PHA112 Drug Receptor Concepts PDF

WEEK 11 MPharm Programme Drug-Receptor Concepts 1 Dr Gabriel Boachie-Ansah [email protected] Dale 113 ext. 2617 MPharm PHA112 Drug Receptor Concepts WEEK 11 Outline of Lectures What is Pharmacology? Drug-Receptor Concepts Drug-Receptor Interactions Drug-Drug Interactio...

WEEK 11 MPharm Programme Drug-Receptor Concepts 1 Dr Gabriel Boachie-Ansah [email protected] Dale 113 ext. 2617 MPharm PHA112 Drug Receptor Concepts WEEK 11 Outline of Lectures What is Pharmacology? Drug-Receptor Concepts Drug-Receptor Interactions Drug-Drug Interactions Variation in Drug Responsiveness Clinical Selectivity Slide 2 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Learning Outcomes 11 At the end of this lecture, you should be able to: Define and distinguish between ‘pharmacology’, ‘pharmacodynamics’ & ‘pharmacokinetics’ Explain how drugs act to produce their effects Describe the various types of drug targets or receptors Define and distinguish between an agonist, a partial agonist & an antagonist Describe how drug-receptor binding translates into a biological effect Define & discuss the importance of therapeutic index Slide 3 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 Basic Principles of Drug Action What is Pharmacology? Drugs & The Human Body Drug taking initiates 2 processes action of the drug on the body action of the body on the drug Slide 4 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Drug-Body Interactions 11 “When we have a headache, we take for granted that after taking some aspirin, our headache will probably disappear within 15 to 30 minutes. We also take for granted that, unless we take more aspirin later, the headache may recur within a few hours. This familiar scenario reveals the primary events of pain relief: The first is the administration and absorption of the drug into the body; the second is the distribution of the drug throughout the body; the third is the interaction of the drug with relevant functional components of the body, which are responsible for the drug’s actions; and the fourth is the elimination of the drug from the body”. RM Julien Slide 5 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 Basic Principles of Drug Action What is Pharmacology? Drugs & The Human Body Drug taking initiates 2 processes action of the drug on the body action of the body on the drug Study of this ‘drug-body interaction’ called ‘pharmacology’ Slide 6 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 Branches of Pharmacology (Traditional) Pharmacology ‘The study of the interaction between drugs & the living body’ Pharmacodynamics Pharmacokinetics ‘The study of the effects of drugs on the living body and how the effects are produced’ ‘The study of how the body deals with or handles drugs’ Pharmacotherapeutics ‘The study of the use of drugs in the treatment & prevention of disease’ Slide 7 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 Drug-Receptor Concepts How Do Drugs Act? Slide 8 of 76 MPharm PHA112 Drug Receptor Concepts WEEK How Do Drugs Act? 11 Drugs interact with biological systems in ways that mimic, or otherwise affect, the natural chemical messengers or processes of the body Two types of drug action Non-specific drug action Some drugs act in a simple physical or chemical manner – e.g. antacids, osmotic diuretics, osmotic laxatives lack any specific structure-activity relationship require large doses of drug for effect Slide 9 of 76 MPharm PHA112 Drug Receptor Concepts WEEK How Do Drugs Act? 11 Two types of drug action Specific drug action Most drugs act in a highly specific manner – e.g. phenylephrine, salbutamol, atropine, digoxin they interact with or bind to specific macromolecular or cellular targets in the body, called ‘receptors’ show clear-cut structure-activity relationship produce biological effects at very low doses Slide 10 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 The Idea of the Drug ‘Receptor’ The Drug Receptor Concept ‘Most drugs produce their biological effects by interacting with specific macromolecules in the body, called receptors’ The Receptor ‘The specialised component of the cell or organism that interacts with a drug, and initiates the chain of biochemical events leading to the drug's observed biological effects’ Slide 11 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 The Drug Target or ‘Receptor’ Drug receptors are protein or glycoprotein molecules Most drug receptors are located on the cell membrane e.g. atenolol, chlorphenamine, cimetidine, codeine Some drug receptors are located inside the cell e.g. oestrogen, testosterone, vitamin D, etc Slide 12 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 Drug Targets or Receptors are Protein or Glycoprotein Entities 1-adrenergic Receptor GABAA Receptor Slide 13 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Drug Targets or ‘Receptors’ 11 Multiple Types of Drug Targets or ‘Receptors’ ‘Classical’ receptors – regulatory protein or binding sites for endogenous or natural chemical messengers, such as neurotransmitters & hormones Ion channels – e.g. lidocaine, diazepam, amiodarone Enzymes – e.g. NSAIDs, Statins, ACE inhibitors Carrier or transport proteins – e.g. digoxin, PPIs, SSRIs Slide 14 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 Drug-Receptor Interactions How do drugs and receptors interact? Slide 15 of 76 MPharm PHA112 Drug Receptor Concepts Drug-Receptor Interactions WEEK 11 Drugs bind to receptors because the drug’s molecular structure & shape are similar to those of the natural chemical messengers the body produces to target those receptors There must be a complementary fit between the drug molecule & the binding site on the receptor Drug & receptor interact to form a drug-receptor (D-R) complex via a reversible chemical reaction The drug-receptor interaction is governed by the Law of Mass Action We can relate drug concentration & biological effect to the fraction of receptors occupied by the drug Slide 16 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 Drug-Receptor Interactions The ‘Lock and Key’ Relationship basis of selectivity of drug action chemical selectivity biological or tissue selectivity Slide 17 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 Drug-Receptor Interactions Drug & receptor interact to form a D-R complex via a reversible chemical reaction Drug + Receptor  Drug-receptor complex k1 D + R == DR k2 The fraction of receptors occupied by the drug is a function of: the concentration of drug in the biophase the equilibrium dissociation constant (KD) for drugreceptor complex Slide 18 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Fractional Occupancy 11 Drug Concentration-Receptor Occupancy Curve Concentration of drug Slide 19 of 76 MPharm PHA112 Drug Receptor Concepts Drug-Receptor Interactions WEEK 11 The ‘Receptor Occupancy Theory’ Assumptions drug effect is proportional to the fraction of receptors occupied maximum drug effect (Emax) occurs when all receptors in the system are occupied by the drug Slide 20 of 76 MPharm PHA112 Drug Receptor Concepts Drug Concentration-Effect Curve WEEK 11 E Emax Drug Concentration Slide 21 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 Log Drug Concentration-Effect Curve E Emax Log Drug Concentration Slide 22 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 MPharm Programme Drug-Receptor Concepts 2 Dr G Boachie-Ansah [email protected] Dale 113 ext. 2617 MPharm PHA112 Drug Receptor Concepts WEEK 11 Drug-Receptor Interactions What happens when a drug binds to its receptor? Slide 24 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 Consequences of Drug-Receptor Interaction One of four possible things can happen The drug may mimic a natural, endogenous chemical messenger  produce the same effect as the natural chemical messenger (called an ‘Agonist’ drug) The drug may ‘block’ the receptor, i.e. prevent the natural chemical messenger from binding  produce no effect (called an ‘Antagonist’ drug) Slide 25 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 Consequences of Drug-Receptor Interaction One of four possible things can happen (cont’d) The drug may bind to a site near the binding site for a natural, endogenous chemical messenger & influence its binding   or  the effect of the natural chemical messenger (called an ‘Allosteric modulator’) The drug may bind to the site normally occupied by a natural, endogenous chemical messenger  produce an opposite effect to the natural chemical messenger (called an ‘Inverse Agonist’ drug) Slide 26 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 Types of Drug-receptor Interaction Agonist Antagonist Positive Allosteric Modulator Negative Allosteric Modulator Slide 27 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 Drug Agonism & Antagonism What is the basic distinction between ‘Agonist’ & ‘Antagonist’ drugs? Both have ‘affinity’ for their receptors ‘a measure of the ease with which a drug binds to its receptor’ ‘a measure of the probability that a drug molecule will interact with a receptor to form a drug-receptor complex’ ‘Affinity’ expresses the chances of the drug binding to its receptor (By analogy, a key fitting into a lock) affinity is measured by the KD of the drug Affinity = 1/KD Slide 28 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 Drug Agonism & Antagonism What is the basic distinction between ‘Agonist’ & ‘Antagonist’ drugs? Agonist drugs have ‘efficacy’, whereas antagonist drugs have no ‘efficacy’ ‘a measure of the ability of the drug-receptor complex to couple or transduce the drug binding into a biological response’ ‘Efficacy’ expresses the ability of the drug to ‘activate’ or cause a conformational change in the receptor that will lead to a biological response. (By analogy, the key turning the lock) ‘efficacy’ (e) may be 0, low or very high Slide 29 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Agonist Drug Action 11 Agonist ‘a drug that binds to its receptor, activates the receptor, and elicits a biological response’ Two types of Agonist Full agonist Partial agonist Slide 30 of 76 MPharm PHA112 Drug Receptor Concepts Agonist Drug Action WEEK 11 Full Agonist binds to its receptor, activates the receptor & is capable of eliciting the maximum possible response has high efficacy (e) e.g. dobutamine, salbutamol Partial Agonist binds to its receptor and activates the receptor, but can only elicit less than the maximum possible response intermediate efficacy (e) reduces the response elicited by the full agonist e.g. buprenorphine, oxymetazoline Slide 31 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 Slide 32 of 76 Full vs Partial Agonist MPharm PHA112 Drug Receptor Concepts Antagonist Drug Action WEEK 11 Antagonist (pharmacological) ‘a drug that binds to its receptor but fails to activate the receptor, and so fails to elicit a response’ it has an efficacy (e) of 0 it competes with the agonist drug (or natural chemical messenger) for binding to the receptor its biological ‘effect’ results from preventing the agonist drug (or natural chemical messenger) from binding to its receptor e.g. atenolol, chlorphenamine, naloxone Slide 33 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Drug-Receptor Interactions 11 The 'Spare Receptor' / 'Receptor Reserve' concept exceptions to the ‘receptor occupancy theory’ full agonists may elicit maximum response without full receptor occupancy system is said to have ‘spare receptors’ or a ‘receptor reserve’ enables economy of hormone / transmitter secretion allows low affinity drugs to elicit maximum possible response Slide 34 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 Drug-Receptor Interactions Characteristics of the Graded Dose-Response Curve Slide 35 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Intensity of Effect 11 Log Drug Concentration Slide 36 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Characteristics of the Graded Dose-Response Curve 11 Potency A measure of amount of drug needed to elicit a specified response reflected in the location of D-R curve along dose axis experimentally expressed as ED50 or EC50 clinically expressed as absolute or relative potency not a critical characteristic of the drug e.g. morphine vs diamorphine (heroin) Slide 37 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Potency 11 Slide 38 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Characteristics of the Graded Dose-Response Curve 11 Maximal efficacy maximal response / effect produced by the drug reflected as a plateau in the log D-R curve the most important characteristic of drug e.g. paracetamol vs morphine maximal efficacy may be determined or limited in clinical practice by the onset of adverse side effects! Slide 39 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 11 Slide 40 of 76 Maximal Efficacy MPharm PHA112 Drug Receptor Concepts WEEK Characteristics of the Graded Dose-Response Curve 11 Slope slope of curve varies from drug to drug reflects the magnitude of change in response per unit change in dose the slope may be an important consideration in clinical practice under certain circumstances! Slide 41 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Characteristics of the Graded Dose-Response Curve 11 Biological variability different responses to same dose of drug in different individuals different responses to same dose of drug in same individual possible sources of variation in drug response Age Gender Genetic factors Polypharmacy Pathological state Slide 42 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 12 Slide 44 of 76 Drug-Drug Interactions MPharm PHA112 Drug Receptor Concepts WEEK Drug-Drug Interactions 12 Drug Antagonism Summation / Additivity Synergism / Potentiation Slide 45 of 76 MPharm PHA112 Drug Receptor Concepts Drug-Drug Interactions WEEK 12 Drug Antagonism ‘interaction between two drugs such that the effect of one is diminished or completely abolished in the presence of the other’ Types of drug antagonism Competitive antagonism (pharmacological / receptor) Non-competitive antagonism Chemical antagonism Pharmacokinetic antagonism Physiological or Functional antagonism Slide 46 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Drug Antagonism 12 Competitive Antagonism the agonist & antagonist drugs compete for same receptor binding site antagonist drug binding reduces chances of agonist binding   agonist effect 2 subtypes, depending on nature of antagonistreceptor interaction Reversible or surmountable Irreversible or insurmountable Slide 47 of 76 MPharm PHA112 Drug Receptor Concepts Competitive Antagonism WEEK 12 Reversible Competitive Antagonism both the agonist & antagonist drugs bind reversibly to the receptor the fraction of receptors occupied depends on 2 drugs’ relative receptor affinities & concentrations antagonism can be overcome by increasing concentration of agonist drug this leads to two effects on the agonist log D-R curve (in the presence of an effective dose of the antagonist drug) a parallel shift to the right no reduction in the maximal response Slide 48 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Competitive Antagonism 12 100 E Emax 50 0 Agonist Concentration Slide 49 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Competitive Antagonism 12 Irreversible Competitive Antagonism the antagonist drug binds irreversibly to the receptor (due to high affinity or covalent bonding) a fraction of receptors rendered permanently unavailable for agonist drug binding the antagonism cannot be overcome by increasing concentration of agonist drug this leads to two effects on the agonist log D-R curve (in the presence of an effective dose of the antagonist drug) a reduction in the slope of the curve a reduction in the maximal response Slide 50 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 12 Irreversible Competitive Antagonism System Without Spare Receptors 100 E Emax 50 0 Log Agonist Concentration Slide 51 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 12 Irreversible Competitive Antagonism System with Spare Receptors 100 Irreversible Antagonist Log Agonist Concentration Slide 52 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Drug Antagonism 12 Non-competitive Antagonism the antagonist drug does not compete with agonist drug for same receptor binding site the antagonist drug may bind to a different site on the receptor or interfere with response coupling the antagonism cannot be overcome by increasing concentration of agonist drug this leads to two effects on the agonist log D-R curve (in the presence of an effective dose of the antagonist drug) a reduction in the slope of the curve a reduction in maximal response Slide 53 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Non-competitive Antagonism 12 100 E Emax 50 0 Log Agonist Concentration Slide 54 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Drug Antagonism 12 Chemical Antagonism results from direct interaction between the antagonist & agonist drugs the ‘antagonist’ drug binds to / combines with the active drug (‘agonist’) in solution the active drug is rendered inactive or unavailable to interact with its target receptors typical examples protamine vs heparin dimercaprol vs heavy metals (Cd, Pb) Slide 55 of 76 MPharm PHA112 Drug Receptor Concepts Drug Antagonism WEEK 12 Pharmacokinetic Antagonism the ‘antagonist’ drug acts to reduce the effective concentration of the active drug (‘agonist’) at its site of action possible mechanisms reduced absorption from the GIT ferrous salts vs tetracycline antibiotics increased metabolic degradation phenobarbital vs warfarin increased renal excretion NaHCO3 vs aspirin Slide 56 of 76 MPharm PHA112 Drug Receptor Concepts Drug Antagonism WEEK 12 Physiological / Functional Antagonism interaction of two opposing agonist effects in a single biological system  cancelling out of each other’s effect the two drugs elicit opposing responses by acting on different receptors typical examples acetylcholine vs noradrenaline (heart rate) glucocorticoids vs insulin (blood sugar levels) Slide 57 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Summation / Additivity 12 Summation When the combined effect of two drugs which elicit the same overt response, regardless of their mechanism of action, is equal to the algebraic sum of their individual effects Additivity When the combined effect of two drugs, which act by the same mechanism, is equal to that expected by simple addition of their individual effects Slide 58 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Synergism / Potentiation 12 Synergism or Potentiation When the conjoint effect of two drugs is greater than the algebraic sum of their individual effects the synergist may act to increase the concentration of the other drug at its receptor sites tyramine & MAO inhibitors increase the responsiveness of the other drug’s receptor-effector protein benzodiazepines & GABA (GABAA receptor) Slide 59 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 12 Synergism / Potentiation GABAA Receptor Ion channel Slide 60 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 12 Synergism / Potentiation Potentiation By Benzodiazepines Slide 61 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 12 MPharm Programme Drug-Receptor Concepts 4 Dr G Boachie-Ansah [email protected] Dale 113 ext. 2617 MPharm PHA112 Drug Receptor Concepts WEEK 12 Slide 63 of 76 Variation in Drug Responsiveness MPharm PHA112 Drug Receptor Concepts WEEK 12 Variation in Drug Responsiveness Definitions, Types & Mechanisms Drug Tolerance Definitions Mechanisms Slide 64 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 12 Variation in Drug Responsiveness Scope inter-patient variation intra-patient variation Possible consequences lack of efficacy unexpected side effects Possible mechanisms pharmacokinetic pharmacodynamic Slide 65 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 12 Variation in Drug Responsiveness Possible types of variation qualitative variations quantitative variations Quantitative variations Hyper-responsiveness Hypo-responsiveness or tolerance Tolerance Innate vs acquired tolerance Tolerance vs Tachyphylaxis Slide 66 of 76 MPharm PHA112 Drug Receptor Concepts Acquired Tolerance WEEK 12 Definitions ‘An acquired state of progressively decreasing responsiveness to a drug as a result of prior or repeated exposure to the drug or another drug with a similar action’ Mechanisms Pharmacodynamic Metabolic Exhaustion / Depletion of mediators Physiological adaptation Slide 67 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 12 Mechanisms of Acquired Tolerance Pharmacodynamic Receptor ‘down-regulation’ reduction in receptor density e.g. 1-adrenergic receptor Receptor ‘uncoupling’ uncoupling of receptors from their effector systems e.g. 2-adrenergic receptor Metabolic enhanced metabolism of the drug due to induction of metabolising enzymes e.g. alcohol, barbiturates, etc Slide 68 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 12 Mechanisms of Acquired Tolerance Exhaustion / Depletion of mediators common with indirectly-acting drugs due to depletion of endogenous stores of mediators of the drug’s action Amphetamine, nitrates Physiological adaptation evoked compensatory or homeostatic mechanisms blunts or cancels the drug’s effects Diuretics, nitrates Slide 69 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 12 Clinical Selectivity Slide 70 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 12 Clinical Selectivity Absolute vs Relative Selectivity Therapeutic or Desirable effects vs Adverse or Undesirable or Side effects Concept of Therapeutic Index Slide 71 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Clinical Selectivity 12 Absolute vs Relative Selectivity no drug has only one single, specific effect drugs produce a spectrum of effects hence ‘relative’, not ‘absolute’, selectivity of drug action relative selectivity ‘the degree to which a drug acts upon a given site relative to all possible sites of interaction’ Slide 72 of 76 MPharm PHA112 Drug Receptor Concepts WEEK Clinical Selectivity 12 Therapeutic vs Undesirable / Side effects drug effects split into therapeutic & undesirable undesirable effects may be minor or serious how do undesirable effects come about? both effects may be mediated via same receptor-effector mechanism – e.g. nitrates, insulin, warfarin both effects may be mediated via identical receptors located in different tissues – e.g. haloperidol, verapamil both effects may be mediated via different types of receptors – e.g. salbutamol, propranolol Slide 73 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 12 Concept of Therapeutic Index Therapeutic Index Determined by the ratio of toxic to therapeutic dose median toxic dose (TD50) Therapeutic Index = median effective dose (ED50) Provides a useful measure of the margin of safety of the drug the benefit to risk ratio of the drug e.g. penicillin vs warfarin Slide 74 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 12 Slide 75 of 76 Concept of Therapeutic Index MPharm PHA112 Drug Receptor Concepts WEEK Recommended Reading 12 Katzung BG, Trevor AJ. Basic & Clinical Pharmacology, 15th Edition. New York: McGraw-Hill Education, 2021. ISBN: 978-1260452310. Ritter JM et al. Rang & Dale’s Pharmacology, 10th Edition. London: Elsevier, 2023. ISBN: 978-0323873956. Slide 76 of 76 MPharm PHA112 Drug Receptor Concepts WEEK 13 MPharm Programme Enzymes 1 Dr Gabriel Boachie-Ansah [email protected] Dale 113 ext. 2617 MPharm PHA112 Enzymes WEEK Outline of Lectures 13 What enzymes are, and why they do Enzyme structure & classification, and enzyme co-factors Enzymes & cellular metabolism How enzymes work, and the factors that affect enzyme function How enzymes interact with their substrates Enzyme kinetics Enzyme inhibition Slide 2 of 69 MPharm PHA112 Enzymes Learning Outcomes WEEK 13 At the end of this lecture, you should be able to: Describe the structure, classification & function of enzymes Describe the metabolic processes that are catalysed by enzymes, and why enzyme catalysis is needed Describe the nature of the interaction between enzymes & their substrates Describe enzyme kinetics & the associated MichaelisMenten & Lineweaver-Burk Plots Describe the various types of enzyme inhibition Appreciate the relevance of enzymes & enzyme inhibition in medicine Slide 3 of 69 MPharm PHA112 Enzymes Enzymes WEEK 13 What Are Enzymes? Specialised, catalytically active biological macromolecules Act as specific, efficient and active catalysts of chemical reactions in aqueous solution Most enzymes are Globular proteins Some are RNA – e.g. ribozymes and ribosomal RNA Slide 4 of 69 MPharm PHA112 Enzymes Enzymes WEEK 13 How Are Enzymes Named and Classified? Enzymes are named by adding the suffix “-ase” to: the name of their substrate, or a word or phrase describing their catalytic action Classification based on the type of reaction catalysed Classification 1. Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases Slide 5 of 69 Type of Reaction Catalysed Oxidation–reduction reactions Transfer of functional groups Hydrolysis reactions Group elimination to form double bonds Isomerization Bond formation coupled with ATP hydrolysis MPharm PHA112 Enzymes WEEK 13 International Classification of Enzymes Slide 6 of 69 MPharm PHA112 Enzymes Enzymes WEEK 13 Naming of Enzymes Each enzyme is assigned a ‘four-part classification number’, and ‘a systematic name’, which identifies the reaction it catalyses – e.g. for hexokinase: Formal name: ATP:glucose phosphotransferase Enzyme Commission number is 2.7.1.1 2 = the class name (transferase) 7 = the subclass (phosphotransferase) 1 = phosphotransferase with a hydroxyl group as acceptor 1 = D-glucose as the phosphoryl group acceptor Slide 7 of 69 MPharm PHA112 Enzymes Enzymes WEEK 13 What Are The Key Structure-Function features of Enzymes? Enzymes are protein They have a globular shape & a complex 3-D structure They have an ‘active site’ – it’s unique shape & chemical environment determine which substrate(s) will bind Some enzymes require additional non-protein chemical component(s), called cofactor(s), in order to function properly Cofactors act as non-protein "helper" molecules – may be metal ions or organic / metallo-organic molecules Slide 8 of 69 MPharm PHA112 Enzymes WEEK 13 Slide 9 of 69 Enzyme Cofactors MPharm PHA112 Enzymes WEEK Enzyme Cofactors 13 Metal ion cofactors small inorganic ions – Mg++, K+, Ca++, Zn++, Cu++, Co, Fe may be free (e.g. Na+, K+) or held in coordination complexes with the enzyme protein (e.g. Zn++, Ca++) assist with enzyme catalysis Slide 10 of 69 MPharm PHA112 Enzymes WEEK Enzyme Cofactors 13 Organic / metallo-organic cofactors Coenzymes – organic cofactors that are loosely bound and easily released from the enzymes Prosthetic groups – organic cofactors that are tightly bound to the enzymes Coenzymes usually act as ‘co-substrates’ or as transient carriers of specific functional groups Most are derived from vitamins – organic nutrients that are required in small amounts in the diet Examples include: NAD (niacin; B3) FAD (riboflavin; B2) Coenzyme A Slide 11 of 69 MPharm PHA112 Enzymes WEEK 13 Slide 12 of 69 Examples of Coenzymes MPharm PHA112 Enzymes WEEK Enzyme & Cofactors 13 The complete, catalytically active enzyme together with its bound coenzyme and/or metal ion is called a holoenzyme The protein part of such an enzyme is called the apoenzyme or apoprotein Slide 13 of 69 MPharm PHA112 Enzymes Enzymes WEEK 13 Why Are Enzymes So Important? They catalyse (accelerate) biochemical reactions in the body – by speeding up chemical reactions Most biochemical & physiological reactions in the body proceed at very slow pace Enzymes act to speed up these so-called ‘chemical reactions of life’ Without enzymes, most chemical reactions of life would proceed so slowly (or not at all) that life could not exist Slide 14 of 69 MPharm PHA112 Enzymes Enzymes WEEK 13 What Are These ‘Chemical Reactions of Life’ That Are Catalysed By Enzymes? Enzymes catalyse cellular metabolic reactions Metabolism is the sum of the chemical reactions that take place in an organism Two types of metabolism or metabolic reactions Anabolism or Anabolic reactions – involve the formation of bonds between molecules Catalysed by Anabolic enzymes Catabolism or Catabolic reactions – involve the breaking of bonds between molecules Catalysed by Catabolic enzymes Slide 15 of 69 MPharm PHA112 Enzymes WEEK Enzymes & Cellular Metabolism 13 Anabolism or Anabolic reactions Biosynthetic – building of complex molecules from simpler ones Involve the formation of bonds between molecules Energy-utilising processes / reactions Involve dehydration synthesis reactions (reactions that release water) – e.g. carbohydrate/protein synthesis Endergonic – consume more energy than they produce Slide 16 of 69 MPharm PHA112 Enzymes WEEK Enzymes & Cellular Metabolism 13 Catabolism or Catabolic reactions Degradative – breakdown of complex molecules into simpler ones Involve the breaking of bonds between molecules Energy-releasing processes / reactions Involve hydrolytic reactions (use water to break chemical bonds) – e.g. digestion of carbohydrates Exergonic – produce more energy than they consume Slide 17 of 69 MPharm PHA112 Enzymes WEEK 13 Slide 18 of 69 Cellular Metabolism MPharm PHA112 Enzymes WEEK 13 Enzyme-catalysed Metabolic Reactions Anabolic - dehydration synthesis (synthesis) enzyme Catabolic - hydrolysis (digestion) enzyme Slide 19 of 69 MPharm PHA112 Enzymes WEEK Enzymes & Cellular Metabolism 13 So, Why Do Cellular Metabolic Reactions Require The Intervention of Enzymes? All chemical/metabolic reactions require the initial input of energy (Activation Energy, EA) in order to proceed EA is needed to increase collisions between reactant molecules to shift the reactant molecules into a ‘transition state’, where existing bonds can be broken & new ones formed EA is usually too high for the metabolic reactions to proceed significantly at ambient temperature Enzymes, as catalysts, help to lower the EA and enable metabolic reactions to proceed at a faster rate Slide 20 of 69 MPharm PHA112 Enzymes WEEK 13 Endergonic Slide 21 of 69 MPharm Exergonic PHA112 Enzymes WEEK 13 Enzyme-catalysed Metabolic Reactions How Do Enzymes Work? They act as catalysts – speed up metabolic/biochemical reactions without being consumed or chemically altered they provide an alternative pathway or mechanism for the reaction & lower the activation energy, EA they bind to & form an intermediate with the reactant (substrate), which is released later on during the product formation step Enzymes accelerate the rate of the reaction without shifting or changing the equilibrium of the reaction!!! equilibrium is reached faster with enzyme!!! Slide 22 of 69 MPharm PHA112 Enzymes WEEK 13 Enzyme-catalysed Metabolic Reactions Slide 23 of 69 MPharm PHA112 Enzymes WEEK 13 Enzyme-catalysed Metabolic Reactions Slide 24 of 69 MPharm PHA112 Enzymes WEEK 13 Enzyme-catalysed Metabolic Reactions Slide 25 of 69 MPharm PHA112 Enzymes WEEK How Do Enzymes Work? 13 Enzymes bind their substrates with high specificity Binding specificity is governed by 3D arrangement of atoms ‘Lock and Key’ Model ‘Induced-Fit’ Model (E. Fisher, 1890) (D.E. Koshland, 1958) Active site is complementary to shape of substrate Slide 26 of 69 MPharm Active site forms a complementary shape of substrate after binding substrate PHA112 Enzymes WEEK How Do Enzymes Work? 13 ‘Lock and Key’ Model Simplistic model of enzyme action Substrate fits into 3-D structure of enzyme active site weak chemical bonds formed between substrate & enzyme like a “key fits into lock” Slide 27 of 69 MPharm PHA112 Enzymes WEEK How Do Enzymes Work? 13 ‘Induced Fit’ Model More accurate model of enzyme action Substrate binding causes the enzyme to change shape (‘conformational change), leading to a tighter fit this brings chemical groups in position to catalyse reaction Slide 28 of 69 MPharm PHA112 Enzymes WEEK How Do Enzymes Work? 13 ‘Induced Fit’ Model More accurate model of enzyme action Substrate binding causes the enzyme to change shape (‘conformational change), leading to a tighter fit this brings chemical groups in position to catalyse reaction Hexokinase (a) without (b) with glucose substrate Slide 29 of 69 MPharm PHA112 Enzymes Enzymes WEEK 13 What Are The Factors That Affect Enzyme Function? Enzyme concentration Substrate concentration Temperature pH Salinity Slide 30 of 69 MPharm PHA112 Enzymes WEEK Factors That Affect Enzyme Function 13 Effect of Enzyme Concentration Initially, as  enzyme concentration   reaction rate more enzymes  more frequent collisions with substrate Then, reaction rate levels off with further increase in enzyme concentration Reaction rate substrate concentration becomes the limiting factor not all enzyme molecules can find a substrate Enzyme concentration Slide 31 of 69 MPharm PHA112 Enzymes WEEK Factors That Affect Enzyme Function 13 Effect of Substrate Concentration Initially, as  substrate concentration   reaction rate more substrate  more frequent collisions with enzyme Then, reaction rate levels off with further increase in substrate concentration Reaction rate all enzyme active sites become engaged (saturated) maximum rate of reaction has been reached Substrate concentration Slide 32 of 69 MPharm PHA112 Enzymes WEEK Factors That Affect Enzyme Function 13 Effect of Temperature  temperature   reaction rate molecules move faster   collisions between enzyme & substrate  temperature   reaction rate molecules move slower   collisions between enzyme & substrate Optimum T° – peak effect on enzyme-catalysed reaction greatest number of molecular collisions of enzyme & substrate  temperature beyond optimum T°  enzyme denaturation disrupts bonds in enzyme & between enzyme & substrate enzymes lose their 3D shape (3° structure) Slide 33 of 69 MPharm PHA112 Enzymes WEEK 13 Effect of Temperature on Enzyme Function Optimum Enzyme Activity Increasing number of collisions (Q10) 0 10 20 Denaturation 30 40 50 Temperature (C) Slide 34 of 69 MPharm PHA112 Enzymes WEEK 13 Effect of Temperature on Enzyme Function Optimum Temperature hot spring bacteria enzyme Reaction rate human enzyme 37°C 70°C Temperature Slide 35 of 69 MPharm PHA112 Enzymes WEEK 13 MPharm Programme Enzymes 2 Dr Gabriel Boachie-Ansah [email protected] Dale 113 ext. 2617 MPharm PHA112 Enzymes WEEK Factors That Affect Enzyme Function 13 Effect of pH Changes in pH add or remove H+  small changes in the charges on the enzyme & substrate molecules (altered critical ionization states) affect the binding of the substrate with the enzyme active site Optimum pH – peak effect on enzyme-catalysed reaction pH 6-8 for most human enzymes – depends on localised conditions e.g. pepsin (stomach) = pH 2-3; trypsin (small intestines) = pH 8 Extreme pH levels  enzyme denaturation disrupt attraction between charged amino acids disrupt bonds & the enzyme’s 3D shape active site is distorted  loss of substrate fit Slide 37 of 69 MPharm PHA112 Enzymes WEEK Effect of pH on Enzyme Function 13 Optimum pH trypsin Reaction rate pepsin pepsin trypsin 0 1 2 3 4 5 6 7 8 9 10 11 12 pH Slide 38 of 69 MPharm PHA112 Enzymes 13 14 WEEK Factors That Affect Enzyme Function 13 Effect of Salinity (Salt Concentration) Changes in salinity add or remove cations & anions Extreme salinity  enzyme denaturation Enzymes are intolerant of extreme salinity disrupts attraction between charged amino acids affects 2° & 3° enzyme structure Reaction rate disrupts bonds & the enzyme’s 3D shape Salt concentration Slide 39 of 69 MPharm PHA112 Enzymes WEEK Enzyme Kinetics 13 Enzyme kinetics is the study of the rates of chemical reactions that are catalysed by enzymes It provides insight into the mechanisms of enzyme catalysis & their role in metabolism how the activity of enzymes is controlled in the cell how drugs and poisons can inhibit or modulate the activity of enzymes In 1913, Michaelis and Menten proposed the model known as Michaelis-Menten Kinetics to account for & explain how enzymes can increase the rate of metabolic reactions how the reaction rates depend on the concentration of enzyme & substrate Slide 40 of 69 MPharm PHA112 Enzymes WEEK Michaelis-Menten Kinetics 13 All enzymes show a ‘saturation effect’ with their substrates At low substrate concentration [S], the reaction rate or velocity, V, is proportional to [S] However, as [S] is increased, the reaction rate falls off, and is no longer proportional to [S] On further increase in [S], the reaction rate or velocity becomes constant and independent of [S] At this stage, the enzyme is saturated with substrate A plot of initial reaction velocity, V, against substrate concentration, [S], gives a rectangular hyperbola The Michaelis-Menten equation or kinetics model was developed to explain or account for this ‘saturation effect’ Slide 41 of 69 MPharm PHA112 Enzymes WEEK 13 Relationship between Reaction Velocity & Substrate Concentration Slide 42 of 69 MPharm PHA112 Enzymes WEEK Michaelis-Menten Kinetics 13 Michaelis and Menten proposed the following mechanism for a saturating enzyme-catalysed single substrate reaction: According to this postulate/scheme In an enzyme-catalysed reaction, a free enzyme, E, binds its substrate, S, to form an enzyme-substrate complex, ES ES either undergoes further transformation to yield a final product, P, and the free enzyme, E, or breakdowns via a reverse reaction to form the free enzyme, E and substrate, S Slide 43 of 69 MPharm PHA112 Enzymes WEEK Michaelis-Menten Kinetics 13 Rate of formation of ES = k1[E][S] Rate of breakdown of [ES] = k-1[ES] + k2[ES] = (k-1 + k2)[ES] At equilibrium, k1[E][S] = (k-1 + k2)[ES] Re-arranging, [ES] = [E][S]/{(k-1 + k2)/k1} But (k-1 + k2)/k1 = KM (Michaelis constant) Therefore, [ES] = [E][S]/KM Slide 44 of 69 MPharm PHA112 Enzymes WEEK Michaelis-Menten Equation 13 By re-arranging the equations, and making several assumptions, they derived the Michaelis-Menten Equation Vmax S v= K M + S Where, Vmax = the maximum velocity or rate of reaction, at maximum (saturating) concentrations of the substrate KM = (k-1 + k2)/k1 = substrate concentration at which the reaction velocity is 50% of the Vmax (Michaelis constant) [S] = concentration of the substrate, S Slide 45 of 69 MPharm PHA112 Enzymes WEEK Michaelis-Menten Kinetics 13 A graph of initial reaction velocity, V0, against substrate concentration, [S], results in a rectangular curve, where Vmax represents the maximum reaction velocity Slide 46 of 69 MPharm PHA112 Enzymes Linear Transformations of Michaelis-Menten Equation WEEK 13 It is not easy to accurately determine Vmax at high substrate concentrations from the Michaelis-Menten curve Algebraic transformation of the Michaelis-Menten equation into linear forms for plotting experimental data Lineweaver-Burk Plot (also called Double Reciprocal plot) Eadie-Hofstee Plot Slide 47 of 69 MPharm PHA112 Enzymes WEEK Lineweaver-Burk Plot 13 Also called the Double Reciprocal Plot – derived by taking the reciprocal of both sides of the Michaelis-Menten equation, and separating out the components of the numerator on the right side of the equation: 1 1  KM = + v vmax  vmax Slide 48 of 69  1   [ S ]0 MPharm PHA112 Enzymes WEEK Eadie-Hofstee Plot 13 Derived by inverting the Michaelis-Menten equation, and multiplying both sides of the equation by Vmax A plot of V against V/[S] yields Vmax as the y-intercept, Vmax/Km as the x-intercept, and Km as the negative slope v v = −KM + vmax [S ]0 Slide 49 of 69 MPharm PHA112 Enzymes WEEK 13 Significance of KM (Michaelis Constant) It has same unit as the substrate (M) The substrate concentration [S] at which the reaction proceeds at half maximal velocity (50%), i.e. KM = [S] at ½ Vmax A measure of an enzyme’s affinity for its substrate – the lower the KM value, the higher the enzyme’s affinity for the substrate and vice versa Provides an idea of the strength of binding of the substrate to the enzyme molecule – the lower the KM value, the more tightly bound the substrate is to the enzyme for the reaction to be catalysed Indicates the lowest concentration of the substrate [S] the enzyme can recognise before reaction catalysis can occur Describes the substrate concentration at which half the enzyme's active sites are occupied by substrate Slide 50 of 69 MPharm PHA112 Enzymes WEEK 13 Significance of KM (Michaelis Constant) KM Values for Some Key Enzymes Slide 51 of 69 MPharm PHA112 Enzymes WEEK 13 Significance of Vmax Gives an idea of how fast the reaction can occur under ideal circumstances Reveals the turnover number of an enzyme, i.e. the number of substrate molecules being catalysed per second Magnitude varies considerably – from  10 in the case of lysozyme to 600,000 in the case of carbonic anhydrase Slide 52 of 69 MPharm PHA112 Enzymes WEEK 13 Significance of Vmax Turnover Numbers of Some Key Enzymes Slide 53 of 69 MPharm PHA112 Enzymes WEEK Enzyme Inhibition 13 Enzymes are required for most of the processes required for life They catalyse biological reactions by reducing the activation energy needed for the reactions to occur They bind to specific substrates in the reaction pathway & speed up the reaction, but are released unchanged to be used again Activity of enzymes needs to be tightly regulated to maintain homeostasis Regulation is accomplished via enzyme inhibition Enzyme inhibition is widely exploited in clinical therapeutics Slide 54 of 69 MPharm PHA112 Enzymes WEEK Types of Enzyme Inhibition 13 Two types of inhibition Irreversible Reversible Reversible inhibition Competitive Non-competitive Uncompetitive Mixed Slide 55 of 69 MPharm PHA112 Enzymes WEEK Competitive Inhibition 13 The inhibitor (I) is structurally similar to the substrate (S) The inhibitor (I) competes with the substrate (S) for the substrate binding site The inhibitor has virtually no affinity for the enzymesubstrate complex (ES), since the substrate (S) already occupies the inhibitor binding site when bound In the presence of an effective concentration of inhibitor the apparent KM is increased there is no change in the Vmax The inhibition can be reversed by increasing the concentration of the substrate (S) Slide 56 of 69 MPharm PHA112 Enzymes WEEK 13 Slide 57 of 69 Competitive Inhibition MPharm PHA112 Enzymes WEEK 13 Slide 58 of 69 Competitive Inhibition MPharm PHA112 Enzymes Non-competitive Inhibition WEEK 13 The inhibitor has similar affinity for both the free enzyme (E) and the enzyme-substrate complex (ES) The inhibitor may bind with the free enzyme as well as the enzyme-substrate complex The inhibitor binds with the enzyme at a site which is distinct from the substrate binding site The binding of the inhibitor does not affect the substrate binding, and vice versa However, the inhibitor binding to enzyme or enzymesubstrate complex prevents enzyme from forming its product there is no effect on, or change, in the KM the Vmax for the reaction is decreased Slide 59 of 69 MPharm PHA112 Enzymes WEEK 13 Slide 60 of 69 Non-competitive Inhibition MPharm PHA112 Enzymes WEEK 13 Slide 61 of 69 Non-competitive Inhibition MPharm PHA112 Enzymes WEEK Uncompetitive Inhibition 13 The inhibitor has affinity for the enzyme-substrate complex (ES), but not the free enzyme (E) The inhibitor binds only the enzyme-substrate complex (ES), but not the free enzyme (E) An inactive ESI complex is formed when the inhibitor reversibly binds to the enzyme-substrate complex The inactive ESI complex does not form a product (P) the apparent KM is decreased – due to the selective binding of the inhibitor to the enzyme-substrate complex (ES) the Vmax for the reaction is also decreased inhibition cannot be reversed by increasing the substrate concentration Slide 62 of 69 MPharm PHA112 Enzymes WEEK 13 Slide 63 of 69 Uncompetitive Inhibition MPharm PHA112 Enzymes WEEK 13 Slide 64 of 69 Uncompetitive Inhibition MPharm PHA112 Enzymes WEEK 13 Slide 65 of 69 MPharm PHA112 Enzymes WEEK 13 Summary of the Effect of Enzyme Inhibition on the KM and Vmax Type of Inhibition Apparent KM Apparent Vmax Increased Unchanged Competitive Non-competitive Unchanged Uncompetitive Decreased Slide 66 of 69 MPharm PHA112 Decreased Decreased Enzymes WEEK 13 The Inhibitor Constant (Ki) A measure of the affinity of an inhibitor drug for an enzyme Ki values are used to characterize and compare the effectiveness of inhibitors relative to KM Useful and important in evaluating the potential therapeutic value of inhibitor drugs for a given enzyme reaction In general, the lower the Ki value, the tighter the binding, and hence the more effective an inhibitor is Slide 67 of 69 MPharm PHA112 Enzymes WEEK Graphical Determination of the Inhibitor Constant (Ki) 13 Non-competitive inhibitor Slide 68 of 69 MPharm PHA112 Competitive inhibitor Enzymes WEEK 13 Enzymes as Important Drug Targets Disease Enzyme Drug Mechanism Hypertension Angiotensin Converting Enzyme (ACE) Lisinopril Inhibitor Alzheimer’s Disease Acetylcholinesterase (AChE) Donepezil Inhibitor Parkinson’s Disease Inflammation Dopamine β-hydroxylase L-DOPA Substrate Cyclooxygenase 2 (COX 2) NSAIDs, e.g. aspirin, ibuprofen, naproxen, celecoxib Inhibitors Gout Parkinson’s Disease Xanthine oxidase Allopurinol Inhibitor Monoamine oxidase Selegiline Moclobemide Inhibitor Statins Inhibitor Cardiovascular HMG CoA reductase disease Slide 69 of 69 MPharm PHA112 Enzymes MPharm Programme Introduction to Microbiology Dr Callum Cooper [email protected] Microbiology textbooks • Willey J.M. et al. Prescott’s Microbiology (8th ed.) Publisher: McGraw- Hill Higher Education. • Madigan M.T. et al. Brock Biology of Microorganisms (14th ed.) Publisher: Pearson. • Denyer S.P et al. Hugo & Russell’s Pharmaceutical Microbiology (8th ed.) Publisher: Blackwell publishing • Russell, Hugo & Ayliffe's: Principles and Practice of Disinfection, Preservation and Sterilization, (5th ed.) Publisher Wiley-Blackwell EVERYTHING CAN BE USED FOR EXAM QUESTIONS! Learning Objectives • Gain awareness of basic microbiology principles • • • • What is microbiology What are microorganisms How do we study microbes Describe and provide examples of relevant areas in which microbiology plays an important role • • Human health and wellbeing Pharmaceuticals and biotechnology What is microbiology? • Microbiology is the generic term for the study of microorganisms (microbes) • Includes but is not limited to bacteria, viruses, fungi, archea, parasites and protozoa • Studies individual microbes and also communities of microbes • Microbiology has a huge impact in a variety of fields… • • • • • Fermented foods, baking, brewing, wine making Water and sewage treatment Agriculture and spoilage Study of pathogens and infectious disease Pharmaceuticals and biotechnology What are microbes? • Microorganisms (microbes) are microscopic (<1mm) and often unicellular • Viruses are acellular • Fungi can form multicellular structures Viruses Bacteria RBC (~100nm) (1-2µm) (~8µm) • In this module we will generally be talking about bacteria 1nm Chicken Egg (40-50mm) 10nm 100nm 1µm 10µm 100µm 1mm 10mm 100mm • Among the first organisms to evolve on Earth • Can adapt rapidly to changing conditions • Greatest genetic diversity of all groups of living organisms The Tree of Life Letunic and Bork (2016) Nucleic Acids Res doi: 10.1093/nar/gkw290 http://itol.embl.de What are microbes? • They are present in nearly every environment How is this relevant to me? • Total number of human cells: 1012 or 10 000 000 000 000 • Total microbes in healthy human gut: 1013 or 100 000 000 000 000 Are they all “friendly” and harmless? • Simple answer: NO Ref: By NASA/Crew of STS-132 - http://spaceflight.nasa.gov/gallery/images/shuttle/sts-132/hires/s132e012208.jpg(http://spaceflight.nasa.gov/gallery/images/shuttle/sts-132/html/s132e012208.html), Public Domain, https://commons.wikimedia.org/w/index.php?curid=10561008 Top causes of death in the UK by age and sex, 1915 to 2015 https://www.ons.gov.uk/peoplepopulationandcommunity/birthsdeathsandmarriages/deaths/articles/causesofdeatho ver100years/2017-09-18 Microbes can directly affect our health • Not all microbes are “friendly” (pathogenic) • Major causes of mortality • Introduction of Penicillin G (1st antibiotic) in the late 1940s revolutionised modern medicine • Made many fatal infections treatable • Microbes increasingly shown to have roles in non-infectious diseases; • Stomach ulcers • Obesity Microbes can directly affect our health Ref: Häggström, Mikael (2014). "Medical gallery of Mikael Häggström 2014". WikiJournal of Medicine 1 (2) Microbes can directly affect our health “Friendly” bacteria Commensals E. coli Nissle 1917 “Unfriendly” bacteria Pathogens E. coli O157:H7 Normal (Commensal) Flora • Normal flora are those organisms living in benign symbiosis with host: • e.g. E. coli (non toxigenic), lactic acid bacteria, Staphylococcus aureus, Candida yeast & many more • Some may be pathogenic but unable to enter disease process (lack attachment to suitable surface) But if conditions change: • Microbes may grow more extensively & cause infections, possibly fatal – e.g. MRSA • Changed conditions include hormonal changes, climatic, environmental, stress Medical microbiology, public health and epidemiology • Microbiology plays an important role in public health • Identification of diseases • Tracking and control of disease outbreaks • Tracking of antimicrobial resistance • Identifying appropriate treatments • Vaccination • Allows for the mathematical modelling of disease progression and spread • Alemi, A.A., et al. 2015. You can run, you can hide: The epidemiology and statistical mechanics of zombies. Physical Review E, 92(5), p.052801; http://mattbierbaum.github.io/zombies-usa/ Challenges in medical microbiology: Antibiotic resistance • Misuse has accelerated the development of resistance… • Use as a growth promoter in livestock • Non-prescription use • If resistance not tackled estimated 10 million deaths/year due to AMR infections by 2050 • More than cancer and diabetes combined • Economic loss of $60-100 Trillion USD Microbiology in Biotechnology and Industry • Biotechnology is the broad area of biology which uses living systems/organisms to produce products • Molecular biology developments have greatly increased scope of biotechnology Ref: Ingrid Moen, Charlotte Jevne, Jian Wang, Karl-Henning Kalland, Martha Chekenya, Lars A Akslen, Linda Sleire, Per Ø Enger, Rolf K Reed, Anne M Øyan and Linda EB Stuhr: Gene expression in tumor cells and stroma in dsRed 4T1 tumors in eGFP-expressing mice with and without enhanced oxygenation. In: BMC Cancer. 2012, 12:21. doi:10.1186/1471-2407-12-21 What is Biotechnology? • Different types of Biotechnology are colour coded Colour Green White Yellow Blue Red Dark Application Agriculture Industrial Food technology Aquatic Medical and Pharmaceutical Bioterrorism • Industrial use of microbes often termed biotechnology Microorganism Saccharomyces cerevisiae Lactobacillus spp. Aspergillus niger Bacillus licheniformis Penicillium chrysogenum Streptomyces griseus Escherichia coli (GMO) Product(s) Bread, beer, wine Yoghurt, bread Citric acid Alkaline proteases Penicillin Streptomycin Insulin (Human identical) Biotechnology: Industrial chemicals Citric acid • Aspergillus niger (fungus) • Uses raw materials such as brewery waste, coconut oil, rapeseed oils • Part of its normal metabolism • >1,000,000 tonnes/year produced • Uses as an acidity regulator or flavouring Proteases • Bacillus licheniformis (bacterium) • Produces alkaline protease in response to nutrient limitation • Enzymes used in laundry detergent • Alkaline proteases digest protein-based stains • > 1,000 tonnes/year produced Biotechnology: natural medicinal products Natural products of microbial origin include: • Antimicrobials • Penicillins, streptomycin (Fungi) • Endolysins (Viral) • Cholesterol lowering-agents • Lovastatin (Fungi) • Neurotoxins • Botulinum toxin (Bacteria) Biotechnology: recombinant medicinal products • Bacteria can be genetically modified to produce therapeutic proteins; • e.g Insulin (humulin) • First produced in 1978 by Genetech then licenced to Eli Lilly • In use > 25 years • Recombinant Vaccines e.g. Gardasil • Targets Human papilloma virus (HPV) • Contains recombinant viral protein produced in a bacterium Microbial Spoilage • Microbes can utilize almost anything to gain nutrients… • Including NUCLEAR WASTE • Microbial spoilage can be defined as the deterioration of a product by a contaminating microbe • Pharmaceutical products can also be spoiled by microbes • Can target raw materials or finished products • Causes economic loss • Increases risks to patients by affecting safety and quality Practical microbiology: Growing microbes • In general, microbes can be grown in the laboratory using solid or liquid media; • Nutrient rich • Non-selective • Specialist media exists… • Identify unknown microbes • Selectively culture specific microbes Practical microbiology: Looking at microbes Viruses Bacteria RBC (~100nm) (1-2µm) (~8µm) 1nm 10nm 100nm 1µm 10µm 100µm Chicken Egg (40-50mm) 1mm 10mm Naked eye Light microscope Electron microscope 100mm Practical microbiology: Molecular microbiology • Extract genetic components • DNA • Targeted regions e.g PCR • Whole genome sequencing • RNA • Gene expression • Extract cell components • Protein • Immunological Profiling • Structural studies • Sequencing Summary • What is microbiology and what are microbes ? • Areas in which microbiology is important and relevant to pharmacy; • Health and disease • Biotechnology and industry • How microbes are studied in the laboratory Extra reading • Chapter 1: Hugo and Russell’s Pharmaceutical Microbiology • Brock Microbiology Part I section 1 • Prescott's Microbiology Part IX MPharm Programme Microbial Classification Dr Callum Cooper [email protected] Learning Outcomes • Describe the differences between cellular and acellular microorganisms • Describe the main taxonomic groups of microorganisms and their features • Describe how microbes can be classified and provide examples Biological systematics • Studies how life changes through time and how living things relate to one another • Taxonomy: define systems by shared characteristics • Classification: arrange organisms into groups • Nomenclature: assign names Classification of Biological entities Biological Entities Cellular Eukarya Acellular Bacteria Archaea The Tree of Life Tree of Life website: tolweb.org/tree/ Development of classification systems: cellular entities Linnaeus Haeckel Chatton Whittaker Woese 1735 1866 1925 1969 1977 2 Kingdoms 3 Kingdoms 2 Empires 5 Kingdoms 3 Kingdoms Not described Protista Prokaryota Monera Bacteria Archaea Protista Vegetablia Plantae Animalia Animalia Eukaryota Plantae Fungi Animalia Eucarya Classification of Biological entities Biological Entities Cellular Eukarya Acellular Bacteria Archaea Viruses Viroids Virusoids Prions Classification systems: Acellular entities • Viruses and prions are anomalous entities • Non-living • Viruses are parasitic (require hosts and resources to reproduce) • Viral classification is an ongoing source of debate • 2 competing systems; • Baltimore system • International committee for taxonomy of viruses (ICTV) system • Classically based on phenotype and nucleic acid type • Prions (PrP) do not reproduce • Misfolded proteins • Can exist as multiple isoforms By Emw - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=8821061 Taxonomic ranks Domain Eukaryota Bacteria Phylum Chordata Proteobacteria - Class Mammalia γ-proteobacteria - Order Primates Enterobacteriales Caudovirales Family Hominidae Enterobacteriaceae Subfamily Genus - - Homo Escherichia Species H. sapiens E. coli N.B. Genus and species are normally written in italics Podoviridae Autographivirinae Phikmvvirus Pseudomonas virus phiKMV Taxonomic ranks: Species • What is a species? • A group of living organisms capable of interbreeding, even if geographically isolated; Tigon Liger Panthera tigris Panthera leo WHAT ABOUT ORGANISMS THAT DO NOT REPRODUCE SEXUALLY? (e.g. bacteria) Taxonomic ranks: Species in microbiology • Species: collection of strains that share stable properties but differ significantly from other groups of strains Staphylococcus aureus Staphylococcus epidermidis • Strain: is a genetic variant or subtype of a bacterial species that varies slightly from other members of the same species Staphylococcus aureus E. coli Nissle 1917 Methicillin resistant Staphylococcus aureus E. coli O157:H7 Taxonomic ranks: Species/Strains in microbiology • These organisms are assigned on the basis of phenotype, serotype or genotype; • Phenotype: an organism's observable characteristics or traits (e.g morphology, development, biochemical or physiological properties) • Serotype: distinct variations in cell surface antigens within a species • Genotype: is the part of the genetic makeup of a cell, which determines one of its characteristics Phenotype classification in microbiology • Morphology • Cell shape (bacilli or cocci) • Cell structure (gram staining) • Biochemistry • Enzyme production • Transport proteins activity • Life cycle • Vegetative or spore forming Phenotype classification in microbiology • Ecological niche • Temperature • Thermophile • Psychrophile • Interactions with other organisms • Susceptibility to bacteriophages • Pathogenicity (ability to cause disease) Provides the least detailed information Serotype classification in microbiology • Serotype: distinct variations in cell surface antigens within a species A B • Salmonella genus has over 2600 different serotypes • Kauffman-White classification • O antigen: oligosaccharides on the cell surface • H antigen: flagellar proteins ?