2. Drug Metabolism: Individual Enzymes Flashcards
1
Q
Why study drug metabolism?
A
-Predict the formation of active/toxic metabolites
-Understand how interpersonal differences affect drug action
-Drug licensing
-Understand the pharmacokinetics of a drug
-Changes in metabolism can predict toxicity (eg drug drug interactions)
-Prodrug activation
2
Q
What is xenobiotic metabolism?
A
The process by which the body transforms foreign compounds to facilitate their elimination
3
Q
What are xenobiotics?
A
Non endogenous compounds, eg
-Drugs
-Toxins
-Pollutants
-Environmental chemicals
4
Q
Describe the process of enterohepatic recirculation
A
-Compounds are digested and absorbed from the ileum into the bloodstream
-These are transported via the hepatic portal vein to the liver
-These are secreted into the bile, which are secreted back into the duodenum via the bile duct
5
Q
What is the action of enterohepatic recirculation on xenobiotics action?
A
Prolongs the effects of drugs and xenobiotics
6
Q
Give some sites of metabolism
A
-Liver
-GI
-Kidney
-Skin
-Lungs
-Plasma
-Brain
7
Q
Give the functions of the liver
A
-Detoxification
-Metabolism of carbohydrates, lipids and proteins
-Synthesis of plasma proteins
-Storage of glycogen, vitamins and minerals
-Bile production
8
Q
Give some cell types in the liver
A
-Hepatocytes
-Cholangiocytes
-Kupffer cells
-Stellate cells
-Endothelial cells
-Fibroblasts
9
Q
What is liver regeneration?
A
-Process where the liver recovers following damage
-Involving hepatocyte proliferation, requiring liver stem/progenitor cells
10
Q
What may repeated liver damage result in?
A
-Chronic exposure to harmful xenobiotics can result in fibrosis (excessive scar tissue)
-This hinders the regenerative capacity, leading to cirrhosis
11
Q
Where in the hepatocyte is the main area of xenobiotic metabolism?
A
Smooth endoplasmic reticulum
12
Q
What are the phases of drug metabolism?
A
-Phase I (catabolic)
-Phase II (anabolic)
-Phase III (transport)
13
Q
Describe Phase I metabolism of drugs
A
-Functional groups are introduced
-Creating a more polar molecule
-Completed by a range of enzymes (eg CYP450s, esterases, oxidases, dehydrogenases)
14
Q
Describe Phase II metabolism of drugs
A
-Drug is conjugated with an endogenous substance
-Detoxify, create more polar compounds and prepares for excretion
-Completed by a range of transferase enzymes
15
Q
What are prodrugs?
A
-Pharmacologically inactive compounds that undergo transformation
-Releasing an active drug
16
Q
What are the benefits of using prodrugs over normal drugs?
A
-Improved bioavailability
-Enhanced lipophilicity
-Targeted delivery
-Improved stability and half life
-Bypassing first pass metabolism
-Reduced irritation during administration
17
Q
Give some examples of prodrugs
A
-Diacetylmorphine
-Codeine
-Cyclophosphamide
18
Q
What are cytochrome P450 enzymes?
A
-Superfamily of heme containing monooxygenases
-Involved in the metabolism of drugs, xenobiotics and endogenous compounds
-Most important superfamily in phase I metabolism
19
Q
Where are cytochrome P450s found in the cell?
A
Found in the endoplasmic reticulum and mitochondria
20
Q
Describe the cytochrome P450 nomenclature
A
-Root CYP followed by family (eg CYP1)
-then subfamily (eg CYP1A)
-Then form number (eg CYP1A1)
-Then allelic variant (eg CYP1A1*4)
21
Q
What is the sequence similarity between families of cytochrome P450s versus between form numbers?
A
<40% versus >95%
22
Q
What are the functions of cytochrome P450s?
A
-Xenobiotic metabolism (oxidation to hydrophilic metabolites, prodrug activation, detoxification)
-Endogenous metabolism (steroidogenesis, eicosanoid metabolism)
-Regulation of vitamin and hormone levels (metabolism of retinoid acid and vitamin D)
23
Q
Which families of cytochrome P450s are involved in the metabolism of xenobiotics?
A
CYP1 - 3
24
Q
Describe the structural characteristics of CYP450s
A
-Between 40000 and 50000 MW
-Contains protoporphyrin IX heme group, coordinated with an iron atom
-Heme iron is ligated to a conserved cysteine residue in protein
-Contain a hydrophobic active site (varying between specificities) which interacts with lipophilic molecules
25
Describe protoporphyrin IX
-P450 prosthetic group
-Highly conjugated tetrapyrrole macrocycle
-Pyrrole rings linked via methine bridges forming a planar system
-Central Fe is coordinated by the four nitrogen atoms in porphyrin ring
-Central Fe is in (III) rested state or binding oxygen state (II)
26
Why are Cytochrome P450 enzymes called that?
-Protoporphyrin IX gives CYP450 its characteristic absorption peak at 450nm when Fe2+ is bound to CO
27
Give the general CYP450 catalytic reaction
-Monooxygenation where one oxygen atom is incorporated into substrate (RH)
RH + O2 + NADPH + H+ -> ROH + H2O + NADP+
28
What are the components required for the cytochrome P450 reaction?
-Cytochrome P450 enzyme
-NADPH cytochrome P450 reductase
-Phosphatidylcholine
29
What is the role of NADPH in the cytochrome P450 reaction?
Supplies 2 protons and 2 electrons in an electron transfer process
30
Describe the structure of NADPH-Cytochrome P450 reductase
Flavoprotein containing two flavin cofactors:
-FAD (Flavin Adenine Dinucleotide)
-FMN (Flavin mononucleotide)
31
Other than NAPDH-Cytochrome P450 reductase, what other flavoproteins are used?
Adrenodoxin in mitochondrial P450s
32
Describe the function of NAPDH-Cytochrome P450 reductase in the CYP450 catalytic cycle (forming the monooxygenation reaction)
-Enables the two step reduction of CYP450 heme iron
-Electrons flow from NADPH -> FAD -> FMN -> CYP450 Fe(III), reducing to Fe(II), increasing its affinity for oxygen
-Electron is transferred from NAPDH-CYP450 reductase to Fe2+-O2, generating a Fe3+-O2- Superoxide intermediate
-Leading to O-O bond cleavage, forming Fe4+=O, which hydroxylates the RH to ROH.
33
Describe the mechanism of CYP450 induction
-Occurs via (xenobiotic or drug) ligand binding to nuclear receptor
-This complex is translocated to the nucleus, which in turn binds to xenobiotic response elements in CYP450 gene promoters, enhancing mRNA transcription of CYP enzymes
-mRNA is translated into functional CYP450 enzymes, increasing metabolic capacity
34
What are the consequences of CYP450 induction?
-Increased drug metabolism, leading to decreased therapeutic effect
-Drug drug interactions as CYP450 inducers can alter the metabolism of coadministered drugs
-Toxin and carcinogen activation
-Hormonal disruptions as certain CYPs alter hormonal metabolism
35
Describe the distribution of cytochrome P450s in the body
-70-80% of total CYP450 activity is in the LIVER, embedded in the ER of hepatocytes
-SMALL INTESTINE in enterocytes
-KIDNEYS in renal proximal tubule cells
-LUNGS in bronchial epithelial cells and alveolar macrophages
-BBB in endothelial cells
-SKIN in keratinocytes and dermal fibroblasts
-PLACENTA
-ADRENAL GLANDS
36
What is the function of CYP450 in the adrenal glands and gonads?
Synthesis of steroid hormones
37
Give examples of reactions catalysed by P450s
-N dealkylation
-O dealkylation
-Aliphatic hydroxylation
-Aromatic hydroxylation
-N oxidation
-S oxidation
-Deamination
38
What is each cytochrome P450 isoform a product of?
Different genes
39
What do different CYP450 isoforms vary in?
-Substrate specificity
-Tissue distribution
-Polymorphisms
-Regulatory mechanisms
-Clinical significance
40
How many drugs subject to metabolism are P450 substrates?
70-80%
41
Which CYP450 isoforms are most drugs metabolised by?
Almost 90% of all drugs metabolised by CYPs are metabolised by
-CYP3A4
-CYP2D6
-CYP2C9
42
Give some methods used to identify which P450 isoform is responsible for the metabolism of a drug
-Correlation analysis using human liver microsome bank
-Inhibition studies
-Studies using purified or expressed enzymes
43
Describe using correlation analysis with human liver microsomes to identify P450 isoform specificity
-Uses HLMs from different donors with known CYP activities
-Drug metabolism rates are correlated with known CYP isoform activities
-High correlations suggest involvement of this CYP.
44
Describe using inhibition studies to identify P450 isoform specificity
-Specific inhibitors are used in microsomal or recombinant enzyme assays
-Rate of baseline drug metabolism is measured, and a selective inhibitor is added
-If inhibition occurs, that CYP isoform is likely involved
45
Describe using purified or recombinant CYP enzymes to identify P450 isoform specificity
-Drugs are incubated with individual CYP enzymes
-Metabolite formation is measured
-Metabolic rates are compared, with metabolism occurring signifying a specific isoform is responsible
46
Give the xenobiotic substrates of CYP2C9
Warfarin and Ibuprofen
47
Give the xenobiotic substrates of CYP2D6
Codeine, Metoprolol, Propranolol, Tricyclic antidepressants
48
Give the xenobiotic substrates of CYP3A4
Nifedipine, Cyclosporin and many others
49
Give the xenobiotic substrates of CYP1A1
Benzopyrene and Estradiol
50
Give the xenobiotic substrates of CYP1B1
Benzopyrene, Benzathracene, Estradiol
51
What is special about CYP1A1 and CYP1B1
-They have overlapping substrate specificity
-They are both induced by the aryl hydrocarbon receptor
52
Give the xenobiotic substrates of CYP2C19
Omeprazole and Clopidogrel
52
Give the xenobiotic substrates of CYP2C8
Taxol and Retinoic acid
53
Give the xenobiotic substrates of CYP2E1
Ethanol, Carbon tetrachloride
54
Describe CYP2D6
-Highly specialised for basic compounds, meaning typical substrates have a basic nitrogen
-Position of oxidation is usually 5-7 Å from the basic nitrogen
-5 to 10% of the population lack this enzyme due to genetic mutations
-Most substrates are either cardiovascular agents, antipsychotics or antidepressants
-Not inducible
-Mainly uses hydroxylation reactions
55
Describe CYP2C9
-Substrates tend to have areas of strong hydrogen bond forming potential or ion pair formation 5-10 Å from the site of metabolism
-Substrates include NSAIDs, anticoagulants, anti epileptics, antihypertensives
-Subject to genetic polymorphism
-Inducible by barbiturates and rifampicin
56
Describe CYP3A4
-Highly inducible by glucocorticoids, rifampicin, and other compounds
-Most abundant and clinically significant CYP
-Considerable structural diversity, but have limited effects due to CYP3A5 compensation
-N dealkylation reactions particularly common, also aromatic hydroxylation
-Substrates include Erythromycin, Lidocaine, Tamoxifen, Cocaine, Verapamil
57
Give the factors determining metabolism by specific P450 isoforms
-Enzyme active site topography that influences substrate binding and orientation within the catalytic pocket
-Steric hindrance of access to active site (substrates must fit correctly
-Ease of hydrogen abstraction affects the reactivity, with different isoforms differing in preference for hydrogen abstraction sites
58
Describe P450 isoforms role in metabolising xenobiotics other than drugs
-CYP1A1 has major role in activation of polycyclic aromatic hydrocarbons
-CYP2E1 contributes to ethanol metabolism
-CYP1A2 can activate arylamine compounds to carcinogens and has an important role in caffeine metabolism
59
What is autoinduction of cytochrome P450 enzymes?
The process where a drug induces the expression of the CYP450 enzyme responsible for its own metabolism
60
Give some non P450 enzymes involved in oxidation (phase I)
-Flavin linked monooxygenases (FMO)
-Prostaglandin H synthase dependent cooxidation
-Amine oxidases
-Oxidoreductases
61
Give some non P450 enzymes involved in hydrolysis (phase I)
-Esterases
-Epoxide hydrolases
-Paraxonases
62
Instead of a heme prosthetic group (like in CYPs), what prosthetic group do Flavin-linked monooxygenases contain?
Flavin Adenine Dinucleotide (FAD)
63
Describe the two step mechanism through which flavin linked monooxygenases oxidise?
1) NADPH reduces FAD within the FMO active site, forming an FADH2 intermediate
2) Oxygen binds to FADH2, generating a species that oxidises substrates before releasing H2O
64
Are Flavin linked monooxygenases (FMOs) inducible?
-Most isoforms are thought to be non inducible
-But FMO5 is an exception
65
What is FMO5 inducible by?
Rifampicin in human hepatocytes
66
How many isoforms are there of Flavin linked monooxygenases?
5 (FMO1-5)
67
Describe the physiological distribution of Flavin linked Monooxygenases (FMOs)
-Highly expressed in liver, lungs and kidneys, with FMO3 being the major hepatic isoform in humans
-FMO1,2,4 and 5 exhibit tissue specific expression patterns
68
Which reactions do Flavin Linked Monooxygenases catalyse?
-Oxidation of nucleophilic nitrogen, phosphorus and sulfur in xenobiotics during phase I metabolism
-P oxidation of organoPHOSPHATES
-S oxidation of thioesthers to SULFOXIDES
-N oxidation to tertiary AMINES
69
Where in the cell are flavin linked monooxygenases found?
In the endoplasmic reticulum
70
Describe peroxidase dependent cooxidation
-Enzymatic process where peroxides catalyse the oxidation of substrates in the presence of Hydrogen peroxide during Phase I metabolism
-Completed either through hydrogen peroxide activation, substrate oxidation, electron transfer or reactive species formation
71
Give some examples of enzymes with peroxidase activity
-Prostaglandin H synthase (COX!)
-Myeloperoxidase
-Lactoperoxidase
-Glutathione peroxidase
72
In which cells is peroxidase dependent cooxidation important?
Cells that lack other xenobiotic oxidising enzymes
73
Describe both the cyclooxygenase activity and the peroxidase activity of prostaglandin H synthase
-Cyclooxygenase activity: Converts arachidonic acid into prostaglandin G2 (PGG2)
-Peroxidase activity: Reduces PGG2 to Prostaglandin H2
74
Describe the cooxidation mechanism that prostaglandin H synthase uses
-During peroxidase activity/step, PGHS can cooxidase other substrates (eg drugs and xenobiotics) through redox in the presence of H2O2
75
Describe amine oxidases
-Enzymes involved in the phase I oxidative deamination of amines
-Catalyse primary, secondary and tertiary amines to aldehydes or ketones (releasing ammonia
76
Give some key classes of amine oxidases
-Monoamine oxidases
-Diamine oxidase
-Histamine N methyltransferase
77
Describe Monoamine oxidases
-Membrane bound Flavin containing enzymes facilitating oxidation of amines
-Main role is oxidising neurotransmitters but also oxidises some xenobiotics eg propranolol
-2 classes: MAO-A and MAO-B
78
Describe the MAO-A subtype of Monoamine oxidases
-Metabolises serotonin, norepinephrine, dopamine and tyramine
-Distributed in brain, liver, gut, placenta and kidneys
-Found in serotinergic, noradrenergic and dopaminergic neurones
79
Describe the MAO-B subtype of Monoamine oxidases
-Metabolises mostly dopamine, but also phenylethylamine and trace amines
-Highly concentrated in dopaminergic neurones in the brain (particularly basal ganglia)( but also in platelets and astrocytes
80
What are MAO-A gene variations linked to?
Mood disorders
81
What are MAO-B gene variations linked to?
Neurodegenerative diseases (particularly Parkinson's)
82
Give examples of oxidoreductases/dehydrogenases
-Alcohol dehydrogenase
-Aldehyde dehydrogenase
-Carbonyl reductase
-NAD(P)H quinone oxidoreductase
83
Describe what oxidoreductase/dehydrogenase enzymes are dependent on in each direction
-Oxidising direction: NAD(P) dependent
-Reducing direction: NAD(P)H dependent
84
Describe alcohol dehydrogenase
-Involved in the metabolism of alcohols including ethanol and other alcohols from drugs/xenobiotics
-Oxidise alcohol to aldehyde
-Normal method of ethanol detoxification
-eg Ethanol to Acetaldehyde
85
Describe aldehyde dehydrogenase
-Involved in the oxidation of aldehydes to their corresponding carboxylic acids
-eg converts Acetaldehyde to acetic acid, which is further metabolised to acetyl-CoA
86
Give the reaction that alcohol dehydrogenase catalyses
Alcohol + NAD+ -> Aldehyde + NADH + H+
87
Give the reaction that aldehyde dehydrogenase catalyses
Aldehyde + NAD+ -> Carboxylic acid + NADH + H+
88
Describe carbonyl reductases
Reduce carbonyl compounds (such as ketones and aldehydes) in phase I metabolism
89
Give some examples of carbonyl reductase substrates
-Quinones
-Prostaglandins
-Menadione
-Various xenobiotics
90
Describe NAD(P)H quinone oxidoreductase 1
-Flavoprotein involved in reduction of quinones (or quinone like compounds) using NADH or NAPDH
-Involved in detoxification of reactive quinone intermediates and prevention of oxidative damage
-Helps target UV generated damage
-Helps metabolise xenobiotics
91
Describe esterases
-Catalyse hydrolysis of ester bonds in phase I metabolism
-Only require water
-Found in Liver, plasma, kidneys, intestine, nervous system and muscle tissue
92
Give some examples of esterase enzymes used in phase I metabolism
-Butrylcholinesterases (AKA serum cholinesterase)
-Carboxylesterase 1 (hCE1) and 2 (hCE2)
-Paraoxonase (PON1)
93
Describe Carboxylesterases
-Catalyse the hydrolysis of a wide range of chemicals eg cocaine (hCE1) and procaine (hCE2) in phase I metabolism
-Widely distributed, including liver, intestine, brain, plasma
-hCE1 prefers substrates with a small alcohol group, large acyl group
-hCE2 prefers substrates with a large alcohol group, small acyl group
94
Describe cholinesterases
-Hydrolyse choline esters
-Include Acetylcholinesterase (hydrolysing ACh in synaptic cleft) and Butyrylcholinesterase (hydrolysing aspirin to salicylate)
95
Describe epoxide hydrolyses (EPH)
-Esterase that cleaves epoxides to diols in phase I metabolism
-Exist either in microsome or cytosol, with different substrate specificities
-Responsible for drug metabolism and benzopyrene activation to genotoxicity
96
Describe Paraoxonase enzymes
-Family of hydrolase (esterase) enzymes involved in detoxification, lipid metabolism and antioxidant defence in phase I metabolism
-Xenobiotic targets include pesticides, nerve agents (PON1)
-Have roles in degradation of oxidised lipids (PON1 and 3)
-Have roles in reducing ROS (PON2)
97
Give some conjugation reactions found in phase II metabolism
-Glucuronidation
-Sulfation
-Amino acid conjugation
-Glutathione conjugation
-Methylation
-Acetylation
98
Which conjugation reactions aim to increase charge/water solubility of a compound?
-Glucuronidation
-Sulfation
-Amino acid conjugation
-Glutathione conjugation
99
Which conjugation reactions aim to inactivate a compound?
-Methylation
-Acetylation
100
What enzyme carries out glucuronidation?
Uridine diphosphate-glucuronosyltransferase (what a mouthful)
AKA UDP-GTs
101
What enzyme carries out sulfation?
Sulfotransferase
102
What enzyme carries out methylation?
Methyltransferase
103
What enzyme carries out acetylation?
Acetyltransferase
104
What enzyme carries out glutathione conjugation?
Glutathione S transferase
105
What is the purpose of conjugation reactions in phase II metabolism?
-Compounds are transformed from lipophilic to more hydrophilic
-Neutralise toxic compounds
-This facilitates easier and more efficient excretion of xenobiotics
106
What is required as a cofactor for glucuronidation?
UDP glucuronic acid
107
Which endogenous compounds undergo glucuronidation?
-Steroids
-Vitamins
-Bile acids
-Bilirubin
108
Where are UDP-GTs found?
Only in endoplasmic reticulum
109
Describe UDP-GTs
-Monomeric proteins of Mw 50-60,000
-Contain no prosthetic groups
-Have a large number of isoforms
110
Where are UDP-GTs found?
-Found highest in the liver
-Also present in kidney, lung, small intestine, skin and adrenal gland
111
Why are phase II enzymes found in the same space as phase I enzymes?
To reduce the effect of reactive/toxic phase I products
112
Give the effect of glucuronidation
Normally promotes excretion and a loss of biological activity
113
Give some of the other effects of glucuronidation, outside of metabolising for excretion
-Some glucuronides have biological activity (eg morphine glucuronide)
-Some are reactive and can bind irreversibly to cellular proteins (eg acyl glucuronides)
-Some can trigger an immune response
114
What are the two main UDP-GT families in humans?
UGT1 and UGT2
115
Describe what all UGT1 isoforms are products of?
The UGT1A gene complex consists of multiple first exons that combine with shared downstream exons to produce different UGT1A isoforms.
116
Describe what all UGT2 isoforms are products of?
Separate genes scattered throughout the human genome similar to other enzyme systems such as the P450 superfamily
117
Give some compounds that UGT1A1 is specific for?
-Bilirubin
-Ethinyl estradiol
118
Give some compounds that UGT1A4 is specific for?
Amines such as
-Imipramine
-Amitriptyline
-Chlorpromazine
119
Give some compounds that UGT1A6 is specific for?
Paracetamol
120
Give some compounds that UGT2B7 is specific for?
-Morphine
-Ibuprofen
121
Give some compounds that UGT2B10 is specific for?
Nicotine
122
Which glucuronidated compounds are excreted in the bile?
Those with a MW > 400
123
Which glucuronidated compounds are excreted in the urine?
Those with a MW < 400
124
What may occur to glucuronides excreted in the bile?
They may undergo enterohepatic recirculation (potentially increasing half life), reducing ability to effectively predict elimination
125
Which functional groups are UDP-GTs specific for?
-Phenols: e.g., acetaminophen
-Alcohols: e.g., morphine
-Carboxylic Acids: e.g., ibuprofen
-Amines: e.g., sulfonamides
-Thiol groups: e.g., thiol-containing drugs or toxins
126
Give some examples of UDPGT inducers
-Phenobarbitol
-Aromatic hydrocarbons
-Estrogen and Testosterone
127
Describe sulfotransferases
-Soluble cytosolic enzymes
-Contain two subunits of MW ~34000
-Distributed widely
-At least 11 separate isoforms
128
What do the SULT1 family mostly sulfate?
Phenolic compounds (e.g., hormones, drugs), alcohols, and catecholamines
129
What do the SULT2 family mostly sulfate?
Steroids, including estrogens, androgens, corticosteroids, and bile acids.
130
What do the SULT4 family mostly sulfate?
Unclear, but appears to be neurotransmitters in the CNS
131
Describe the mechanism of action of Sulfotransferases?
Transfer a sulfonate group (typically from PAPS) to a hydroxyl, amine, or other nucleophilic group of the substrate
132
Describe substrates processed by sulfotransferases
-More soluble
-Anionic
-Inactive
133
Why does sulfation play a more significant role when substrate levels are low?
-Glucuronidation and glutathione conjugation are less effective, meaning when not saturated sulfotransferases may be the primary mechanism
-Cofactors (eg PAPS) are limited
134
What is required for sulfation?
-Sulfonate donors
-eg PAPS
135
Does sulfation always lead to detoxification?
-No, as it may activate carcinogens
-eg forming reactive nitrenium compounds such as N-hydroxyacetylaminofluorene
136
Give some substrates that are sulfated leading to carcinogenesis
-Polycyclic Aromatic Hydrocarbons (PAHs) eg cigarette smoke, grilled meats
-Aromatic Amines eg dyes and tobacco smoke
-Catechol estrogens
137
Describe the regulation of sulfotransferase through ligand binding
Dioxins and other AhR ligands activate the aryl hydrocarbon receptor (AhR), leading to changes in gene expression, including the induction or repression of sulfotransferases
138
Where in the cell do amino acid conjugation reactions occur?
Mitochondria
139
What are the major conjugating amino acids in humans?
-Glycine
-Taurine
-Glutamine
140
What enzyme catalyses amino acid conjugation
Amino acid transferase
141
Describe the mechanism of amino acid conjugation
-Compound is activated by Acyl Coenzyme A
-Amino acid is conjugated forming an amide
142
What carries out the initial reaction in amino acid conjugation (activation)
Mitochondrial xenobiotic Medium chain fatty acid: CoA ligases (MACS)
143
What are CoA ligase MACS also known as?
Mitochondrial acyl-CoA synthetase
144
Describe the selectivity of Amino acid/N acyl transferase?
While many CoA derivatives are produced, only certain ones are conjugated with amino acids (like glycine or glutamine) for detoxification
145
What can the selectivity of amino acid/N acyl transferase lead to?
This poor conjugation can lead to the sequestration (trapping) of CoASH in its thioester form, reducing its availability for other cellular processes and possibly contributing to toxicity
146
What substrates does acetylation focus on?
-Compounds containing amine or hydrazine groups
-Also focuses on hydroxyl or sulfhydryl groups
147
What enzymes carry out acetylation?
N-acetyltransferases
148
Could you describe the acetylation phase II reaction?
NAT enzymes transfer an acetyl group (-COCH3) from acetyl-Coenzyme A (acetyl-CoA) to the xenobiotic or drug
149
How is coenzyme A produced in the body?
Obtained through diet
150
Where in the cell are N-acetyltransferase enzymes found?
NAT1 and NAT2 are found in the cytosol
151
Are N-acetyltransferases inducible?
NO!
152
Where in the body are N-acetyltransferases expressed?
-NAT1 expressed in most tissues
-NAT2 expressed mainly in liver and intestine
153
Describe polymorphisms in acetylation
-Polymorphisms found mostly in NAT2
-NAT1 also shows some, but not as much as NAT2
-Can be classified as slow, intermediate or fast acetylators, based on the number of alleles (homozygous vs hetero) for the functional NAT2
154
Give some drug substrates for N-acetyltransferases?
-Isoniazid (NAT2)
-Sulfamethoxazole (NAT1)
155
Describe the effects of acetylation
-Masking of amine group, resulting in decreased toxicity
-May activate or detoxify certain carcinogens
156
Describe the link between N-acetyltransferases and cancer
-Marginal associations with NATs and cancer, cancers are multigenerational diseases
-NATs catalyse inactivation of drugs and arylamine carcinogens
-Also activate some heterocyclic and aromatic arylamines
-May also produce reactive intermediates that initiate carcinogenesis
157
Describe glutathione
-Tripeptide consisting of glutamate, cysteine and glycine
-Important in maintaining reduced environment within the cell
158
What is the concentration of Glutathione in human liver cells?
Approx. 10mM
159
Describe glutathione S trasferases
-Catalyse conjugation of glutathione to electrophilic compounds through nucleophilic cysteine thiol group
-Found in most human tissues, with a wide range of different isoforms
-Consist of homo or heterodimers of subunits of Mr approx 25,000
160
Give the isoforms of Glutathione S transferases
-Alpha (GSTA)
-Mu (GSTM)
-Pi (GSTP)
-Theta (GSTT)
-Omega (GSTO)
-Zeta (GSTZ)
161
Which classes of glutathione S transferases are inducible?
-Alpha (GSTA)
-Mu (GSTM)
162
What are the functions of Glutatione S transferases
-Glutathione conjugation (Phase II metabolism) ie Xenobiotic detoxification
-Neutralisation of Reactive Oxygen Species (ie oxidative stress)
-Regulation of apoptosis
163
Give drug substrates conjugated by Glutathione S transferases
-Anticancer drugs (eg Cyclophosphamide)
-Vasodilators (eg Nitroglycerine)
-Analgesic (eg Paracetamol)
-Diuretic (eg Ethacrynic acid)
164
Describe Glutathione S transferase's role in paracetamol metabolism
~5% of paracetamol is metabolised by CYP450s to NAPQI
-NAPQI is highly reactive and detoxified by GST
165
How can NAPQI cause damage in high concentrations?
-In overdose, Glutathione is depleted, meaning GST is unable to detoxify NAPQI
-NAPQI covalently binds to proteins in centrilobular regions leading to cell death and necrosis
166
Describe Methyltransferases
-Target amines, thiols and alchohols
-Utilises S-adensoylmethione as cofactor
-Focus on reducing pharmacological activity
-Do not improve solubility
167
Give types of methyltransferases
-O methyl transferases (eg catechol-O-methyltransferase)
-N methyl transferases (eg histamine-N-methyltransferases)
-S methyl transferases (eg thiopurine methyltransferase
