chemistry . 94 The role of biologically active molecules in biochemical systems In this section, you will read about several important drug molecules and their mechanism of action in biological systems. Some of them (for example, penicillin-based antibiotics) have already appeared as case studies in the context of other aspects of medicinal chemistry in Topic guides 9.1–9.3. You will also revisit some of the ideas about drug toxicity encountered in Topic guide 9.2, and learn how toxicity is assessed. On successful completion of this topic you will: •• understand the role of biologically active molecules in biochemical systems (LO4). To achieve a Pass in this unit you need to show that you can: •• discuss the development and role of selected biologically active molecules (4.1) •• explain clinical toxicological terms citing suitable examples (4.2) •• explain the principles of clinical toxicity (4.3). 1 Unit 9: Medicinal Chemistry 1The development of selected drug molecules Penicillins Figure 9.4.1: Penicillin G, the molecule extracted from the Penicillin mould, has R = C6H5–CH2–. The β-lactam ring is circled. H H N R O S O N CH3 CH3 COOH The penicillin group of molecules are antibiotics used to treat a range of bacterial infections. The story of the accidental discovery of the antibiotic nature of penicillin is well known – a Petri dish used to culture Staphylococcus was accidently contaminated with Penicillium mould. This produces the molecule we now call penicillin as a secondary metabolite. All penicillins have the core structure shown in Figure 9.4.1; the nature of the R group is different for each member of the group. Mode of action Penicillins act on bacteria by interfering with the construction of the bacterial cell wall. This structure consists of a network of polymer chains (known as peptidoglycan, and consisting of amino acids and sugars bonded together). One of the key steps in the completion of this strong network is the cross-linking of the peptidoglycan chains. This occurs when two short peptide chains are linked by a transpeptidase enzyme (see Figure 9.4.2). If this process of cross-linking is prevented, the cell wall is therefore weakened and cannot withstand the pressure within the cell, which therefore bursts, killing the bacterium. O Figure 9.4.2: The structure of the peptidoglycan network present in bacterial cell walls. The crosslink formed by the transpeptidase enzyme is shown in the diagram. O O Peptide chains Key terms Secondary metabolite: A molecule produced by a living organism that has no known role in its biochemistry. These substances are frequently used as possible starting points from which to develop lead compounds. Nucleophile: Group of atoms with a lone pair of electrons that can be donated to an electron deficient atom. Polysaccharide chain O O O O O O This cross-link is formed by the action of the transpeptidase enzyme O O O O O O O O O O Penicillin inhibits the transpeptidase enzyme, which forms the cross-links, by permanently binding to the active site. The key structural feature of the penicillin molecule is the β-lactam ring. This is attacked by nucleophiles, such as –OH groups, and attaches to a serine residue in the active site. 9.4: The role of biologically active molecules in biochemical systems 2 Unit 9: Medicinal Chemistry Figure 9.4.3: The mechanism by which the β-lactam ring is opened up, involving nucleophilic attack by the OH of a serine residue. proton transfer N O – O OH ser N hydrolysis –O + OH ser O N H + NH O O ser NH O OH + OH ser ser Activity Research how chemists have developed new penicillins to overcome bacterial resistance. Explain how the structural features of these adapted molecules achieve this. Key terms Hydrolysis: A reaction in which a molecule is broken down by the action of water. Peptide (or oligopeptide): Molecules consisting of short chains of amino acid residues. Figure 9.4.4: The angiotensin-converting enzyme catalyses the formation of angiotensin II from angiotensin I, by removing two amino acids. ACE inhibitors angiotensin I 0000000000 ACE angiotensin II 00000000 + 00 hypertension This opens up the β-lactam ring and the serine residue is later regenerated by a final hydrolysis step (see Figure 9.4.3). Antibiotic resistance Some bacteria possess a gene that enables them to synthesise an enzyme called penicillinase or β-lactamase. This enzyme causes the hydrolysis of the amide group in the β-lactam ring, inactivating penicillin. These bacteria are therefore resistant to penicillin. ACE inhibitors Angiotensin (more correctly known as angiotensin II) is a small peptide hormone involved in the system that regulates blood pressure. When present in raised concentrations it causes high blood pressure (hypertension) which, if left untreated, has serious health consequences. Angiotensin II is formed by the removal of two amino acids from the inactive compound angiotensin I (see Figure 9.4.4). The reaction is catalysed by the angiotensin-converting enzyme (ACE). Inhibition of this enzyme will therefore decrease the rate of formation of angiotensin II. Development of ACE inhibitors The development of this group of drug molecules is a good example of how structure-activity relationships can be used in the development of an effective drug. The importance of ACE first came to be realised from a study of the venom of the Brazilian Pit Viper; its fatal effects were due to a massive and very rapid drop in blood pressure caused by inhibition of ACE. However, this inhibitor was itself a peptide. This meant it would not be suitable as an orally-administered drug because it would be rapidly hydrolysed in the stomach. This peptide had a proline amino acid at the carboxyl end of the peptide chain: H2N–Glu–Trp–Pro–Arg–Pro–Gln–Ile–Pro–Pro–COOH Researchers created thousands of molecules based on proline and evaluated their effectiveness using animal-based tests. Initially, a succinyl derivative was found to be the most effective inhibitor and became the lead compound (see Figure 9.4.5). Figure 9.5.5: The structures of (a) proline (b) the general structure of the proline derivatives that were investigated (c) succinyl proline. = a single amino acid (a) (b) HN (c) R N OH 9.4: The role of biologically active molecules in biochemical systems HO OH N OH 3 Unit 9: Medicinal Chemistry Further screening suggested that adding a methyl group in the 2-position of the succinyl group further increased inhibitory strength by a factor of about 15. This created a chiral carbon at the 2 position and only one enantiomer showed the enhanced inhibitory effect. Figure 9.4.6: The structure of captopril. The presence of the methyl and SH group increases the inhibitory effect enormously compared to succinyl proline. HS N O O OH At this point, structure-activity relationships were used to optimise the lead compound. ACE functions in the presence of zinc ions, which were thought to complex to the penultimate amino acid of the substrate. Replacing the second carboxyl group from succinic acid with an SH (sulfhydryl) group would increase the strength of binding to this zinc ion. The result of this drug development process was to produce the first marketable ACE inhibitor, captopril (see Figure 9.4.6). Figure 9.4.7 shows the binding of captopril to the active site of ACE. CH3 H S CH2 CH C O : : Figure 9.4.7: Captopril binds to the active site of ACE using covalent bonds to the Zn2 ion as well as hydrogen bonding and ionic bonding. N C O– + NH3 O H Zn2+ N Activity Describe the similarities and differences in the structures of captopril and succinyl proline. Take it further The Wikipedia entry for ACE inhibitors, found at http://en.wikipedia.org/wiki/ACE_inhibitors_drug_design tells the story of the development of ACE and gives an excellent insight into the practical application of structure-activity relationships. Anticancer agents Cancer, the uncontrolled division of cells, is not a single disease but a spectrum of more than 100 identifiable types, usually specific to a particular tissue or organ. The different types of cancer respond in different ways to drugs and, as a result, the number of anticancer drugs that have been developed is huge. The majority of drug treatments are designed to disrupt the processes that occur during cell division. This makes it very difficult to achieve high levels of potency without also observing very significant toxic effects, since the drugs will also disrupt the activities of other cells that undergo rapid cell division such as skin cells, blood cells in the bone marrow and cells lining the gastrointestinal tract, creating unpleasant and potentially fatal side effects. More recently, drugs have been developed that target specific receptors present in cancer cells and offer hope for effective treatment without the risk of toxicity to normal cells. A discussion of the full range of anticancer agents is beyond the scope of this publication; two case studies will illustrate some of the key principles. 9.4: The role of biologically active molecules in biochemical systems 4 Unit 9: Medicinal Chemistry Methotrexate Methotrexate disrupts the synthesis of the base thymine, which is one of the four bases present in the nucleotides that make up DNA. One of the key steps in the synthesis of thymine is methylation of a uracil base (see Figure 9.4.8). This methylation step requires the involvement of a molecule called tetrahydrofolate (shown in the diagram as tetrahydrofolic acid, THF), which is eventually oxidised to dihydrofolate (shown as dihydrofolic acid, DHF). In order for synthesis of thymine to continue, the tetrahydrofolate must be regenerated from dihydrofolate by the action of dihydroreductase. Dihydroreductase Figure 9.4.8: Synthesis of a uracil group requires regeneration of THF. THF DHF Methylene THF + CH3 N O CH3 N N O Uracil monophosphate N Thymine monophosphate It is this latter step that is targeted by methotrexate – it acts as a competitive inhibitor of dihydroreductase. It is easy to see why when you examine the structures of methotrexate and DHF in Figure 9.4.9. Within a constant regeneration of DHF, the synthesis of thymine will cease. Figure 9.4.9: The structures of dihydrofolate (top) and methotrexate (bottom) show how similar in structure the competitive inhibitor is to that of the normal substrate. O O O N N H N N N H H N NH3 Cl Pt Cl NH3 O H O OH O OH N N N OH N NH2 Figure 9.4.10: The structure of cisplatin. The ‘cis’ prefix in the structure indicates that the chloride ligands are adjacent to each other on the same side of the Pt ion. OH N H O N Cisplatin Cisplatin is an example of a transition metal complex, in which small molecules and ions (in this case ammonia and chloride ions) form dative covalent bonds to a central transition metal ion (platinum) – see Figure 9.4.10. It was discovered to have anticancer properties in the 1960s. The mechanism of its action is now known to involve disrupting both replication and transcription of DNA. 9.4: The role of biologically active molecules in biochemical systems 5 Unit 9: Medicinal Chemistry Activity Write a short report describing the mechanism of action of cisplatin, making it clear how the mechanism relates to its structure. Take it further More details of the action of cisplatin can be found at http://chemcases.com/cisplat/cisplat12.htm, including some excellent graphics showing the disrupted structure. Antiviral drugs Viruses, which cause diseases such as influenza (‘flu), chickenpox and AIDS, have proved far more difficult to target by drug therapy than bacterial agents. The main reason for this is that viruses use their host cell’s metabolism to produce all the molecules required for replication and assembly of new viruses, hence it is very difficult to target specific metabolic reactions of viruses without also affecting the host’s metabolism. Vaccines Vaccines provide a way of protection against infection by viruses, by stimulating the body’s immune system to recognise virus particles. Case study Viruses mutate rapidly, and this is particularly significant in the production of seasonal ‘flu vaccine, often taken by at-risk groups of patients at the onset of each winter. Each year in February, the World Health Organization (WHO) recommends the three strains of ‘flu virus that should be included in that year’s vaccine. From this point, small laboratories, such as that run in the UK by the National Institute for Biological Standards and Control, create a ‘seed’ vaccine strain. They incubate small amounts of the virus mixture in a hen’s egg, creating a sample large enough to work with. Surface proteins from the viruses are attached to a harmless virus particle to create the seed vaccine strain. From this point it may take three to five months to produce and market sufficient quantities of the vaccine to meet demands. Clearly the time frame of this process is very different from that of the development of a new drug; clinical safety trials must also be carried out within the same time frame. 1 How do you think the WHO decides which strains to include in the annual vaccine? 2 Why is it difficult for the producers of vaccines to produce vaccines rapidly enough to combat pandemics (such as the swine flu pandemic in 2009)? Activity Research the mechanism by which oseltamivir prevents the infection of host cells and write a short report to explain it. Tamiflu (oseltamivir) Whereas vaccines make use of the body’s immune system, antiviral drugs work by targeting the activity of proteins specific to viruses. One such protein is known as viral neuraminidase, which has a role in allowing the virus to infect host cells. Antiretroviral drugs Link The structure of DNA is covered in detail in Unit 1: Biochemistry of Macromolecules and Metabolic Pathways. The influenza virus is able to be replicated and assembled because its RNA genome is able to be replicated and transcribed in the host cell’s nucleus (usually it is only DNA that undergoes these processes). Some viruses have a different mechanism of replication. This is known as reverse transcription, whereby the viral RNA is transcribed into a DNA molecule that then directs protein synthesis in the conventional way. 9.4: The role of biologically active molecules in biochemical systems 6 Unit 9: Medicinal Chemistry Take it further If you are unfamiliar with the structure and function of DNA, and particularly the processes of replication and transcription referred to in this section, there are some useful animations with descriptions available at websites such as www.johnkyrk.com/DNAanatomy.html. These types of viruses, known as retroviruses, can be treated by antiretroviral drugs that target the stages in this process. AZT (azidothymidine, zidovudine) Figure 9.4.11: The structures of AZT (top) and thymidine (bottom) show how similar the two molecules are. O NH HO – O N O AZT acts as a competitive inhibitor of the reverse transcriptase molecule because it bonds strongly to one of the active sites of the enzyme. As a result, reverse transcription is stopped and the virus is inactivated. Nitric oxide + N=N=N O H3C HO The nucleotides in DNA consist of three groups: a phosphate, sugar and a base. AZT has a structure similar to a naturally occurring combination of sugar and base known as thymidine – it differs only by having an azido (–N=N+=N−) group attached in place of a hydroxyl group (see Figure 9.4.11). NH O OH N O Nitric oxide (NO) is produced by all cells in the body and has a range of functions, mostly connected with signalling between cells; being so small it rapidly diffuses across cell membranes and within the cytoplasm. There specifically, it has a direct role in neurotransmission. One important effect is to cause dilation of blood vessels (vasodilation); nitric oxide stimulates the enzyme guanylyl cyclase to produce cyclic GMP (guanosine monophosphate) – another important signalling molecule that causes smooth muscle fibres to relax. Because of this effect, it has important medical uses (and it must surely have the simplest structure of any drug molecule!) and can be used directly in emergency treatment of heart failure. In cases of severe angina (chest pains), nitroglycerin is given, which reacts in the body to form NO in situ. Portfolio activity (4.1) Research a drug molecule, which could be one of the examples from the activity sections in this topic guide. Write a detailed report describing: •• the disease or condition against which it acts, including any significant biochemical details •• how the drug was discovered and developed •• the mechanism of the drug’s action. 9.4: The role of biologically active molecules in biochemical systems 7 Unit 9: Medicinal Chemistry 2 Clinical toxicology Toxicology is the study of the toxic effects of substances; clinical and preclinical tests are used to assess the toxicity of drugs and the same principles are used to assess toxicity of other chemical substances. Toxicity You met the concept of toxicity (the ability of a substance to cause harm to a person) in Topic guide 9.2. The toxic effects can be short term or long term; hence several types of toxicity need to be considered. Acute toxicity This is toxicity due to the effects of a single exposure to a substance, or several exposures over a short period (for example, 24 hours). A measurement of the acute toxicity of a drug must be made before proceeding to clinical trials – this is done by carrying out studies in at least two animal species. These studies will lead to a calculation of the LD50 value for a drug, which is the dose required to cause mortality in 50% of a group of test animals (frequently rats or mice). The LD50 is expressed as the number of grams of a substance per kilogram of the animal being tested. Some values of LD50 for a range of drugs that have been discussed in this unit are given in Table 9.4.1 – the smaller the number, the more toxic the substance. Table 9.4.1: Table of some LD50 values. Activity Use research to find LD50 values for three other drugs encountered in this unit. Compare the values from different sources – what do you notice? Drug LD50 g/kg–1 for mice, oral route of administration paracetamol 1.9 captopril 3.1 AZT 3.0 methotrexate 0.13 For comparison, the Botulinum toxin type A (used in cosmetic treatment) has an LD50 value of around 5 × 10–8 g/kg–1. There is considerable controversy about how valid these animal-based LD50 values are in predicting the acute toxicity of a drug in humans, as well as the ethical issues raised by the use of animals in this way. Chronic toxicity This is toxicity due to the effect of repeated or continuous exposure, sometimes lasting for the entire life of the exposed organism (effects of exposure of less than one year are sometimes referred to as sub-chronic toxicity). Unlike acute toxicity data, no single measure of chronic toxicity is used; the research data will highlight specific issues of toxicity to certain tissues or systems. Again, some of the data will be obtained from animal studies but clinical trials will also monitor the chronic effect of drug treatment if this is appropriate. 9.4: The role of biologically active molecules in biochemical systems 8 Unit 9: Medicinal Chemistry Take it further More details of the testing regimen used to establish chronic toxicity and other long-term effects of drugs can be found at www.meti.go.jp/english/information/data/TESTmammal.html. A database of toxicological studies for a wide range of drugs can be accessed at http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?HSDB. Specific chronic tests Table 9.4.2 summarises some specific chronic tests. Table 9.4.2: Specific chronic tests. Teratogenic toxicity tests To assess the potential of the drug to cause foetal abnormality (teratogenicity) Reproductive toxicity tests To assess the potential of the drug to cause fertility problems or developmental problems in offspring Mutagenicity To assess the potential of the drug to cause genetic mutations Activity A well-known example of a drug that was widely used before its teratogenicity was fully appreciated was thalidomide. Research the effects that this drug had and the mechanism of its teratogenic effect. 3 Clinical toxicity Faced with the toxicological information about the toxicity of a drug, what principles will a medical practitioner use to manage or evaluate the use of a drug in a clinical setting? Risk assessment This approach is common to any setting where a hazardous substance or process is being used. Essentially it seeks to answer three key questions: •• What is the hazard which might be encountered? •• What is the risk of that hazard occurring? •• How can the risk be reduced or managed? In the case of a drug, the possible toxic effects (as covered above) will be the hazard that may be encountered – the toxicological studies will give some quantitative information about the probability of harm occurring. The management of the risk is the point at which the practitioner will need to take decisions – about dosage, route of administration, length of treatment, and so on. In many cases there will be recommendations available on databases but, in some cases, the practitioner’s own experience and judgement will be called into play. 9.4: The role of biologically active molecules in biochemical systems 9 Unit 9: Medicinal Chemistry Risk-benefit analysis In many cases a decision may need to be taken about whether the benefit of the drug treatment outweighs the risks of harm that may result from the treatment. In the case of life-threatening diseases such as cancer, where the risks of harm from treatment are high and the potential for benefit may be measured only in terms of a short extension of life, such analyses may be difficult for both practitioner and patient and may raise difficult ethical issues about the rights of patients to access such treatment against the judgement of the practitioner. Portfolio activity (4.2, 4.3) Choose a drug (or drugs) that you have been reading about in this topic guide. Use this drug (or these drugs) to illustrate the principles of clinical toxicity. In your report: •• provide information about the acute and chronic toxicity of the drug, clearly distinguishing between the two terms •• explain what data would be needed to assess teratogenic toxicity, reproductive toxicity and mutagenicity, providing any data for your chosen drug •• include a risk-benefit analysis of the use of the drug by comparing the known hazards with the benefits it can provide. Checklist At the end of these two sections you will understand the following ideas: acute toxicity is measured by the LD50 value assessing chronic toxicity requires detailed long-term studies these studies will also look at the potential for causing birth defects, reproductive problems and genetic mutations in taking decisions about the use of a drug, a risk assessment should be carried out and the benefits considered against the risks of identified hazards. Acknowledgements The publisher would like to thank the following for their kind permission to reproduce their photographs: Shutterstock.com: isak55 All other images © Pearson Education In some instances we have been unable to trace the owners of copyright material, and we would appreciate any information that would enable us to do so. About the author David Goodfellow studied Natural Sciences at Cambridge and spent 20 years teaching A-level Chemistry in a sixth-form college. He was lead developer for the OCR AS Science in 2008 and for several years was chief examiner for the course. He now works as a freelance writer and examiner alongside part-time work as a teacher. Publications include a textbook for the AS Science course, teaching materials to accompany Chemistry GCSE courses and contributions to textbooks for BTEC First Applied Science. 9.4: The role of biologically active molecules in biochemical systems 10