PHA4011-E Fundamentals of Pharmacy Science Unit 1 Study Pack PDF

PHA4011-E Fundamentals of Pharmacy Science Unit 1: Fundamental Chemistry of Drugs and Molecules of Life Unit Team Required References Required: Unit Lead...

PHA4011-E Fundamentals of Pharmacy Science Unit 1: Fundamental Chemistry of Drugs and Molecules of Life Unit Team Required References Required: Unit Lead Study pack Dr Richard Wheelhouse Videos Web links Recommended: Unit Facilitators Chemistry in context Dr Sriharsha Kantamneni Marks' Basic Medical Dr Maria Azmanova Biochemistry An Introduction to Medicinal Chemistry Learning Outcomes By the end of this unit, you are expected to be able to: 1. Identify important organic functional groups in any molecular structure 2. Apply basic rules of chemical nomenclature to drug substances 3. Understand how atomic hybridisation directs bonding and the shape of atoms and molecules 4. Understand the importance of 3D shapes of molecules, including stereochemistry 5. Know what makes an organic molecule ‘drug-like’ 6. Identify important interactions between drugs and their receptors 7. Describe the basic structure and function of amino acids and proteins, and describe examples which demonstrate their role in biological systems 8. Describe the basic structure and function of nucleic acids and explain their widespread importance in biological systems 9. Describe the basic structure of monosaccharides and polysaccharides and describe their key functions 10. Describe the basic structure of lipids and explain their functional roles in the human body 11. Describe the major classes of vitamins and their functional roles, and explain how their deficiencies compromise human health 12. Describe the process of drug discovery 13. Understand what structure-based design is 1 STUDYING IN THIS UNIT - Please read carefully This reading pack consists of some easy-to-use explanatory notes (including videos and diagrams) and some detailed directed reading, both of which should be covered in preparing for your assessments. The pack has been designed to provide you with additional background information which you can come back to, to supplement your learning, while the lectures for this unit will cover the basic concepts. As you study, keep in mind the learning objectives written above, and be sure to make notes of your own. If you have trouble understanding a concept, please consult these resources before posting a question on the discussion board. Introduction You cannot divorce Biology from Chemistry; the former is a special extrapolation of the latter. After all, our body is a collection of chemicals. The proteins that form our hair, nails, and muscle fibres are chemicals; the minerals that are the basis of our bones and teeth are chemicals; even the food and drink we consume every day are chemicals. In fact, any and every object that we see around us is an example of Chemistry in action: all are formed from a collection of millions of tiny atoms. Biology and Chemistry explore the same thing – the world around us – but simply at different scales. Chemistry explores life at the level of atoms and molecules: it is really all about understanding how atoms interact to form larger, more complicated substances, how these substances react with each other to form new substances, and how these substances behave – whether they are flexible or rigid; liquid or solid; stable or reactive. Biology then looks at how these substances behave when they are combined on a larger scale – the scale of cells, tissues, organisms, populations, or ecosystems. This reading pack is designed to reinforce and develop understanding of fundamental Chemistry concepts introduced in pre-university (e.g., ‘A’-level or equivalent) study. These include concepts such as depiction, functional groups, nomenclature, bonding, electronics, stereochemistry of organic molecules. The emphasis here is very much on the application of fundamental chemistry concepts to the use of drugs. This knowledge also serves as a base for the second part of the pack which focuses on the four fundamental macromolecular building blocks: proteins, nucleic acids, lipids and polysaccharides. While their exact structures can differ, the basic chemistry of those building blocks is essentially similar in all biological systems. Vitamins are also essential to life, however what constitutes a true vitamin, is dependent on species. The aim here is to introduce the fundamental structure and functions of the above-mentioned macromolecules, in order to provide a molecular understanding of how biological cells and systems work. Later in the pack, we introduce you to molecular recognition. Every biological event involves molecular interactions. Molecules must interact to initiate an action, and then separate. Whether it is an enzyme binding to its substrate, a drug interacting with its receptor site localised in a macromolecule, a hormone binding to its receptor on a membrane, all require to be bound in a precise orientation for a short time. Such ‘recognition’ events are made possible through intermolecular interactions. At the end of the pack, we have introduced the drug discovery process and a case study to showcase one type of drug design – structure-based design. Later on in the module, in other units, you will be introduced to other case studies showcasing more examples of drug design. 2 1. Simple molecules 1.1. Organic structures Interactive periodic table Before we look into drug molecules and the molecules of life, we need to remind ourselves the language of organic chemistry. Chemical structures can be represented graphically in a wide range of ways (Table 1). A simple molecular formula provides information on the atoms present in a molecule but there is no information concerning connectivity or structure. Structural and skeletal formulae both provide additional information concerning the arrangement of atoms within a molecule and their connectivity. Whereas the structural formula displays all atoms present, the skeletal formula focuses on the carbon framework and omits hydrogen atoms attached to carbon, though those attached to other atoms are still included, therefore helping us to focus on the 2D shape of the molecule and its functional groups. Molecular models are 3D representations and are useful when considering chemical reactivity and how molecules might interact with each other or with a protein. Table 1. Different ways of representing a molecule Water Ammonia Ethanol Acetone Molecular H2O NH3 C2H6O C3H6O formula Structural formula Skeletal formula Molecular model (ball- and-stick type) Molecular model (space- filling type) Organic molecules are usually represented using skeletal formulae to simplify the structures and emphasise the features that are important to understanding the properties of the molecules. In these diagrams, the line ends and vertices represent carbon atoms. Hydrogen atoms are ‘implied’ – that is, they are not usually shown, but each carbon must have four bonds, and it is assumed they have the required number of hydrogens for this to be the case. Atoms other than carbon or hydrogen are always shown, and hydrogen atoms are shown if 3 they are bonded to one of these ‘heteroatoms’. Aspirin is given as an example below in Figure 1. Figure 1. Aspirin represented in its skeletal formula This way of drawing drug molecules is used in research papers, advanced textbooks, databases and reputable web sites. It has several advantages over the way you have learnt to draw organic molecules at A-level. It is very important to become comfortable with this way of drawing molecular structures. Most drugs are more complicated than the examples given so far, and the components of their receptors that you will meet later on in this unit, even more so. 1.1.1. Functional groups A molecule essentially consists of a hydrocarbon skeleton decorated with various functional groups. The skeleton gives the drug its shape in 3 dimensions and the functional group is an atom or a group of atoms within a molecule that serves as a site of chemical reactivity. Carbon combines with other atoms such as H, N, O, S and halogens to form functional groups. A reaction is the process by which one compound is transformed into a new compound. Thus, functional groups are important in chemical reactions. It is important that you are able to recognise these functional groups because they dictate the physical, chemical and other properties of organic molecules, including various drug molecules. Since the atoms that comprise functional groups are generally more electronegative than carbon, functional groups contribute to the polarity of a drug molecule. They also confer hydrogen bond donor (NH, OH) and acceptor properties (lone pairs). Some groups are acidic or basic and may be charged, especially at physiological pH. All of these interactions are important in defining how a drug molecule behaves in the body and interacts with its receptor. Figure 2 shows some of the most important functional groups in organic chemistry, alongside some common ring systems. Rings are extremely common in drugs, especially heterocycles that include one or more nitrogen, oxygen and sulphur atoms. Numbering of the heterocycles starts from a heteroatom and proceeds to other heteroatoms and then any substituents bear the lowest possible numbers. Beyond the simple guidelines here, we recommend that you look these up when needed. There are also software tools to interconvert chemical structures and systematic nomenclature and we will practice using such software in the computer workshop for this unit. 4  Figure 2. Functional groups and common ring systems in organic chemistry. R represents the rest of the molecule and X represents any halogen atom Exercise 1 Draw structures for each of the following: 1. 4-aminobutanoic acid; 2. but-2-ene; 3. alanine, NH2CH(CH3)CO2H; 4. methylpropanoate, N-methyl-propionamide 5. benzoic acid and cyclohexane carboxylic acid; 6. five different benzenoid compounds with molecular formula C9H11NO2 which illustrate the functional groups: (i) alcohol (ii) amine (iii) ester (iv) phenol (v) amide YOU WILL NEED THE ANSWERS TO Q6 FOR THE COMPUTER WORKSHOP SESSION FOR THIS UNIT. 5  Practice identifying functional groups 1.1.2. Nomenclature A basic understanding of organic chemical nomenclature is useful in communication and understanding: if you do not have a name for something, it is difficult to communicate about it! Any organic chemical name takes the form of prefix-parent-suffix as shown below in Figure 3. Figure 3. Prefix-Parent-Suffix method of naming a molecule Naming a compound is a multi-step process: Step 1. Parent - How many carbons are in the longest chain? This refers to the length of the main chain of carbon atoms. Below is a table for you to fill in to correlate the chain length with the corresponding parent you need to use in the naming process. Note that you have to find the longest carbon chain when assessing the structure. If an additional carbon chain is attached to the main parent chain, then the substituent name is to be used. Table 2. Carbon chain lengths and their corresponding parent or prefix (if used as substituent) Parent (prefix if used as Parent (prefix if used as Chain length Chain length substituent) substituent) 1 carbon meth (methyl) 6 carbons 2 carbons eth (ethyl) 7 carbons 3 carbons prop (propyl) 8 carbons 4 carbons 9 carbons 5 carbons 10 carbons Step 2. Suffix – What is the highest priority functional group present? The most oxidised functional group in the structure takes precedence when we decide on the suffix. The most weighted of all is the carboxylic acid functional group, for a list of functional 6 groups and their priority, alongside their corresponding suffix, see Table 3. If there is more than one functional group in the structure, the highest priority group determines the suffix, and the other groups (if present) will be written as a prefix. Note if there are only carbons and hydrogens in the structure, the suffix depends on whether there are any double or triple bonds. Table 3. List of functional groups, their priority and corresponding suffix or prefix Suffix Prefix Functional group (when primary) (when secondary) Carboxylic acid -oic acid carboxy- Ester -oate alkoxycarbonyl- Acid halide -oyl halide halocarbonyl- Amide -amide carbamoyl- Nitrile -nitrile cyano- Aldehyde -al formyl- Ketone -one oxo- Alcohol -ol hydroxy- Thiol -thiol mercapto- Amine -amine amino- Alkene -ene - Alkyne -yne - Alkane -ane - Step 3. Prefix – What is attached to the parent chain, and where? The prefix describes any additional substituents in the structure. Groups such as F (fluoro), Cl (chloro), Br (bromo), I (iodo), NO2 (nitro), almost always appear in the prefix. In addition, we have to determine their positions on the parent carbon chain and include this number in the prefix. Numbering should be done by counting all of the carbons in the parent carbon chain consecutively so that the primary functional group has the lowest possible number. Worked example: Figure 4. Structure and chemical name of 4-hydroxyhexanal Step 1. Parent (How many carbons are in the longest chain?) – 6 carbons as shown in Figure 4. 7 Step 2. Suffix (What is the highest priority functional group present?) - This molecule contains two functional groups, an aldehyde (in blue) and an alcohol (in green). The aldehyde has a higher priority and therefore it is the primary functional group of the molecule and determines that the suffix will be -al. Step 3. Prefix (What is attached to the parent chain and where?) – There is an alcohol attached to the carbon chain and this functional group has lower priority therefore the prefix hydroxyl- will be added to the name. Determining the position of this group is done by starting the numbering so as the primary functional groups has the lowest possible number (in this case 1), therefore giving the alcohol group position 4 as shown in Figure 4. Exercise 2 Give systematic names for the following compounds Practice naming molecules 1.1.3. The importance of acids and bases in Pharmacy The majority of drugs contain weakly acidic or weakly basic functional groups. There are many too that contain both types of groups and such compounds are described as amphoteric compounds. The ionisation state of a drug that contains acidic and/or basic functional groups will depend on the pH of the medium in which it is dissolved. This in turn will influence the solubility of the drug, the way in which it is formulated as a medicine, its absorption and distribution within the body and its pharmacological activity. Many of the pharmaceutical excipients that are used in formulating drugs as medicines also contain acidic and/or basic functional groups. In general, acidic and basic functional groups are capable of ionisation, where an acidic functional group is able to give a proton away becoming negatively charged, O O R R +H OH O 8 and a basic functional group is able to gain a proton becoming positively charged. R NH2 + H R NH3 The pH at which the ionisation is exactly 50% is called the pKa. Table 4 shows the chemical structures and pKa values for some acidic and basic functional groups which are commonly found in drug substances, with examples of drugs in which they are found. The concept of acids and bases, and ionisation of molecules, will be revisited again in Unit 2 in more detail. 9  Table 4. Acidic and basic groups commonly found in drug substances Acidic groups (red) Ibuprofen (analgesic) Carboxyl R-COOH (pKa 2-4) Phenol (used in antiseptic mouth spray) Phenol Ar-OH (pKa ~10) Diclofenamide (antiepileptic) Sulfonamide R-SO2NH2 (pKa 8-10) Phenytoin (antiepileptic) Imide R-CO-NH-CO-R (pKa 8-10) Basic groups (blue) Primary Amphetamine (for treatment of attention deficit hyperactivity disorder) aliphatic R-NH2 amine (pKa 9-10) Isoprenaline (β2-agonist) Secondary aliphatic R-NH-R amine (pKa 9-10) Amitriptyline (antidepressant) Tertiary aliphatic R-N(R)2 amine (pKa 9-10) Benzocaine (local anaesthetic) Aromatic amine Ar-NH2 (pKa 2-4) Pyridoxine (vitamin B6) Pyridine (pKa ~5.5) Dacarbazine (anticancer agent) Imidazole (pKa 6-7) 10  1.1.4. Bonding of atoms and shapes of molecules The shape of a molecule affects how it interacts with other molecules and that in turn can give rise to all kinds of interesting phenomena. One important area is how drugs work in the body. Using this as an example, the drug – a molecule with a specific shape – will commonly interact with a larger molecule, such as protein. This causes a chain of events which give the drug its specific medicinal properties. Understanding how molecules have their particular shapes is therefore very important, especially when we want to design a new drug to fight a particular disease. Note, the information below gives you an explanation of the shapes of molecules and bonding of atoms. The working knowledge that you need is summarised in Table 5. If you find this section difficult, there are several online videos that cover orbitals and hybridisation. Molecular orbitals It was mentioned earlier that at A-level, carbon atoms tend to be drawn with 90° bond angles whereas you (should) already know that the geometry of an alkyl carbon is tetrahedral (triangle-based pyramid). That is, the four bonds are evenly arranged in space to be as far apart from each other as possible (Figure 5). Figure 5. The tetrahedral shape of methane You should have also learned previously that carbon has four electrons in its outer shell available for bonding and that these occupy s (spherical) and p (dumbbell-shaped) orbitals (Figure 6). Orbitals describe where an electron can be found within a certain degree of probability. Think about an orbital as a balloon that represents 90-95% probability of finding an electron. 11  Figure 6. s- and p-orbitals Considering the individual atomic orbitals described above, orbital hybridisation is the concept of mixing these atomic orbitals to form new hybrid orbitals (with different shapes, energies, etc., from the component atomic orbitals) suitable for the pairing of electrons to form covalent bonds in molecules. Adding the one s + three p orbitals together will give us a set of four hybrid orbitals. The new hybrid orbitals are called sp3 after the orbitals from which they are derived and exist in a tetrahedral array (Figure 7). Overlapping sp3 orbitals on adjacent carbon atoms will create an alkane molecule where the carbon atoms all have the requisite tetrahedral geometry and bond angles of 109º 28'. The groups at the ends of an ordinary single bond are free to rotate. Figure 7. sp3 orbital hybridisation resulting in a tetrahedral geometry What about alkenes (carbon-carbon double bond present) where the bond angles at carbon are 120º? This time one of the p orbitals is omitted from the hybridisation, so that: 1  s orbital + 2  p orbitals → 3  sp2 orbitals The result is shown in Figure 8. Three sp2 orbitals protrude around the equator of the carbon atom and the unused p orbital remains at 90º to the sp2 orbitals. 12  Figure 8. sp2 orbital hybridisation resulting in trigonal geometry Figure 9 illustrates what happens when two sp2 hybridised atoms form a bond: sharing electrons by overlap of an sp2 orbital from each atom forms the strong single bond (called a sigma bond, ). Overlap of the parallel p-orbitals forms an orbital that places a cloud of electrons above and below the -bond, this is called a pi-bond (). The  (or “double”) part of the bond is weaker than the  so is much more reactive. Another consequence of the -cloud orbital structure is that the double bond cannot rotate so lends rigidity to the molecule. Figure 9. Bonding of sp2 hybridised atoms to form sigma () and pi () bonds Triple bonds are formed by sp hybridised atoms, that is, two of the p-orbitals are left out of the hybridisation process. sp orbitals lie at 180º to each other to give linear geometry (Figure 10). 1  s orbital + 1  p orbital → 2  sp atomic orbitals (bond angle: 180°) 13  Figure 10. sp hybridisation found resulting in linear geometry, found in triple-bonded structures such as alkynes When sp hybridised atoms bond, the two pairs of “spare” p-orbitals overlap to produce two - bonds that give an electron cloud that encases the -bond (Figure 11). Figure 11. An orbital representation of triple bond formation The hybridisation principles apply to all atoms. Drug molecules typically contain only C, H, N, O, S, F, Cl, P. Nitrogen and oxygen can also be sp2 or sp hybridised in double or triple bonds – but remember the roles played by lone pairs (Figure 12). 14  Figure 12. sp3 hybridisation and bond angles in water and ammonia The oxygen atom of water, has two bonds to H and two lone pairs and so adopts a slightly distorted tetrahedral geometry: since the lone pairs are not formally shared with another atom, the electron density is closer to the nucleus, so repels the bonds slightly more than the bonds repel each other and compresses the bond angle. A similar situation applies in ammonia. Think about hybridisation in an ester or nitrile. Summary of Orbitals and Bonding (what you need to know from this section): -bonds formed by overlap of spn hybrid orbitals – the “single” bond framework of a molecule, electron density is concentrated between nuclei. -bonds made by overlap of p-orbitals. The double and triple bonds – electron cloud around single bonds. These restrict rotation about the bond. Table 5. Summary of atomic hybridisation Number of -bonds Hybridisation Geometry (shape) 4 sp3 tetrahedral 3 sp2 trigonal 2 sp linear You will not be asked to explain orbital hybridisation. You do need to know and understand the information in Table 5. The shapes of individual atoms contribute significantly to the 3D shapes and flexibility (or rigidity) of drugs. A ring of sp2 hybridised atoms (e.g., benzene) has to be planar and rigid. A chain of sp3 hybridised atoms is quite flexible; a ring of 5 or 6 sp3 hybridised atoms is less flexible but all the atoms cannot lie in the same plane. sp hybridised atoms tend to make very straight rigid structures (we will see the ethinylestradiol crystal structure in the computer workshop). 15  1.1.5. Stereochemistry Stereochemistry is concerned with the spatial arrangements of atoms in molecules, and the effects that these have on the physical and chemical properties of the molecules. A detailed understanding of stereochemistry is vital in pharmaceutical science: The clinical efficacy of a drug is critically dependent on its three-dimensional structure The stereochemistry of a drug can impact upon its toxicity and side effects All drugs are chemical entities and a great majority of them contain stereocentres, show stereoisomerism and exist as enantiomers. It is therefore important to understand how drug chirality affects the interaction of the drug with its target and be able to use proper nomenclature in describing the drugs themselves and the nature of the forces responsible for those interactions. Most often, only one form shows correct physiological and pharmacological action. For example, only one enantiomer of morphine is active as an analgesic, only one enantiomer of glucose is metabolised in our body to give energy and only one enantiomeric form of adrenaline is a neurotransmitter. An interesting property of sp3 atoms is that if all four attached groups are different, the molecule cannot be superimposed onto its mirror image (i.e., the two isomers are different compounds) and they are said to be chiral (Figure 13). The separate chiral isomers are a pair of enantiomers. The property of chirality is not unique to molecules: your hands are pair of enantiomers (check this), as are your feet and various other body parts that come in pairs (ears, toes). The only easily detectable physical difference between a pair of enantiomeric molecules is their ability to rotate the plane of polarised light, so they are often referred to as optical isomers. Figure 13. Illustrations of chirality 16 Most natural molecules have chiral centres and occur as single optical isomers, in particular the amino acids which make up proteins. This means that all drug receptors and enzymes are chiral. As a consequence, if a drug is chiral, usually only one enantiomer will interact fully with the receptor, so in theory such drugs should optimally be administered as single enantiomers. The mirror image isomers can be inactive or may have other effects including: producing side effects, countering the effect of the drug or being metabolised to a toxic product. In practice, chiral synthesis or separation can be very expensive so companies market racemic mixtures of synthetic drugs if they can. This can be risky. One of the most notorious cases involving adverse side effects from a supposedly inactive isomer concerns thalidomide. Thalidomide was developed in the 1950s as a sedative and was used to counter nausea during pregnancy (morning sickness). It was found that the drug inhibited the incorrect growth of blood vessels, resulting in abnormalities to the children of mothers taking thalidomide - the most common being limb malformation. Further investigation showed that the (S) enantiomer causes the sedation whereas the (R) enantiomer causes birth defects by interfering with the growth of blood vessels (Figure 14). However, the enantiomers interconvert in vivo, so a single enantiomer drug could never be safe. A variant, lenalidomide, is now under clinical trials as an anticancer drug that works by inhibiting the growth of blood vessels that feed tumours. Figure 14. Enantiomers of thalidomide Another case is methadone. It is relatively cheap to manufacture methadone as the racemic mixture. The (R) enantiomer is a powerful analgesic and is used in treatment of heroin addiction; the (S)-enantiomer is cardiotoxic at high doses and accounts for death from methadone overdose. Diastereoisomers These are stereoisomers of each other but they are not specifically mirror images of each other. Cis-trans diastereoisomers - found in molecules with double bonds and saturated rings (Figure 16). Figure 15. Examples of cis and trans isomers Chiral diastereoisomers - chiral isomers which are not enantiomers, i.e. not mirror images of each other. These differ in physical, chemical & biological properties. Found only in molecules with multiple chiral centres, e.g., sugars. Absolute stereochemistry (R or S designation) is the same at some centres but must differ in at least one. 17 Summary of definitions: Chiral centre: sp3 atom bearing four different substituents Enantiomers: isomers that are mirror images of each other and cannot be superimposed Diastereoisomers: stereoisomers that are not enantiomers Racemic mixture (racemate): an equal mixture of both enantiomers of a compound, has no measurable optical rotation Exercise 3 Draw and label enantiomers of 1. 1-chloroethanol 2. methadone 1.2. Interactions between molecules Every biological event involves molecular interactions. Molecules must interact (bind) to initiate an action, and then separate. Whether it is an enzyme binding to its substrate, a hormone binding to its receptor on a membrane, or RNA being transcribed on a DNA template, all require to be bound in a precise orientation for a short time. Such ‘recognition’ events are made possible through intermolecular interactions. Dipole interactions Bond polarity is a useful concept for describing the sharing of electrons between atoms. The shared electron pairs between two atoms are not necessarily shared equally and this leads to a bond polarity. Atoms, such as nitrogen, oxygen and halogens, that are more electronegative (pulling the electron density) than carbon, have a tendency to have partial negative charges. Atoms such as carbon and hydrogen have a tendency to be more neutral or have partial positive charges. Thus, bond polarity arises from the difference in electronegativities of two atoms participating in the bond formation. Figure 16. Distribution of electrons in bonds from perfect covalent to ionic. In medicinal chemistry, most bonds fall into the polar covalent category. Pink shading shows the electron cloud. 18 The difference in electronegativity between atoms comprising functional groups causes partial charge separation resulting in dipoles. When the dipoles of a drug and receptor come in close proximity (more stringent distance requirement), they produce dipole interactions. The more electronegative oxygen atom attached to carbon atom in carbonyl groups (ketones, esters, amides) results in such dipoles, and thus dipole interactions, with the biological receptors when they occur in drugs. Such interactions are very common since most drugs possess carbonyl or other functional groups exhibiting partial charge separation and dipoles. Figure 17. Dipole interactions of active site serine with, A) chlorpheniramine (ion-dipole), and B) captopril (dipole-dipole). Hydrogen bonding Hydrogen bonds are a subtype of dipole interactions which can form whenever a strongly electronegative atom (e.g., oxygen, nitrogen and fluorine) approaches a hydrogen atom which is covalently attached to a second strongly electronegative atom. For example, hydrogen bonding can occur between a carbonyl and amino group (Figure 17). Figure 18. An example of hydrogen bonding between a carbonyl and amino group. A variety of such groups, containing an electronegative atom, occur in biological molecules (e.g., hydroxyl, carbonyl, amino). In every hydrogen bond there is the acceptor, a hydrogen atom attached to an electronegative atom, and the donor, an electronegative atom (O, N or F) with one or more lone pairs of electrons. The hydrogen bond is of particular importance in biological systems and it is said to be the ‘bond of life’. It may look like a simple charge-charge interaction, but the hydrogen bond has special characteristics. With this type of intermolecular interaction, the hydrogen bond has an optimum length and is linear. For example, in the hydrogen bond between a carbonyl and amino group shown above, we could draw a straight line between the oxygen and the nitrogen, along which the bond would be aligned. In other words, the direction of the bond is relatively constrained. Compared to other intermolecular interactions, the hydrogen bond is also relatively strong. These properties of the hydrogen bond have important consequences in biology, constraining the distances and orientations between molecules. Therefore, hydrogen bonds are significant in determining the structures of proteins, and in holding together the double-stranded DNA molecule. 19 Charge-charge (ionic) interactions Charge-charge interactions are electrostatic in nature (as indeed are hydrogen bonds) but, unlike hydrogen bonds, the distance between interacting groups is not ‘fixed’ and the alignment of the interaction is not so important. Any group or atom in a molecule which carries a charge, as a result of either the electronegativity difference between atoms, the presence of an acidic or basic group, or an atom with a lone pair of electrons, can engage in charge-charge interactions. That interaction can be attractive (with an oppositely charged group, Figure 18) or repulsive (with a similarly charged group). Both are important in the interaction of biological molecules. Figure 19. An example of a charge-charge interaction between ionised carbonyl and amino groups. Interaction between charged groups is likely to be very pH-dependent. For example, proteins consist of amino acids, linked together through peptide bonds to produce long polypeptide chains. The side groups of amino acids in the chain frequently contain carboxyl (-COOH) or amino (-NH2) groups. At physiological pH, these two groups will carry a charge: the carboxyl group dissociates, losing a proton (H+) and becoming negatively charged, -COO-; the amino group associates with a proton to become positively charged (-NH3+). On cellular proteins, therefore, there are likely to be numerous charged groups, where charge-charge interactions become possible between molecules, or indeed within molecules. Hydrophobic interactions Hydrophobic (meaning literally ‘water hating’) interactions differ from those intermolecular interactions already described above, in that they are not electrostatic in nature. Nor is there any attraction, or repulsion, between hydrophobic groups. The basis of hydrophobic interactions resides in the behaviour of water. Hydrophobic groups are so called because they are insoluble in water and do not interact with it. Hydrophilic (‘water loving’) groups are soluble in water, and do interact with it. The functional groups that we have come across so far, such as carboxyl (-COOH), amine (NH2), hydroxyl (-OH), are all polar groups. They all contain an electronegative atom that will induce a dipole and an unequal distribution of charge. Water is a polar molecule and it will interact with other polar groups through electrostatic interactions. On the other hand, hydrophobic groups do not contain any particularly electronegative atoms. For example, the apolar methyl group (-CH3) consists of carbon covalently bonded to three hydrogen atoms. The electronegativities of carbon and hydrogen are similar and there is little tendency for electrons to assume an unequal distribution. The methyl group is therefore ‘neutral’. Neutral groups have no way of interacting with water. The water solvent will respond to such apolar groups by ‘excluding’ them. Apolar groups do not interact or repel each other, but rather come together through the action of the water solvent (Figure 19). 20  Figure 20. An example of a hydrophobic interaction between a benzene ring and a methyl group. The ’hydrophobic effect’ is of major importance in biology. Biological membranes form a hydrophobic barrier which defines a cell, or cellular organelle. Protein molecules fold and adopt specific three-dimensional structures, driven by the ‘expulsion’ of hydrophobic groups from water. The hydrophobic groups in proteins are found buried within the protein’s interior, where there is no water. 21 2. Complex molecules 2.1. Amino acids and proteins For more information about amino acids and their role in protein formation, please study the directed reading in Chapter 6 “Amino Acids in Proteins” of Marks’ Basic Medical Biochemistry, A Clinical Approach Proteins are composed of one or more chains of repeated units (amino acids) connected together by peptide bonds. Proteins are a fundamental component of all cells and are involved in almost every complex function in multicellular organisms, including digestion, neurotransmission and immunity from infection amongst many others. 2.1.1. Amino acids Amino acids are a class of molecules that contain an acidic group (carboxylic acid) and a basic group (amine) (Figure 20). All protein molecules studied to date are composed almost exclusively of only 20 amino acids (standard amino acids) which each have different side chain (“R”) groups (Figure 20). All standard amino acids are classed as “α-amino acids” because they all have an amino group on the first (α) carbon atom adjacent to the carboxylic acid group. Moreover, with the exception of proline, they are primary amino acids since they have a primary amine group (proline has a secondary amine group). Figure 21. General structure of amino acids With the exception of Glycine, the α-carbon atoms in the standard amino acids are chiral (or asymmetric) centres. This means each amino acid could exist in one of two enantiomers. All the 20 standard amino acids are fundamental components of proteins in the human body. However, our bodies can only synthesise 10 of the standard amino acids. The rest can only be synthesised by plants and microorganisms and must therefore be obtained through food. Therefore, these are termed essential amino acids as they are an essential part of a healthy human diet. Arginine is often referred to as an essential amino acid even though it can be synthesised by the human body. This is because the majority of synthesised Arginine is used 22 to produce urea – hence a dietary source of arginine remains essential, especially in infants and growing children. Amino acids can be divided into FIVE major groups according to the nature of their side chain (Figure 21). Side groups can be classified as nonpolar (straight or branched chains, that may or may not be cyclic), aromatic, uncharged polar, charged (negative/acidic or positive/basic side chains) or sulphur-containing. Figure 22. Standard amino acids classified according to the functional groups of the side chain 2.1.2. Acid-base properties of amino acids Amino acids have at least two or, for those with an ionisable side chain, three acid-base groups. Therefore, they are amphoteric, i.e., they can react as either acids or bases depending on the pH of the medium, see the equation below. In acidic media (below pKa1), the carboxylic group is unionised while the amino group is in its ammonium ionised state (left hand structure). In basic media (above pKa2) the opposite is true with the carboxylic acid group in its ionised carboxylate form while the amino group is in its non-ionised state. 23  O pKa1 O O O pKa2 H3N H3N H2N H2N OH O O OH protonated form zwitter ion deprotonated form Does not exist acidic pH neutral pH alkaline pH Around neutral (physiological) pH, both groups are ionised leading to zero net charge for the molecule. Compounds with this property are called zwitterions (or sometimes dipolar ions). Exercise 4 1. What structural elements constitute an amino acid? 2. What differentiates polar and non-polar amino acids? 3. Which pairs of amino acids might contribute to protein conformation by forming ion pair interactions between their side chains at physiological pH? 2.1.3. Peptide bonds and nomenclature Amino acids can be joined together by losing a water molecule. The resulting molecule is called a dipeptide and consists of two amino acids linked though a CO-NH amide linkage also known as a peptide bond (Figure 22). More amino acids can be present to either side of the dipeptide to form a linear polypeptide chain. Polypeptides of more than 100 amino acid residues are called proteins. As each residue could be derived from one of 20 different amino acids, the number of different protein molecules that could theoretically be formed is enormous. H2O O O O H2N + H2N H2N OH OH OH N H O A dipeptide the amide bond is shown in blue Figure 23. Formation of a peptide bond Amino acids are typically identified using either a three letter or single letter code for their names (Figure 21). For example, Alanine is referred to as “Ala” or simply “A”. Meanwhile, short peptide chains are named by the amino acid residues starting with the first three letters of the name of the first amino acid at the amino terminus (also known as N-terminus) and then the name of the subsequent amino acids till the carboxylic acid terminus (known as C-terminus). 24 For example, a tripeptide composed of Alanine, Glycine and Valine would be named “Ala-Gly- Val”. 2.1.4. Modified/non-standard amino acids As stated above, the standard 20 amino acids are almost exclusively used to make proteins in all living cells. However, after the formation of a new protein (translation) a few amino acid residues may be modified in order to establish a specialised protein structure (post- translational modification). Examples of this type of modification are 4-hydroxyproline and 5- hydroxylysine (modified version of the amino acids proline and lysine, respectively). Both of these compounds are essential components of collagen (a very important structural protein in our bodies). Moreover, proteins that form complexes with nucleic acids (known as histones) usually contain modified amino acids in their structure that regulate the ability of genes to be turned on. In addition to their role in polypeptides and proteins, amino acids and their derivatives function as chemical messengers between body cells or as intermediates in various metabolic processes. Examples include the roles of γ-aminobutyric acid (also known as GABA which is a derivative of the amino acid glutamate) and dopamine (a derivative of tyrosine) as neurotransmitters. 2.1.5. Proteins (an introduction) Proteins are the most abundant organic matter in living organisms. Proteins have several functions that can be divided in seven categories: a. Structural proteins provide structural support and framework for the cells and the body. Example – collagen. b. Movement proteins are the main components in muscles. Example - myosin. c. Transport: specialised transporter proteins in cell membranes allow the movement of molecules (including lipids and gases) in and out of cells. Example – GLUT family of glucose transporters. d. Metabolic: enzymes that catalyse the breakdown and synthesis of biological macromolecules to maintain cellular function. Example – hexokinase, which converts glucose to glucose-6-phosphate and is the first step in glycolysis. e. Communication: hormones and neurotransmitters control various biological activities by binding to specific cellular receptors. Example – insulin. f. Defence and protection: proteins contribute to the barrier properties of skin. Example - keratins; proteins that play a major role in the immune system, e.g., antibodies; and coagulation factors, e.g., thrombin. The functionality of any protein is dictated by its amino acid sequence which ultimately governs its structure. There are FOUR levels of protein structural complexity (Figure 23): 25  Figure 24. Protein structure levels i. Primary structure is the simple linear sequence of amino acids in the polypeptide chain of a protein. Secondary structure is the local arrangement of amino acids with respect to each other to form defined, hydrogen-bonded structures. This usually leads to either -helix or -sheet secondary structures (Figure 24). In an -helix, hydrogen bonds form between the oxygen atom of a carbonyl group, and the amide NH hydrogen of the amino acid residue 4 places down the strand. Side chains of the individual amino acids point outwards from the densely populated core - this improves the stability of the helix by preventing steric hindrance. In the -sheet, hydrogen bonds are formed between peptide chains lying adjacent to each other. Parallel sheets occur when these strands run from N to C terminals in the same direction. The opposite is true for anti-parallel sheets, an arrangement that often occurs when a protein strand folds back on itself. ii. The form of the fold depends on the amino acids in the polypeptide – i.e., folding is dictated by primary structure. The alpha-helix arrangement is more common but both can exist in the same chain connected by flexible loops. iii. Tertiary structure is the complex coiling and folding of secondary structure elements that establishes the final three-dimensional structure of the protein. The final structure is the result of the interactions between the polypeptide chain and the surrounding water molecules, and also interactions between the R groups of amino acids in different parts of the chain. iv. Quaternary structure describes the interaction between individual polypeptides to form a multi-subunit protein complex. This can also incorporate other molecules: for example, the red blood cell protein haemoglobin is made up of 4 subunits, each with its own iron-containing “haem group” capable of binding oxygen. 26  Figure 25. The alpha-helix and beta-sheet Levels of polypeptide (protein) structure Proteins are categorised in two main groups according to their overall structure. Globular proteins. These proteins typically have an irregular, rounded shape. Examples include myoglobin, haemoglobin, hormones (e.g., insulin) and enzymes (e.g., coagulation factors in the blood, proteases used to digest food in the stomach). Their core is typically made up of non-polar amino acid residues, whilst charged and polar residues coat the outer surface. This means that these proteins are readily soluble in the cytoplasm and other aqueous environments (e.g., blood, digestive tract). Globular proteins make extensive use of alpha helices. Fibrous proteins: These have linear helical or sheet-like structures. This is typically due either to association of secondary structure domains of individual strand/beta- sheet structures (e.g., collagen triple helix), or due to quaternary association of several individual sheets (e.g., in keratin). Fibrous proteins are tough and usually water insoluble, playing a protective or structural role in the body. The relationship between protein shape and functionality is absolutely critical. Changes in protein structure can lead to a diminishing or loss of function. Example. Sickle cell anaemia arises due to the substitution of a single amino acid in haemoglobin (termed HgbS) resulting in a mutated haemoglobin with a different primary 27 sequence. Normal red blood cells are quite elastic, which allows the cells to deform to pass through capillaries. In sickle-cell disease, low oxygen tension alters the shape of HgbS, and promotes red blood cell sickling (bending to a curved shape) which damages the cell membrane and decreases elasticity. These cells fail to return to normal shape when normal oxygen tension is restored. As a result, these rigid blood cells are unable to deform as they pass through narrow capillaries, leading to vessel occlusion and ischaemia. The actual anaemia of the illness is caused by faster than normal destruction of the red cells because of their altered shape. The tertiary or a quaternary structure of a protein can also be altered by changing conditions such as temperature, pH and ionic composition of the environment. However individual proteins respond differently to changes in these parameters. Example. While digestive enzymes such as trypsin work best at physiological pH (approximately 7.4) found in the small intestine, pepsin works best at the acid environment of the stomach (pH 2) and is virtually inactive at pH 7 (Figure 25). Figure 26. Optimal pH for two enzymes Loss of protein structure can be either reversible or irreversible depending on the severity of change in the conditions. When permanent loss of tertiary structure occurs, the protein is said to be “denatured”. Denaturation can also occur following non-enzymatic modification. For example, glucose can bind to exposed amino groups to form glycosylated proteins, which over time can form large chains known as Advanced Glycosylation End products (AGEs) that are implicated in several chronic diseases such as diabetes and Alzheimer’s. However, it should also be remembered that proteins are also combined enzymatically with either sugars (glycoproteins and proteoglycans), lipids (lipoproteins) or other chemical groups to form new molecules that perform vital roles in the body. Readers wishing to know more details about protein structure are directed to Chapter 7 of Marks' Basic Medical Biochemistry: A Clinical Approach. 28  Case study 1: Stabilisation of injectable protein drugs using sugars The “hydrophobic effect” is where non-polar substances aggregate in aqueous solution, minimising disruption to the hydrogen bonds between water molecules. When proteins are removed from their normal environment, their activity can be lost due to changes in tertiary and quaternary structure. Interactions between hydrophobic amino acid residues minimise their surface area exposure to water. Freeze-drying of proteins can therefore enhance their stability and shelf life. Dried proteins are generally formulated into an “amorphous”/“glassy” (non-crystalline) or a crystalline powder called a “cake”. Protein crystals are a more stable form of storage, but their high molecular weights can make proteins hard to crystallise. Sugars, such as mannitol and sucrose, are added to the protein before freeze-drying. These sugars have a relatively large number of polar functional groups and can repel Normal (left) and collapsed (right) cakes hydrophobic amino acid residues. Therefore, sugars act of freeze-dried product as cryoprotective agents, reducing hydrophobic residue interactions with water, and the overall denaturation of the protein. Sugars also act as a bulking agent, making it easier to weigh and dispense the correct dose into vials. Finally, the addition of sugar enhances the appearance of the cake: a collapsed cake may still be stable but looks relatively unappealing. 29  2.2. Nucleic acids Nucleic acids are essential organic compounds composed of carbon, hydrogen, oxygen, nitrogen, and phosphorous. They encode all the information needed to build various structural and functional proteins in the form of “genes” (specific DNA sequences that carry the instructions to make proteins). Thus, they control all aspects of our bodies from the colour of the eye to the metabolism of various organic compounds in our cells. There are two classes of nucleic acids in our body: DNA (Deoxyribonucleic acid) and RNA (Ribonucleic acid). DNA consists of two biopolymer strands coiled around each other to form a double helix in which specific bases (adenine, guanine, thymine and cytosine [or A, G, T and C]) interact in defined pairs. Thus, A is always paired with T, and G is always paired with C (Figure 26, left). These interactions occur via hydrogen bonding. The expression of genes within cells requires two processes: transcription and translation. Transcription: Double stranded DNA is transcribed to produce single stranded ribonucleic acid (RNA). Three major types of RNA (messenger; ribosomal and transfer) are transcribed from DNA and subsequently participate in the process of translation (the synthesis of proteins). Messenger RNA (mRNA) is the type of RNA that carries the genetic code required to make proteins. Translation: mRNA is translated into proteins in the cytoplasm on ribosomes—structures that contain proteins complexed with ribosomal RNA (rRNA). Transfer RNA (tRNA) carries individual amino acids to the ribosomes, where they are joined in via peptide bonds to form proteins. During translation, the sequence of nucleic acid bases in mRNA is read in sets of three (each set of three bases constitutes a codon). The sequence of codons in the mRNA dictates the sequence of amino acids in the protein. Thus, DNA is said to comprise an organism’s “genetic code”. (Figure 26, right) Figure 27. DNA structure and the genetic code Advances in understanding the structure and the function of those molecules have led to many important applications in several fields including: Biotechnology: defined as the use of molecular methods to modify and engineer the genetic material of living cells so they will produce new substances and/or perform new functions. 30 For example, cloning of the human insulin gene allowed for industrial-scale production of recombinant synthetic insulin proteins in genetically modified bacteria. The purified insulins are then used to make formulations used to treat patients with diabetes. Before the development of this technique, insulin had to be purified from the pancreas of cattle, pigs and other farm animals. Molecular Diagnostics or Genetic Testing: defined as medical tests that identify molecular changes in chromosomes, genes, or proteins. Depending on the test being used, the results can confirm or rule out a suspected genetic condition, help determine a person's chance of developing or passing on a genetic disorder or help predict how quickly they will metabolise specific drugs. Example 1: Conventional prenatal tests for chromosomal abnormalities such as Down's Syndrome rely on analysing chromosome number and appearance of the chromosomes (Down Syndrome is diagnosed by the presence of an additional third copy of chromosome 21). Example 2: Genetic counselling: some diseases, such as sickle cell disease, occur when a person inherits two abnormal copies of the haemoglobin gene, one from each parent. A person with a single abnormal copy does not usually have symptoms and is said to have “sickle-cell trait” and are termed “carriers”. The presence of the abnormal haemoglobin gene is performed by genetic testing of a blood sample. Example 3: Optimal treatments with specific medicines (Pharmacogenomics): an individual patient's “single nucleotide polymorphisms (SNPs)” (defined as slight differences in their DNA sequence) can help predict how quickly they will metabolise particular drugs. For example, the liver enzyme CYP2C19 metabolises several medicines, including the anti- platelet medicine clopidogrel. Clopidogrel is a prodrug (an inactive precursor) which is prescribed to patients at risk of heart attack or stroke. The liver enzyme CYP2C19 catalyses the conversion of clopidogrel into its pharmacologically active form (R-130964), which irreversibly binds P2Y12 purinergic receptors on the platelet membrane to inhibit binding of ADP and thus block platelet activation to reduce the risk of thrombosis (Figure 27). Some patients possess SNPs in specific places on the CYP2C19 gene that make it a poor metaboliser of Clopidogrel. Therefore, by testing for the presence of these SNPs, clinicians can find out whether the drug will be fully effective for that individual patient and adjust the dose accordingly. 31  Figure 28. Anti-platelet action of Clopidogrel Therefore, understanding the structure of nucleic acids is not only essential to understanding how changes in DNA sequence and structure cause disease but can also identify how diseases can be treated. For more information about the structure and function of nucleic acids please study the directed reading in Chapter 12: “Structure of the Nucleic Acids” of Marks' Basic Medical Biochemistry: A Clinical Approach. DNA structure from RNA to protein synthesis 32  2.3. Sugars and Polysaccharides Saccharides (Greek for sugar) are the most common organic molecules on earth, typically being produced from simple molecules (CO2 and H2O to produce compounds of empirical formula C(H2O)n) during photosynthesis. The basic units of carbohydrates are called monosaccharides. Many monosaccharides act as energy substrates that can (largely) satisfy the energy requirements of biological systems. The monosaccharides 2-deoxyribose and ribose are an integral part of DNA and RNA nucleic acids, respectively (Figure 28). O N NH O O OH OH N HO HO N NH2 HO HO O HO OH ribose deoxyribose OH guanosine - example of a nucleotide Figure 29. Structures of ribose, deoxyribose, and guanosine Polysaccharides are carbohydrate macromolecules, consisting of many monosaccharides (typically hundreds) connected together via covalent bonds, in both linear and branched chains. They are important molecules in all biological systems. Glycogen and starch: these are critical energy stores in animals and plants respectively. In mammals, glycogen is synthesised and stored in liver and skeletal muscle. Cellulose: this is the fundamental structural component of plant cell walls. Carbohydrates of intermediate chain length (between 3 and 10 monomers) are called oligosaccharides. Many oligosaccharides can combine with proteins to form glycoproteins or combine with lipids to form glycolipids. Both of these have structural and regulatory functions in the human body. The stereochemistry of monosaccharides affects the physical properties of the sugar. For example, β-lactose is twice as sweet as α-lactose. Meanwhile, α-lactose has better powder- flow properties and therefore is more commonly used as an additive in pharmaceutical industry for tablet production than β-lactose (Figure 29). HO OH HO OH OH OH OH O HO OH O HO HO O O HO O O OH OH galactose OH glucose OH b-lactose a-lactose Figure 30. Structures of α-lactose and β-lactose 33  Stereochemistry of monosaccharides Disaccharides For more about carbohydrate structure and function, please study the directed reading in Chapter 5: “Structures of the Major Compounds of the Body” of the Marks' Basic Medical Biochemistry: A Clinical Approach (read “Carbohydrates” section). 2.4. Lipids Similar to carbohydrates, lipids consist of hydrogen, oxygen and carbon atoms. However, the oxygen content of lipids is much less than that of carbohydrates, meaning that lipids are typically water insoluble. Dietary lipids are classified as fats (solid at room temperature) and oils (liquid at room temperature), and this classification is related to the profile of saturated and unsaturated (containing double bonds) fatty acids within a fat molecule (see video below). Most dietary fats typically exist as triglycerides (also known as triacylglycerols), wherein 3 fatty acids are esterified to a molecular backbone of glycerol. Mono- and diglycerides also exist. In the body, droplets of lipid held in the adipocytes are an excellent long-term energy reservoir, taking up very little space compared to glycogen. Lipids are also important structural compounds with phospholipids being important components of cell membranes. Lipids Fats as fuel Saturated and unsaturated fats Another lipid, cholesterol, is essential for cell membrane fluidity and is a metabolic precursor for bile, the active form of vitamin D and all estrogens. Cholesterol is transported using special carriers called lipoproteins. LDL (low density lipoproteins) essentially transport cholesterol from liver and intestine to the rest of the body. Meanwhile HDLs (high density lipoproteins) remove excess cholesterol and transfer it back to the liver. Cholesterol Different types of lipoproteins 34  Case study 2: Atorvastatin and blood lipids High levels of LDL cholesterol in the blood are a risk factor for atherosclerosis and cardiovascular disease. Statins are a class of drugs used to reduce LDL levels and are the most commonly prescribed drugs in the UK. Atorvastatin (aka Lipitor®) is a potent competitive inhibitor of HMG-CoA reductase. This enzyme plays a pivotal role in producing mevalonic acid, which is an upstream precursor to cholesterol. Inhibition of this rate- Atorvastatin: a competitive HMG-CoA limiting step decreases cholesterol reductase inhibitor production, and also the production of a variety of isoprenoids that may have roles in pro-inflammatory signalling. Atorvastatin is specifically recommended by NICE as the first statin treatment to use in people with CVD. The drug is also recommended as a method of primary prevention in those presenting with a 10% or greater chance of developing CVD over the next 10 years and should be considered for use in all adults with type 1 diabetes (NICE, 2014). For more about carbohydrate structure and function, please study the directed reading in Chapter 5: “Structures of the Major Compounds of the Body” of the Marks' Basic Medical Biochemistry: A Clinical Approach (read “Lipids” section). 2.5. Vitamins Vitamins are organic compounds that (amongst other activities) act as cofactors for enzyme reactions in various essential physiological processes. The word “vitamin” describes how these compounds cannot be synthesized in sufficient quantities by a given organism and must therefore be obtained from the diet. Thus, the term is conditional both on the circumstances and the particular organism. For example, ascorbic acid (vitamin C) is a vitamin for humans but not other animals. Over time, a diet deficient in a vitamin will give rise to a specific vitamin deficiency disease. Vitamins are classified according to their water solubility. Fat-soluble vitamins can be stored long term in the body, whilst water-soluble vitamins are readily excreted. The original names of vitamins (A,B,C etc.) are based upon their order of discovery – today it is common to use the official chemical names. The Reference Nutrient Intake (RNI) is a daily intake calculated to satisfy the requirements of 97.5% of the population, based on a 1991 report by the Department of Health (DoH). 35  2.5.1. Water-soluble vitamins (vitamins B, C) “B complex” vitamins Whilst these vitamins are not physically bound together (i.e., not a true complex) they represent a range of vitamins that typically occur in the same foods and are associated with various aspects of energy metabolism. Vitamin B1 or Thiamin (pictured left) is widespread in both animal and plant kingdoms, particularly concentrated in tissues high in carbohydrate and/or associated with carbohydrate metabolism (e.g., meat, cereal germ). Thiamin occurs in the free form, as thiamine di-phosphate (also known as thiamin pyrophosphate or TPP) or as a tri-phosphate. Thiamin is essential for carbohydrate metabolism, and therefore growth and development. TPP is a prosthetic group (a tightly bound non-protein group forming part of a functional protein) of several enzymes involved in the generation of intermediates in the Krebs cycle. “Beriberi” is a potentially fatal thiamine deficiency disease typified by weight loss, sensory/cognitive dysfunction, weak/painful limbs and heart problems. Vitamin B2 or Riboflavin (pictured right) has a similar distribution to thiamine. In the body, riboflavin exists predominantly as flavin mononucleotide (FMN) or flavin- adenine dinucleotide (FAD) which are prosthetic groups in a variety of respiratory enzymes involved in energy release. Whilst deficiency can lead to ariboflavinosis, it is rare to find this in isolation in humans as sufferers typically present with multiple overlapping deficiencies. Vitamin B3 or Niacin is the name given to both nicotinic acid (pictured left) and its amide. Meats, fruits and vegetables are good sources of niacin. Niacin occurs in vivo as part of the co-enzymes NAD and NADP, and is thus essential for energy metabolism. Niacin deficiency results in pellagra, the pathological impact of which is broad and results in death if not treated. The active form of Vitamin B6 is pyridoxal-5-phosphate (pictured right). However, B6 exists as a variety of inter- convertible forms such as pyridoxine (most common B6 supplement) pyridoxol (only found in plants) and pyridoxamine (only found in foods of animal origin), all of 36 which can also be phosphorylated. Meat, egg yolk, and wheatgerm are abundant sources with less found in milk and cheese. B6 molecules are prosthetic groups to a variety of enzymes involved in amino acid/glucose/lipid metabolism, and the synthesis of haemoglobin. Therefore, whilst deficiency symptoms are similar to those for other “B- complex” vitamins, anaemia is also possible. Vitamin B7 or Biotin (pictured left) can be found in most foods. There is no declared RNI for biotin. Biotin is a prosthetic group in enzymes that play a role in amino acid/glucose/lipid metabolism. Deficiency is rare, but can happen when patients utilise parenteral nutrition for long periods. Vitamin B9 or Folic acid (pictured right) occurs in the diet as a group of polyglutamyl conjugates. Liver and leafy green vegetables are good sources. Tetrahydrofolic acid and its 5-methyl form (5-methyltetrahydrofolate) are major active forms in vivo, and are co-enzymes in the metabolism of one-carbon units, the production of nucleotides, nucleic acids, and the metabolism of haem. Folic acid deficiency is most commonly associated with complications in pregnancy – babies are more likely to be premature, with low birth weight and an increased risk of neural tube defects, such as spina bifida. Thus, it is recommended that folic acid is taken while attempting to get pregnant until 12 weeks pregnant. 37  Vitamin B12 or cobalamin is synthesised by bacteria, meaning there is little to be found in dietary plants. Liver, meat, eggs and milk are good sources. Cobalamin is a co-enzyme responsible for metabolism of one-carbon units, DNA synthesis and formation of red blood cells. When ingested, vitamin B12 combines with a protein in the stomach called “intrinsic factor”. This mix of vitamin B12 and intrinsic factor is then absorbed in the distal ileum. Pernicious anaemia causes your immune system to attack the cells in your stomach that produce intrinsic factor, which makes your body unable to absorb vitamin B12. Vitamin C Vitamin C “activity” refers to both ascorbic acid and its oxidised form dehydro-ascorbic acid (DHAA). Fruits and vegetables are the best source of ascorbic acid. However, the major source of vitamin C in the British diet is the potato. L-ascorbic acid dehydro-L-ascorbic acid Ascorbic acid is perhaps best known (to chemists) as an antioxidant. However, ascorbic acid is an enzymatic co-factor (a molecule that increases the rate of reaction or is required for enzyme function) for hydroxylation reactions, and thus necessary for the formation of collagen (cartilage, scar tissue, blood vessels) and the production of noradrenaline from dopamine. Ascorbic acid also enhances the uptake of dietary non-haem iron. Ascorbate deficiency is responsible for scurvy, whereby insufficient cross linking of collagen results in slow healing of wounds, bleeding gums and mucous membranes, tooth loss, pale skin and nervous dysfunction. 38  2.5.2. Fat-soluble vitamins (vitamins A, D, E, K) Vitamin A or Retinol Retinol (pictured right) is considered the major active form of vitamin A but this can be converted to other retinoids in vivo (retinal and retinoic acid) which also contribute. We do not get retinol directly from food, rather we consume retinol precursors which are converted into retinol. Therefore, in terms of typical consumption we can refer to International Units (IU – 0.3mg) or Retinol Equivalents (RE – 1mg). Vitamin A derived from animal tissues has a high degree of bioavailability and is rapidly absorbed. Retinyl esters of long chain fatty acids (e.g., Retinyl palmitate, pictured left) are hydrolysed then re-esterified by intestinal mucosa during absorption, then bound by retinol-binding protein in the liver. This form can be released from the liver as needed, and subsequently hydrolysed to retinol. Butter, cheese and cod liver oil are good sources. As fat-soluble vitamins accumulate in the body, the over consumption of concentrated animal sources of vitamin A is associated with a risk of toxicity. Vitamin A derived from plant sources will be absorbed after the conversion of carotenoids (yellow/orange pigments) to retinol in the small intestine. For example, β-carotene (pictured above) can be converted into 2 molecules of retinol, as it has 2 beta ionone rings. Conversion is adaptive and inversely related to β-carotene intake, meaning the risk of vitamin A toxicity from consuming plant sources is low. Vitamin A is used to produce rhodopsin (a combination of opsin protein and retinal prosthetic group), which is essential for vision. Vitamin A also plays a role in bone metabolism, whilst retinoic acid is necessary for gene transcription, reproduction and skin health. The carotenoids and vitamin A also have antioxidant activity. Early signs of deficiency are night blindness and skin legions. Acute cases can lead to abnormal bone development, reproductive disorders and death. 39 Vitamin D Vitamin D occurs in the diet chiefly as cholecalciferol. We can make this vitamin ourselves in the skin in sunlight through the conversion of an intermediate in the cholesterol cycle (see below). However, as this can be confounded by many factors (e.g., clothing, ethnicity, seasonal differences in sunlight), dietary sources are important in the UK. Cholecalciferol can be found in dairy products and egg yolk but oily fish (e.g., mackerel) is the best dietary source. Cholecalciferol is converted in the liver to calcifediol which is converted to calcitriol (the active form of vitamin D) in the kidneys. Calcitriol increases blood levels of calcium by increasing its uptake in the intestine (along with its phosphate counter- ion), and through increased resorption of bone mineral. Deficiency can result in rickets, which causes bones to become soft and weak, which can lead to bone deformities. Vitamin E “Vitamin E activity” refers to both tocopherol and tocotrienol, α-Tocopherol (methylated at R1 and R2) has the highest level of vitamin E activity. Vitamin E is a potent antioxidant, responsible for protecting lipids in cell membranes from oxidative damage. Due to their role carrying oxygen around the body, red blood cells are at particular risk of oxidation in vitamin E-deficient patients. 40 Vitamin K “Vitamin K activity” refers to a group of compounds containing an active naphthoquinone ring. Vitamin K1 is phylloquinone produced during photosynthesis and is therefore found at highest levels in leafy green vegetables. The vitamin K2 group are the menaquinones. Whilst K2 compounds are found in meat, eggs and dairy products, our own gut bacteria also produce them. Phylloquinone – K1 Menaquinone structure – K2 Vitamin K is essential for blood clotting/coagulation as it is a co-factor for a liver enzyme (- glutamyl carboxylase) required to carboxylate glutamate residues in clotting factors II, VII, IX and X in order to make them fully functional. Therefore, vitamin K deficiency leads to bleeding disorders. Conversely, the widely used anticoagulant drug warfarin, which is given to reduce risk of coagulation in patients at risk of developing clots, works by binding vitamin K epoxide reductase, the enzyme that recycles oxidized vitamin K1 to its active reduced form after it has participated in -glutamyl carboxylase-mediated clotting factor carboxylation. Exercise 5 1. What are the key roles each of the vitamins fulfil? 2. Which of the vitamins are fat-soluble? What does this mean for their long-term storage and potential for toxicity? 41  2.6. Enzymes and inhibitors Enzymes are proteins that catalyse the conversion of substrates to products by lowering the activation energy required to achieve the “transition state” stage beyond which product formation becomes irreversible (Figure 30). As a result, enzymes increase the rate at which chemical reactions can occur without being consumed or permanently altered themselves (Figure 31). Virtually every chemical reaction within the human body is carried out by enzymes. Therefore, understanding enzymes is crucial to understanding health, disease and treatments for disease. Figure 31. Induced fit theory of enzyme activity Figure 32. Graph illustrating activation energy needed for a reaction with and without an enzyme Enzymes exhibit specificity – only molecules that are the correct shape and can bind to the active site of the enzyme are potential substrates for that enzyme. The shape of this active site is determined by the tertiary/quaternary structure of the protein, and the fit of a substrate can be further improved by hydrogen bonding and other intermolecular forces upon docking – i.e., some enzymes can change the shape of their active site to accommodate certain substrates. Different enzymes work at different rates (e.g., hydrolytic enzymes can typically process 20,000 substrates a second), but this speed also relates to the concentration of available substrates in the surrounding area (i.e., high substrate concentration = faster production rate). However, once an individual enzyme is operating at maximum speed it is 42 said to have met its saturation limit – i.e., further increases in substrate concentration will not improve the rate of the production. It is important to understand that enzyme activity can also be regulated – changes in tertiary/quaternary structure of the enzyme can inhibit/enhance its activity, or even turn it on or off. For example, cofactors are separate (non-protein) molecules or ions that must bind with an enzyme to make it work, either by joining with the active site directly, or by stimulating a conformational change to the protein that (in turn) changes the shape of the active site. Where these cofactors are organic, they are typically referred to as co-enzymes. Enzymes can also be regulated at the level of gene expression – i.e., certain signals can either induce or inhibit the production of that enzyme. This will also influence the total rate of substrate conversion, and might be affected by a variety of parameters including illness, medication use, diet and lifestyle. How enzymes work Six types of enzymes 43 3. Drug discovery and design 3.1. What are drugs? The word “drug” in popular culture is much maligned and frequently used only to imply substances of abuse or illegality. It is often forgotten that the term applies equally to the active ingredients of medicines for the relief and treatment of disease. In a dry technical sense, we can identify any drug as a foreign compound taken into an organism that changes the behaviour of a biological process. For our ethical pharmaceuticals, we can add the rider, “for therapeutic benefit,” to that definition. Typically, there is a minimum concentration (dose) required to elicit the beneficial therapeutic effect and also a maximum concentration above which toxic effects are observed. One glass of wine may make you feel good but a heavy binge leads to a nasty hangover or worse; one paracetamol relieves your headache, swallowing the whole bottle-full may cause fatal liver failure. The difference between the efficacious and toxic dose is the therapeutic index, which should ideally be large. Morphine interacts with the body to bring pain relief Snake venom interacts with the body to cause death Strychnine interacts with the body to cause death LSD interacts with the body to cause hallucinations Coffee interacts with the body to wake you up Penicillin reacts with bacteria to kill them Sugar interacts with the tongue to stimulate taste In reality, most drugs are relatively small organic compounds (relative molecular mass ≤ 500). The origins of drug substances may naively be divided into natural and synthetic compounds. In modern Western medicine, a surprising proportion of drugs (about 50%) is still derived from natural sources or are close synthetic variations on naturally-occurring molecules. Despite their lack of size, drugs are almost never simple molecules in their structures, properties and effects. Understanding them requires a complex and potent mixture of organic, physical and biochemistry. Drugs from natural sources Drugs and medicines can be manufactured in many ways and come from many different sources; you may be aware of classic examples such as the heart drug digoxin, which comes from the foxglove plant or morphine, which is derived from the opium poppy. As well as plant- life, animals, micro-organisms and minerals can be sources of medicines. Gold, platinum and lithium all appear, relatively unmodified, in pharmaceuticals. When medicines were first used, they would be taken directly from the source (e.g., chewing a plant) or preparing a herbal tea. Even today it is estimated that up to 70% of the world’s population depends upon plants as the major source of medicines. Herbal medicines are composed entirely of plant material; a well-known example is senna which consists of the seed pods or leaves of the senna plant. Many important drugs are extracted from plants or other natural sources. Some examples are: Morphine and codeine, extracted from opium 44  Anticancer agents such as vincristine and vinblastine found in the Madagascar periwinkle Antibiotics such as penicillin produced by moulds Statins (cholesterol lowering agents) such as lovastatin are also produced by various species of Aspergillus fungi Semi-synthetic drugs These are usually derived from an active natural product by modification of the molecule to improve it for human use, for example by enhancing potency, reducing toxicity, increasing bioavailability, etc. Examples include: Simvastatin which is made from lovastatin by modification of the ester group. It is 2-3 times more ponent than lovastatin Diacetyl morphine (also known as diamorphine and heroin), is a derivative of morphine that has more potent analgesic activity. However, it is also more addictive than morphine. Diacetylmorphine is NOT a constituent of opium. All of the penicillin and cephalosporin drugs Synthetic drugs These are made entirely by synthesis from chemicals usually derived from coal or oil. Burning oil is a waste of the vast range of small organic molecules that should be put to better use. Examples of synthetic drugs are ibuprofen, propranolol, and temazepam. Although many drugs are manufactured synthetically, some will have depended on natural product for their discovery. An example of this is captopril, used for the treatment of high blood pressure. This drug was developed from a small peptide in the venom of the Brazilian arrowhead viper which causes a marked drop in blood pressure. This is a rare example of an animal-derived drug. Rules of druglikeness - Lipinski’s Rule of Five In 1997, Christopher Lipinski proposed a set of rules for druglikeness. His general observation was that most medicinal drugs were relatively small, lipophilic molecules. This led to the Lipinski’s Rule of Five which says that a molecule of an orally active drug usually obeys at least three of the following criteria: Not more than 5 hydrogen bond donors, e.g., an alcohol group, -OH, or an amine group (primary, -NH2, or secondary, -NHR); Not more than 10 hydrogen bond acceptors, nitrogen atoms, N, or oxygen atoms, O; A relative molecular mass of less than 500; An octanol-water partition coefficient logP not greater than 5. The origin of the rule’s name comes from the fact that all numbers are multiples of five. This became a commonly used way of evaluating the potential of a substance as a medicinal drug. There have been many subsequent refinements and modifications, such as no more than 10 rotatable bonds. 3.2. How do drugs work? In its simplest form, a small drug molecule works by binding to a much larger biological macromolecule (the receptor) to produce a pharmacological effect (Figure 32). Most receptors are proteins but some drugs work by binding or even reacting with nucleic acids. 45  Figure 33. A simplified diagram illustrating drug-receptor binding A simple analogy often used to describe drug action is to imagine a key and a lock. The key fits into the lock and when it is turned the lock opens. Now imagine a key that has only its blade with the notches but lacks the bow end for holding and turning. This key would fit the lock snugly but could not be turned to open the lock. It would also block the entry of a normal key. Such a drug would block the normal function of the receptor, so is described as an inhibitor or antagonist. There are two related alternative scenarios: the first is the skeleton key which takes the place of the normal key and opens a range of locks; the second is analogous to leaving your front door open on the latch, where receptor activity is stimulated or even increased above the usual levels. Such drugs are agonists and a range of effects exists from partial through full to super-agonists; these are considered in detail later in the module. The human body has evolved to resist and remove foreign chemicals. A big difference with drugs is that we want them to get in and stay in. There are many barriers to the uptake and distribution of drugs around the body, for example skin, membranes and cell walls. Once drugs have entered, the body has evolved to eliminate them as soon as possible, usually by metabolism in the liver followed by excretion. As you learn about the science of drugs, you will often hear about PK/PD and ADME (Figure 33). These concern all the underlying organic, physical and biochemistry of drugs and will be recurrent themes throughout the MPharm degree. In particular, you will study them in this module in later units. Figure 34. Concepts of Pharmacodynamics and Pharmacokinetics Molecular recognition: drug-receptor and drug-DNA interactions Most drugs interact with receptor sites localised in macromolecules that have protein-like properties and specific three-dimensional shapes. A receptor is the specific chemical 46 constituents of the cell with which a drug interacts to produce its pharmacological effects. The term receptor is mostly reserved for those protein structures that serve as intracellular antennas for chemical messengers. Upon recognition of the appropriate chemical signal, the receptor proteins transmit the signal into a biochemical change in the target cell via a wide range of possible pathways. A minimum three-point attachment of a drug to a receptor site is essential for the desired effect. In most cases, a specific chemical structure is required for the receptor site and a complementary drug structure. Slight changes in the molecular structure of the drug may drastically change specificity, and thus the efficacy. However, there are some drugs that act exclusively by physical means outside cells, and do not involve any binding to the receptors. These sites include external surfaces of skin and gastrointestinal tract. Drugs also act outside cell membranes by chemical interactions (e.g., neutralisation of stomach acid by antacids). The drug-receptor interaction, i.e., the binding of a drug molecule to its receptor, is governed by various types of chemical bonding that have been already discussed in Section 1.2 Interactions between molecules. A variety of chemical forces may result in a temporary binding of the drug to its receptor. Interaction takes place by utilising the same bonding forces as involved when simple molecules interact (e.g., covalent, charge-charge, hydrogen bonding, hydrophobic interactions). Covalent bonds are strong, and particularly irreversible. Since the drug-receptor interaction is a reversible process, covalent bond formation is rather rare except in a few situations. Some drugs that interfere with DNA function by chemically modifying specific nucleotides are mitomycin C, cisplatin and anthramycin. For example, cisplatin, which is a platinum-based anticancer drug, exerts its therapeutic effect by platinating the N-7 position of guanine on the major groove site of the DNA double helix (Figure 34). This chemical modification of the platinum atom crosslinks two adjacent guanines on the same DNA strand. This blocks replication and inhibits transcription, resulting in DNA damage and subsequently cell death. Figure 35. Cisplatin crosslinking DNA strands through covalent bonding Many drugs are easily ionised at physiological pH and are able to form ionic bonds (charge- charge interactions) by the attraction of opposite charges in the receptor site, for example the ionic interaction between the protonated amino group on salbutamol (inhaler to relieve asthma and breathlessness) and the dissociated carboxylic acid group of its receptor site (Figure 35). 47  Figure 36. Ionic (charge-charge) interaction of salbutamol with adrenergic receptor Hydrogen bonding is also an important binding force in drug-receptor interactions because the drug-receptor interaction is basically an exchange of the hydrogen bond between a drug molecule, surrounding water and the receptor site. These bonds tend to be highly directional, forming straight bonds between donor, hydrogen, and acceptor atoms (Figure 36). Figure 37. Hydrogen bonding between colterol and adrenergic receptor site Formation of hydrophobic bonds between nonpolar hydrocarbon groups on the drug and those in the receptor site is also common. Such interactions occur among hydrophobic molecules (and also among the hydrophobic regions or groups within a macromolecule such as a drug receptor) as they tend to associate with one another while being in a polar solvent (usually water). No actual bond is formed during the process. The polar water molecules preferably interact with each other and therefore exclude the non-polar hydrophobic substances. This preferential interaction of water molecules drives the non-polar molecules to coalesce. The non-polar groups of drug molecules (e.g., alkyl or aryl groups) often interact with the non-polar amino acid residues of the receptor protein including leucine, isoleucine, lysine, valine, tryptophan and phenylalanine in this manner. 3.3. How are drugs discovered and designed? Figure 38 shows an idealised pathway for the discovery and development of a new small molecule drug. The process starts with an opportunity assessment – an unmet clinical need and an estimate of the potential market for a new drug. The molecular biology of the disease must be investigated in detail to identify potential drug targets. Drug targets are usually biological macromolecules which are malfunctioning – mostly proteins but sometimes nucleic acids. Where a disease involves an infectious agent such as a bacterium or virus, there can be large differences between host and infectious agent biochemical pathways that allow for the design of highly selective agents. Some of the most effective antibiotic and antiviral drugs target metabolic steps that do not exist in mammalian biochemistry. Anticancer drugs are much harder as tumour cells and tissues are almost identical to the patient’s own. 48  Figure 38. Flowchart of an idealised drug discovery and development pathway. Once a target is identified and it is shown that by altering its regulation, a useful biological effect can be achieved (validation), it is then necessary to have an assay to measure activity of the experimental agents. Then comes the hard bit – finding new chemical structures to bind the target and make it behave as you intend. Commercial drug discovery now starts with robotic screening of vast libraries of compounds (>100 000) of diverse origins to identify hit compounds with desired activity at a concentration that might be achievable in a patient. [This is generally taken to be about 100 µM which can be achieved by a drug taken in doses around 1 g such as paracetamol; often the newest drugs work at one millionth of that amount and a worthwhile hit should be working below 100 nM]. Hit optimisation is a more rational process. If structural data are available, for example a crystal structure of the receptor with bound ligand (hit), then it is possible to explore space and intramolecular interactions in the binding pocket and propose new compounds for synthesis. A few rounds of design, synthesis and screening should lead to compounds optimised for potency. If structural data are not available, an iterative process of systematically varying the structure of the hit molecule and screening for potency can be used to map the properties of the binding site. At this stage is also common to screen for off-target toxicity and mutagenicity. From this process a small group of candidate compounds should be available for further development. Development involves taking compounds that work in an enzyme or cell culture system and getting them to work in a whole organism. This means dealing with ADME: absorption, distribution, metabolism and excretion. More bluntly, getting the drug to its target and keeping it there long enough to do its job, all at the same time as the body is trying to remove it asap. This can often involve going back to optimisation chemistry to modify solubility, distribution or metabolism. Only once activi