Protein as Drug targets
Carboxypeptidase Mechanism
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lead discovery of ACE inhibitors
- 1965: Mixture of peptides that inhibit bradykininase (vasodilator) activity discovered in pit viper venom - 1965-69: SAR (structure activity studies) on the peptide mixture - 1968: one of these peptides shown to inhibit ACE - More peptides with similar activity isolated from the venom of Japanese pit viper - they did no have the oral bioavailability because of protease degradation - this work led to the design of orally active peptidomimetic ACE inhibitors.
Ligand-Gated Ion Channels: - 4-TM Nicotinic acetylcholine (nAch)
- 4-TM: Nicotinic acetylcholine (nAch) receptor forms a Na+ ion channel and is found at the neuromuscular junction --> two molecules of Ach required to open ion channel (because of two a-subunits) --> Ach binds at the a-subunit - Small number of neurotransmitters produces large biological effect --> opening of a few ion channels mobilizes thousands of ions for each neurotransmitter involve
Neurotransmitters — A Case Study - Adrenalin and noradrenalin acting on which system - acetylcholine acting on which system - example
- Adrenalin and noradrenalin (the catecholamines) control the fight or flight response and act upon the sympathetic nervous system - When 'excited' both compounds are released and act upon adrenoceptors in the heart, lungs, gut, and peripheral blood vessels. As a result heart rate increases, lung capacity is increased and blood is diverted from the gut to the skeletal muscles - In addition pupils dilate, hair may stand on end and loss of blood to surface blood vessels causes pallor - Acetylcholine act on the parasympathetic nervous system and it acts in an opposite manner to the catecholamines (note catecholamines used to inc B.P) - These two systems exist in a delicate balance and any drug that alters this balance will produce a pharmacological response Examples include -adrenergic blockers to control angina
G-Protein Linked Receptors - characteristics
- Alternative mechanism for transmission of neurotransmitter message - Protein on external surface of cell membrane linked to enzyme or ion channel on inner surface via G-proteins - Protein is composed of a single subunit which has 7 transmembrane domains - binding to receptor = --> enzyme activation OR --> enzyme deactivation - Changes in receptor shape (tertiary structure) leads to "start up" or "shut down" of enzyme activity - small number of neurotransmitters produces large biological effect
drug extension - what levels show increased activity and 0 activity - what does a phentyl or hexyl mean in terms of activity - comment on a phenylethyl group
- As the size of the alkyl group is increased from Me to Bu activity drops to 0. Increasing the size still further results in a slight recovery of activity. - Phenethyl group --> 14 fold increase in activity suggesting that the aromatic ring has located a hydrophobic binding pocket
design of agonists - assumption - requirements of the compound
- Assuming we know what binding groups are present in the binding site, we can, in theory, design a molecule to interact with the receptor and induce the necessary conformational change 1. compound must have correct binding group 2. compound must have these groups correctly positioned 3. compound must be right size for the binding site
Medicinal Uses of Enzyme Inhibitors - enzyme inhibitors against micro-organisms
- Bacterial cells and viral cells have some unique enzymes which are crucial for survival of the microorganism eg Penicillins have been widely used as antibacterial agents - These compounds inhibit the transpeptidase enzyme responsible for bacterial cell wall synthesis. - Penicillin G, a natural product was the first to be isolated
Position of the binding groups
- Binding groups must be located in complementary positions to the binding sites in the receptor NOTE; need to take into account steriogenic centre
Base Pairing in DNA - Secondary Structure In DNA's from a large number of sources the ratio of A:T=1 and C:G=1- WHY?
- DNA carries genetic info in such away that it may be duplicated - 1:1 ration suggested that DNA consists of 2 helical strands wrapped around each other but running in opposite directions - Double helix held together by hydrogen bonding between pairs of bases guanine - cytosine adenine - thymine - 10 base pairs in each full term - two strands are not identical but complimentary - wherever a G base occurs in one strand a C occurs in other strand - wherever an A base occurs in one strand a T occurs in other strand. --> Hence G:C = 1 and A:T = 1
DNA - A Valid Drug Target
- DNA important for: cell replication and transfer of genetic info - DNA used for protein coding (via RNA) - many diseases are associated with DNA malfunction and or alteration e.g. cancer, AIDS, Chrons disease, Downs' syndrome, sickle-cell anaemia - Inhibition of DNA function selectively can treat a vast array of disorders - Most current treatments of cancer rely on inhibition of DNA function - stopping the DNA in a cancerous cell from replicating itself, the cell will eventually die - Most forms of chemotherapy utilise the fact that a cancer cell 'turns over' much faster than a healthy cell - It is this increase in cell metabolism and the corresponding increase in substrate uptake that causes the cytotoxic material to accumulate in cancer cells more 'actively' than normal cells
Transition-State Analogues - transition state structure
- Enzyme alters bond lengths, bond angles and charge distribution TRANSITION STATE STRUCTURE - A Transition State Analogue is a compound whose structure mimics the transition state structure of the reaction - Inhibitors that approximate transition state structure bind more strongly to active site than the substrate or product - Works well for enzyme reactions involving two substrates: 2 substrates linked in TS --> more binding interactions
Enzyme Inhibitors Against Viruses
- Enzyme inhibitors (mainly protease inhibitors) are important in battle against viral infections - Successful antiviral drugs are acylovir (herpes) and zidovudine (HIV)
reaction specificity - explain how enzymes show specificity to chemically identical protons
- Enzymes can distinguish between chemically identical protons - enzymes show specificity to chemically identical protons; so if you only want to remove one proton ( e.g. Hb and not Ha) then use an enzyme to do so.
Antigene/Antisense oligonucleotides
- Homopyrimidine oligodeoxynucleotides (~20 bases in length) can bind to homopurine sites on duplex DNA via the formation of Hoogstein-based hydrogen bonds in the major groove - This results in severe structural distortion in the binding region. Once again this results in the inability of the DNA proteins to mediate cell function leading to cell death
Size and Shape for binding sites
- It is possible to have the correct binding groups in the correct positions but still fail to interact effectively - Consider substitution onto the aromatic ring --> The m-methyl group acts as a buffer and prevents the substrate from 'sinking' deep enough into the binding site for effect interaction
Significance of Binding Groups - what does removal of one binding site result in
- It is reasonable to assume that if the neurotransmitter (or hormone) requires three binding interactions then our new compound would need three binding sites - The removal of one of these binding sites would probably result in a significant loss in activity
Ligand-Gated Ion Channels - main role - describe protein
- Movement of Na+ and K+ ions across cell membrane crucial for nerve function - Protein composed of up to 5 subunits --> Each subunit has 4 transmembrane domains --> Centre of complex is hollow and is lined with polar amino acids affording a hydrophilic pore = ION CHANNEL - binding of neurotransmitter of hormone or agonist --> open ion channel - Binding of antagonist --> prevents ion channel opening
Structure of DNA
- Nucleotides join together in DNA by forming a phosphate ester bond between the 5'-OH group on one nucleotide and the 3'-hydroxyl group on the sugar of another nucleotide - One end of the nucleic acid polymer has a free hydroxyl at C3' (the 3' end), and the other end has a phosphate at C5' (the 5' end) - Shorthand notation - start with 5' end of molecule on left hand side and proceed in the 5' to 3' direction across the page
Receptors — A Case Study: Analgesics (alkaloids; opiates) Crude opium contains a complex mixture of some 25 alkaloids, however what is the principal alkaloid and the one responsible for the analgesic activity?
- One class of compounds that are derived from natural sources (termed natural products) are the alkaloids - These materials contain one or more nitrogen atoms enclosed in a ring system - One particular branch of natural alkaloids — the opiates --> have been used as a 'recreational' drug and as materials for the treatment of 'deep pain' - They are extracted from the opium poppy (Papaver somniferum) and are the oldest drugs know to man --> the cultivation use of opium can be traced back to Mesopotamia (~1000 BC) - Crude opium contains a complex mixture of some 25 alkaloids, --> however the principal alkaloid and the one responsible for the analgesic activity is the compound Morphine Morphine is a classic example of the traditional approach to medicinal chemistry, namely:
A Case History —HMGCoA Reductase Inhibitors (also example of transition state mimic)
- Relationship between blood cholesterol and heart disease is well established - Reduction of blood cholesterol levels helps to control the disease - Cholesterol biosynthesis consists of some 26 steps, however, the rate limiting step is the reduction of HMGCoA to mevalonate - Very potent inhibitors of HMGCoA reductase have been isolated by screening natural product extracts - in particular the fungi Aspergillus terreus and Monascus ruber yielded mevinolin - This compound is highly active - The material is administered as the lactone prodrug - Activity is thought to arise because the 2°alcohol acts as a transition state mimic
Example of transition state analogue - the compound needs to be - which enzyme does it target (inhibit) - describe how this analogue works
- The compound must be stable and as such it can only resemble the real transition state of the reaction (remember the transition state of a reaction is the point of most instability on the reaction profile and involves the forming and breaking of bonds) - example of a transition state analogue is 5-fluorouracil that is used to treat breast, liver and skin cancer - targets thymidylate synthetase - 5-Fluorouracil is not the transition state analogue itself --> it is converted in the body to the fluorinated analogue of 2'-deoxyuridylic acid which then combines with the enzyme and another substrate (tetrahydrofolate) in situ to form the transition state analogue. - The required loss of F+ cannot happen and the enzyme (thymidylate synthetase) is blocked - DNA synthesis is halted --> replication and cell division blocked
Lead Modification
- The discovery of ACE inhibitors began with the discovery that one of the peptides in pit viper venom acted as an ACE inhibitor and controlled elevated blood pressure - C-terminal proline; penultimate Alanine and aromatic amino acid in the antepenultimate position best for activity - no crystal structure available, but ACE believed the metalloenzyme (membrane bound protein hard to work with, purify , crystallise) - active site model created based on similarity to another metalloenzyme- Carboxypeptidase A- a Zn-containing peptidase
Neurotransmission
- The message is an electrical 'pulse' that travels down the nerve cell towards the target: a muscle cell or another nerve. - nerves do not connect directly with their targets. --> There is a gap of about 100 Å (the synaptic gap) which the electrical pulse is unable to 'jump.' --> A chemical messenger (neurotransmitter) is released from the nerve cell that diffuses across the synaptic gap to the target cell where it binds with a specific receptor protein embedded on the postsynaptic cell membrane
Chain Cutters - give example - explain how this works
- The neocarzinostatin chromophore is a natural product that is able to intercalate into DNA. Once intercalated the enediyne function is located within the DNA minor groove and undergoes a Bergman-type cyclisation - This cyclisation process results in the formation of highly energetic free radicals which abstract hydrogen atoms from the surrounding DNA chains resulting in strand scission
DNA Structure and Recognition
- The nucleic acids deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are polymeric materials - They control the storage and transfer of genetic information Each monomeric unit - a nucleotide contains a --> nitrogenous base, --> a sugar unit and --> one or more phosphate groups
Non-competitive, reversible (allosteric) inhibitors - importance - how does the cell know to stop producing the product?
- allosteric binding sites important in control/regulation of enzymes --> small molecules bind at a site remote from active site, effect changes in the 3* or 4* quaternary protein structure, render enzyme inactive or more active - Biosynthetic pathways to a given product often involve a series of enzymes - Eventually cell does not require more of the product to be produced how does the cell know to stop producing the product? - uses feedback control where product inhibits the first enzyme in it biochemical pathway - product acts on allosteric site NOT active site because it has been transformed from original substrate and is not recognised by active site -- binds elsewhere
Catalytic role of enzymes - nucleophilic groups
- amino acid cysteine and serine are commonly present in the active sites - contain nucleophilic residues (OH and SH) groups that can participate in the reaction mechanism - they react with substrate to form intermediates which would not be formed in an un-catalysed reaction - these intermediates provide a low energy pathway for the reaction - serine and cysteine provide an alternative pathway for initial step of hydrolysis of peptides and is usually close to histidine residue which is also involved in catalysis of reaction - Example is mechanism of hydrolysis of peptides by 3C protease (5 other amino acids in AS which are crucial)
Design of Competitive Reversible Inhibitors: Transition State Analogue Inhibition
- an inhibitor whose structure resembles the TS of the reaction (transition state: highest point in energy transition)
catalytic role of enzymes - how do enzymes catalyse the reaction
- binding interactions - acid/base catalysis of reaction - nucleophilic groups - Cofactors (non-protein substances needed for reaction eg tetrahydrofolate, also metal ions eg zinc)
Alkylating Agents:: DNA plantations
- cis-diammonia dichloroplatinum (II) (cisplatin) is an important antitumour agent for treatment of testicular and ovarian tumours - co-ordinates predominantly at GN7 (but also AN7) - bifunctional binding --> intrastrand and interstrand crosslinks - GN7-GN7 intrastrand crosslink between neighbouring guanines is major adduct
how do enzymes work in terms of binding specificity?
- could be very broad: (eg. Cytochrome P450 family) - only one enantiomer may for the E.S complex OR - if both enantiomers form E.S complex,only one is converted to product --> this enantiomeric specificity is because of the chirality of enzymes
why inhibit an enzyme? what are modes of enzyme inhibition
- deficiency or excess of a metabolite resulting in a disease normalised by inhibiting the activity of the enzyme involved Modes of enzyme inhibition 1. competitive (with substrate)/ reversible inhibitors (as it is using only non-covalent bonds) 2. Irreversible inhibitors (enzyme inactivator) - covalent bonds 3. non-competitive, reversible (allosteric) inhibitors
Design of Competitive Reversible Inhibitors: Alternative Substrate Inhibition:
- design a molecule that only binds to the enzyme but also acts as a substrate --> inhibitor reacts with the enzyme and gets converted to the product - Disadvantageous if the products formed from the reaction of the alternative substrate and enzyme are toxic.
Dipole-diplole interactions
- dipole movement of substrate aligns with local dipole movement on surface of active site - are parallel but in opposite direction ; max interaction - exist in these molecules because they are polar --> the active site of enzyme will also have a polar group due to polar functional group
what is the role of receptors?
- drugs that interact with receptors most important in medicine --> pain --> depression --> P.D --> psychosis --> heart failure --> asthma - communication between cells is essential if the body is going to operate in a coordinate snd controlled fashion --> cellular communication is facilitated by chemical messengers - receptors are protein binding sites on cell whose function is to receive and pass on chemical messages
Enzyme Inhibitors Against Viruses Enzyme Inhibitors Against Body's Own Enzymes
- enzyme inhibitors (mainly protease inhibitors) are important in battle against viral infections - Successful antiviral drugs are acylovir (herpes) and zidovudine (HIV) Enzyme Inhibitors Against Body's Own Enzymes - Inhibition of specific enzymes can depress metabolism of neurotransmitters: - monoamine oxidase inhibitors prolong life of catecholamines eg adrenaline, therefore can act as stimulant to treat low blood pressure - Anticholinesterases used to treat Alzheimer's disease - Cyclo-oxygenase (COX) inhibitors eg indomethacin used to treat rheumatoid arthritis - Caspase enzymes implicated in process leading to cell death therefore inhibition of caspases potential treatment for stroke patients
how do enzymes work?
- enzymes are macromolecules, substrates could be as small as CO2 - substrate binds only to a small part of the enzyme; the active site --> the remainder of the enzyme maintains the integrity of the AS and channels the substrate to the AS - Specificity and rate of reaction - lower the activation energy of chemical reaction
Design of Competitive Reversible Inhibitors: Slow, Tight-Binding Inhibition:
- equilibrium reached slowly - Substantial inhibition occurs when the concentration of enzyme and inhibitor are comparable - these inhibitors could bind either non-covalently or sometimes even covalently with AS of the enzyme --> therefore they are the bridge between reversible and irreversible and irreversible inhibitors
what are the substrate binding interactions
- interactions which bind substrates to active sites of enzymes same as those that are responsible for tertiary structure of proteins - ionic - hydrogen bonding - van der waals - ionic bonding (of minor importance for protein 3* structure but can be very important for binding substrates) - dipole-diplole interactions - induced dipole interactions
allosteric inhibitor used in treatment of leukaemia - give example
- many enzyme inhibitors have no structural similarity to the natural substrate --> so increasing the amount of substrate has no effect of the level of inhibition - allosteric inhibitors bind to a separate region of the protein and do not compete with the substrate of for the active site - the binding protein changes the shape of the protein and masks the active site from the natural substrate so the substrate can no longer react - inhibition is irreversible if the inhibitor uses non-covalent bonds to bind the allosteric binding site - for enzymes under feedback control can design drugs based on structure of product which bind to allosteric site Example 6-mercaptopurine (allosteric inhibitor used in treatment of leukaemia) 1. inhibits 1st enzyme involved in purine synthesis 2. blockage of purine synthesis 3. blockage of DNA synthesis
Hormones
- messenger compounds that are released by the endocrine system - They are usually carried in the bloodstream to distant cells where they exert their effects Example - hormone oxytocin which is released by the pituitary gland and acts upon the uterus and mammary glands - At full term in pregnancy it is this material that initiates the contractions of the uterus and induces lactation
what are enzymes?
- natural proteins that act as body's catalyst - soluble, found in cytosol of cells - Transform substrates into products via enzyme-substrate complexes - these transformations take place with rate enhancements several orders in magnitude as compared ti uncatalyzed reactions
what are the 3 main ways that communication process can occur?
- neurotransmission, whereby a nerve cell passes a chemical signal onto another nerve cell - hormonal, whereby circulating hormones are released from glands and carried by the blood stream to a distant organ - autacoid transmission, whereby local hormones are released and act on nearby cells
Induced dipole interaction - comment on the aromatic ring
- out of the two things reacting only one has a permanent dipole; the other will be induced - aromatic ring can interact with ionic group such and quaternary ammonium ion - +ve charge of ammonium ion distorts pi bond cloud of aromatic ring to produce a dipole moment such that face of aromatic ring is electron-rich and the edges are electron deficient
pyruvate acid and binding to active site of lactate dehydrogenase - determine the type of functional group which can participate in which non-covalent interactions
- possible interactions: H-bond Van der Waals Ionic - carbonyl= H-bond - carboxylate= ionic bond - methyl group= Van der waals bond
catalytic role of enzymes - acid/base catalysis - why is an acid/base catalysis important in terms of A.S
- provided by the amino acid histidine - histidine is a weak base and can equilibrate between its protonated form and free base form - acts as a 'proton bank'- donates or accepts protons in the reaction mechanism - Important as active sites are often hydrophobic and have low concentration of water and even lower concentration of protons
competitive/reversible inhibitors
- the drug is competing with the natural substrate for the active site - molecule can be designed that is similar to the natural substrates but which binds the active site more strongly - competes with natural substrate for binding, blocks the active site and prevents the natural substrate form binding - clogs up enzyme site, enzyme function is lowered - inhibitor does not undergo chemical reaction or is changed in any way - increased levels of natural substrate displaces a competitive reversible inhibitor and enzyme resumes function NOTE: the longer the inhibitor is present in the active site the greater the inhibition
Active site of enzymes - role of amino acids present in the active site
- the enzyme AS is generally located in a groove or cleft and act as a surface or 'focus' for the reaction, binging the substrates together and holding them in the best position for a reaction to take place - enzyme AS stabilises the sections transition state and consequently lowers the activation energy for the formation; increasing rate NOTE: this is a reversible process - the amino acids present in the AS play a critical role in the enzyme function and have one of the 2 roles --> binding; binds substate to AS --> Catalytic: involved in mechanism of reaction (acid/base catalyses; His or nucleophilic groups; Cys, Ser) NOTE: - removal or mutation of any one key amino acid will result in the loss of activity - due to folding of the enzyme the amino acids close together in AS may be far apart in the primary structure
DNA uses four nitrogenous bases
- the purine and pyrimidine bases are able to associate via hydrogen bonding between H-bond donor and H-bond acceptor
Carboxypeptidase
- the structure of the Carboxypepidase A active site was derived from X-ray crystallographic studies - From the data known about Carboxypeptidase A the medicinal chemist was able to 'construct' a model of the ACE enzyme active site - it was shown that the inhibitors for carboxypeptidase A were also weak but selective inhibitors of ACE - from this model and from a knowledge of carboxypeptidase A inhibitors one compound developed was captopril; contains proline and SH groups
what is the enzyme transpeptidase involved in and describe its structure
- transpeptidase is responsible for bacterial wall syntheses --> the enzyme splits the peptide bond between two D-alanine residues --> once the terminal Ala leaves, the pentaglycyl moiety of another peptide chain forms a bond to the remaining D-Ala linking the two chains together - transpeptidase is similar to serine proteases which catalyse the hydrolysis of peptide bonds
Alkylating Agents: Aziridinylbenzoquinone - agent for what sort of disease - how does it act as an alkylating agent - what is critical for its activity
- undergone clinical trial as anti tumour agent -> efficacy against brain tumours - forms DNA inter strand xlinks -> efficient cross linking -> cell death - aziridines critical for activity
how could the agonist be used in a disease - explain this using an example of a disease
- used when the body cannot produce enough of the natural material (Parkinson's disease — L-Dopa) PD- deficiency of dopamine L-dopa- prodrug (is a carrier protein) - dopamine is polar so cannot cross the BBB so L-dopa (amino acid) used, which reaches target and L-dopa decarboxylates and dopamine is released
how do enzymes work in terms of lowering the activation energy?
--> provides a reaction surface or cavity for binding substrates --> Brings reactants together. --> positions reactants correctly for correct transition state configuration --> weaken bonds -->may participate in mechanism via covalent interactions (most are non-covalent)
Problems with Penicillin G
-Ineffective when taken orally due to acid sensitivity - Sensitive to B-lactamases produced by bacteria - Narrow spectrum of antibacterial activity
SAR Studies example using structure of morphine 1. a phenolic ring (slightly acidic)
1. The phenolic OH: - methylation of the phenolic hydroxyl function results in a 80% loss in activity and generates codeine. - small analgesic effect and is mainly used in anti-cough and anti-diarrhoea formulations (in fact codeine is actually a prodrug for morphine - demethylation takes place in the liver - 3-Acetyl morphine possesses increased analgesic activity over codeine
what are the Drugs Acting on DNA - what are the 5 main groups of drugs that act on DNA
1. intercalating cytostatic agents 2. alkylating agents 3. chain 'cutters' 4. non-covalent groove binders 5. antigene/antisense (triple helix) compounds
what are the 4 main types of receptors
1. ligand gated ion 2. G-protein linked receptors 3. Tyrosine Kinase Receptors (not covered) 4. Intracellular (cytoplasmic) receptors - for steroid hormones (not covered)
SAR Studies example using structure of morphine 2. an alcohol group (comment on the acetylation of both 3 and 6 hydroxyl function)
2. The 6 alcohol: - Replacement of the hydroxyl function on carbon 6 by either an ethyl group (6- ethyl morphine) or an acetyl group (6-acetyl morphine - activity 6x that of morphine) greatly improves the analgesic effect NOTE: activity arises as a consequence of changes in pharmacokinetics and not as a function of their affinity for the receptor i.e. it is a measure of how much of the material can reach the receptor, not how well the material binds once there - Acetylation of both the 3 and the 6 hydroxyl functions led to the formation of diamorphine or heroin. This material has both hydroxyl functions masked and can traverse the blood-brain barrier easily. However, it is still less active than 6-acetylmorphine Why? (hint: codeine injected straight into the brain has NO analgesic activity) --> dimethyl enzyme is not in the brain its in the liver so that OH group is essential for activity so when we changed the OH to OMe; found that the activity was less - The 6-OH group is not required for analgesic activity - its removal can be beneficial to analgesic activity
SAR Studies example using structure of morphine 4. The aromatic ring 5. The ether bridge 6. Stereochemistry (comment on other isomers of morphine)
4. The aromatic ring: Removal of the aromatic ring results in complete loss of activity 5. The ether bridge: The ether bridge is not required 6. Stereochemistry: The other isomer of morphine has no activity at all
Simple Competitive Inhibition: Antihypertensive Drugs - A Case Study - how does ACE inhibition potentiate other actions of other peptides
ACE is a Zn containing exopeptidase--> cleaves of the peptide bond at the terminal of the peptide ( ACE is used for the cleavage of other biologically important peptides) - the Renin-Angiotensin-Aldosterone (RAS) system is a multi-regulated proteolytic cascade of enzyme mediated events that converts Angiotensinogen into Angiotensin I, Angiotensin II (vasoconstriction) and Angiotensin III - cascade of events that controls BP - Key step: cleavage of angiotensin I between Phe and His by the relatively non-specifc enzyme ACE - this enzyme is a glycoprotein found, amongst other places, in the epithelial cells of pulmonary capillaries (note; membrane bound proteins very hard to work worth; not soluble and hard to purify) - the substrate specificity of ACE is minimal requiring at least a tri-peptide, a terminal carboxyl group and any L-amino acid except proline --> thus the substrate specificity is minimal
Pharmacophore Approach to Drug Design - assumption
Assumption - correct positioning of binding groups --> "active" messenger (ie. drug) PHARMACOPHORE = spatial arrangement of functional groups that interact with the receptor --> rest of molecule = framework to hold groups in place
catalytic role of enzymes - binding interactions for allosteric inhibition
Binding interactions - allosteric interaction cannot be explained by a rigid lock and key principle - Induced fit: Small molecule (inhibitor, cofactor, substrate) binding to enzyme forces changes in shape (conformation) of BOTH molecules MOULDING PROCESS - koshland's theory of induced fit: substrate induces the active site to take up ideal shape to accommodate it
Alkylating Agents: CC 1065
CC 1065 is a natural product derived from Streptomyces Zelensis. - It is a known DNA binder and potent anti-tumour drug
Mechanism of Action of Penicillin:
Cross linking of bacterial cell walls inhibited by Penicillin - Transpeptidase is similar to serine proteases which catalyse the hydrolysis of peptide bonds - the enzymes splits the peptide bond between 2 D-Alanine residues. Once the terminal Ala leaves, the pentaglycyl moeity of another peptide chain forms a peptide bond to the remaining D-Ala linking the two chains together. - The shape of penicillin closely resembles that of D-Ala-D-Ala - Transpeptidase reacts with penicillin instead of the polypeptide chain. - The enzyme becomes covalently linked to the drug (irreversible acylated). - New cell wall cannot be produced and therefore the dividing bacterium cannot survive.
Major and Minor Grooves of Double Helix
Duplex b-DNA possesses two distinct grooves in its structure. - major groove (~12 Å wide, 8.5 Å deep) - minor groove (~6 Å wide, 7.5 Å deep) - on the floor of these grooves, the base-pairs that express the genetic info are exposed and can interact with various small molecules - polar sugar-phosphate backbone is on outside of structure where it can form polar interactions with water
Irreversible Inhibitors - Give examples - what are the most effective irreversible inhibitors
EG- penecillin, nerve gases (toxic substances) - irreversible; increased levels of natural substrates cannot dislodge the squatter - the most effective irreversible inhibitors are compounds which react with the nucleophilic -OH and -SH groups of amino acids such serine and cysteine which frequently occur in the enzyme active sites - the inhibitor is an electrophilic drug which fits the active site and locates its electrophilic region close to the nucleophilic residues and forms a covalent bond to the enzyme
Intercalating Agents - what should these agents structure be in order to act as intercalating agents - give example
Intercalating agents are compounds that are able to slip between the layers of nucleic acid base pairs and disrupt the shape of the double helix. - Materials that work in this way must be flat, and therefore aromatic or heteroaromatic in nature. - Several natural compounds such as the antitumour antibiotics Daunomycin and Adriamycin work in this fashion - Antimalarial chloroquine is an intercalating agent
Inhibition of Carboxypeptidase by L-Benzylsuccinic acid
L-Benzylsuccinic acid binds to the active site using the same interactions as the natural substrate but get NO hydrolysis --> no peptide bond so inhibitor binds and blocks active site but no cleaving
example of neurotransmitters
Monoamines - acetylcholine - dopamine - catecholamine - seratonin Amino acids also act as neurotransmitters - glycine - glutamic acid - Y-aminobutanoic acid - 5-hydroxytrptamine
Receptors — A Case Study: Analgesics (alkaloids; opiates) - morphine (why is Morphine a classic example of the traditional approach to medicinal chemistry - list steps of its discovery)
Morphine is a classic example of the traditional approach to medicinal chemistry, namely: Stage 1. Recognition that a natural plant or herb displays a pharmacological action Stage 2. Extraction and isolation of the active compound(s) Stage 3. Synthetic studies (full and partial synthesis) Stage 4. SAR - Structure activity relationships. The synthesis of analogues to determine the structural requirements needed for activity Stage 5. Drug development. The synthesis of (more!) analogues to try and improve activity and or reduce side effects Stage 6. Theories on the nature of the (analgesic) receptors. Synthesis of more analogues to test theories - one of the most effective painkillers available to modern medicine - However it has numerous side-effects including: depression of the respiratory centre, constipation, excitation, euphoria, nausea, pupil constriction, tolerance and dependence - Morphine is used to treat diarrhoea (Codeine & Morphine) - However, some of the other side effects are more sinister. Typically those of tolerance and dependence and the depression of breathing - The former two resulting in severe withdrawal symptoms that typically include; anorexia, weight loss, pupil dilation, chills, excessive sweats, abdominal cramping, muscle cramps and spasms, hyperirritability, lachrymation, tremor, and increased blood pressure and heart rate
Antagonists of Morphine
Nalorphine (Alkyl substitution at 3° N position) - This molecule has virtually no analgesic activity but does bind to the morphine receptors - acts as an antagonist at 2 receptors - Nalorphine does not induce the conformational change in the receptor needed to elicit the 'on' response in 2 of the receptors - Nalorphine is partial agonist at 3rd receptor - weak analgesic but NO side effects
Binding Interactions Between Ligand and Receptor
Neurotransmitter needs all its binding interactions to be effective
Design of Competitive Reversible Inhibitors: Simple Competitive Inhibition
Simple Competitive Inhibition - inhibitor structure resembles that of the natural substrate of the enzyme - most common type of inhibition - the inhibitor does not react with the enzyme, it only blocks the AS of the enzyme and prevent accessibility to the natural substrate eg Antihypertensive drugs: ACE-I
SAR Studies example using structure of morphine 3. The 3° amine:
The 3° amine: - Removal of the nitrogen results in complete loss of activity, and both the dimethyl and N-oxide quaternary salts show no activity in vivo! (cannot cross BBB as drug was made even more polar by N becoming +vely charged) - But, direct injection of the salts into the brain results in analgesic activity about that of morphine - This suggests that the nitrogen is essential and that the compound binds to the target receptor as a protonated salt
base pairing
The bases always associate such that • Adenine is paired with Thymine (A.T) • Guanine is paired with Cytosine (G.C) A.T = forms a 2 point H-bond G.C= forms a 3 point H-bond
Noncovalent Groove Binders - major and minor grooves differ in
These materials bind non-covalently to the DNA minor and major grooves - Major and minor grooves differ significantly in: i. electrostatic potential ii. hydrogen bonding characteristics iii. steric effects iv. hydration - Proteins bind predominantly in the major groove - Small molecules generally bind in the minor groove
Noncovalent Minor Groove Binders - Contributions to minor groove binding (what binding interactions occur) - give example
They are generally long, usually curved, and possess hydrogen bonding sites and charged groups along their length complementary to the DNA Contributions to minor groove binding - Van der Waals contacts through sugar-phosphate backbone - H-bonding (bridging bonds between O2 thymine and N3 adenine) - Selective for AT rich sequences Example Netropsin - Minor Groove Binding
Captopril
Thiol group introduced as it binds to zinc - active site model for carboxypeptidase showed that inhibitors of carboxypeptidase A= weak but selective inhibitors of ACE --> from this developed captopril; contain proline and SH group - captopril had good bioavailability but side effects included rashes, loss of taste, mucosal lesions and a persistent cough - this was because ACE (non-specific enzymes) is used for cleavage of other biologically important peptides; ACE inhibition may potentiate the action of these other peptides
what are the bodies natural pain killers? what is their structural components? which amino acid is essential for their activity?
We know we possess analgesic receptors but what is the natural ligand? The Enkephalins and Endorphins - These are the body's natural painkillers and are composed of short polypeptide chains that range in size from 5-33 amino acids for the 15 or so compounds that have been identified - The key structural component is a Met- or Leu-enkephalin skeleton at their N-terminus. A Tyr is essential for activity
What if the body was producing too much neurotransmitter?
design an antagonist - this material would block the binding site from the natural substrate but not induce the conformational change that switches on the response
resolution of a racemic mixture - comment on the binding energy for E.S complex when the enzyme is exposed to a racemic mixture of a substrate
enzymes are chiral molecules - E + racemic substrate= 2 diastereomic complexes - analogous to the principle behind resolution of racemic mixtures by chiral reagents as shown in the figure above. Diasterioisomers have different physical properties and can be separated. - when an enzyme is exposed to a racemic mixture of a substrate, the binding energy for E.S complex formation with one enantiomer may be much higher than that with the other enantiomer which could be due to differential binding interactions or for steric reasons
Alkylating Agents: nitrogen mustards
mechlorethamine, known as the nitrogen mustards - To reduce mechlorethamine (ie its high activity and poor specificity) compounds such as the phenyl mustards (lower activity) and the uracil mustards (higher selectivity) have been developed
what are the secondary cascade effects of the binding of the neurotransmitter to the receptor?
secondary effects: - flow of ions across cell membrane - enzymes in target cell switched on or off - A biological response then results eg the contraction of a muscle cell, an increase in blood pressure, increase in heart rate, activation of fatty acid metabolism in a fat cell - These receptors are usually glycoproteins of specific 3D structure and are located on the postsynaptic cell membrane - Many types of drug also 'fit' into the various types of receptors: --> agonist: elect a response --> antagonist: block receptor site; no response
when do we need to interfere with cellular communication?
too many messengers -> overheating of cell -> antagonist drug (block receptor) -> normal cell function too few messengers -.> sluggish cell -> agonist drug (replacement messenger) -> normal cell function
Alkylating Agents - describe alkylating agents - the 2 ways they can act as alkylating agents - give example
•Alkylating agents are highly electrophilic compounds that react with a nucleophile to form a covalent bond. - N-7 of guanine, N-7 of adenine, O-6 of guanine, N-3 of adenine, and PO4- - Electrophilic functional groups such as epoxides, cyclopropanes and others can react with these nucleophilic nitrogens forming a DNA/substrate covalent bond 2 ways they can act as alkylating agents - can cross-link the two DNA preventing dissociation during transcription - link two guanines in the same chain so preventing protein access --> Alkylation weakens N-glycosidic bond -> depurination -> strand breakage Example - CC 1065 - mechloroethamine: nitrogen mustard - Aziridinylbenzoquinones (AZQ) - DNA plantation- Cisplatin