1
Q
Glucuronidation
Phase 2 Reaction
A
Most common
Addition of Sugar
detoxification of xenobiotics
–> phenols / alochols
–> aromatic amines / acids
2
Q
Sulfate Conjugation
Phase 2 Reaction
A
Addition of Inorganic Sulfate
sulfate concentration is LIMITED, glucuronidation is more common
–> Phenols (most common) / alcohols
–> aromatic amines
3
Q
Glutathione Conjugation
Phase 2 Reaction
A
Most common route for removal of Electrophiles
Major Detoxification Reaction
GSH - resistant to peptidases due to gamma linkage
Gamma lInkage prevents from peptidase degradation
Glutamine y-linkage + Cysteine + Glycine = GSH
Drug = Electrophile
GSH = Nucleophile
4
Q
Glucuronide Conjugation
Enzymes & Cofactors
A
Conjugates glucuronic acid w/
billirubin / drugs / pesticides / carcinogens
Enzyme = UGT1 / UGT2
Catalyst = UDP
Co-factor = UDPGA
found in: liver / intestines / kidney / skin / brain / spleen/ nasal mucosa
5
Q
Glucoronide Conjugation
Mechanism
A
- Step 1) Formation of UDPGA
- alpha glucose phosphate –> activated glucoronic acid
- Step 2) Sn2 Displacement reaction
- UDPGA = Electrophile
- Drug/Substrate = Nucleophile
- Enzyme = UGT –> UDT
- INVERSION OF ALPHA –> BETA
- product is always beta conjugate
6
Q
Glucorinide Conjugation
UGT Substrates
A
Substrate = Nucleophile
UGT = electrophile
–> Produce ether linkage = polar metabolite
water soluble / excreted in kidney
- Alcohols
- 1* / 2* mainly
- Phenols
- Acids
- Both aliphatic & aromatic
- Amines
- mainly aromatic
- produce –> N-glucuronide
7
Q
Crigler-Najjar Syndrome
Gilberts Disease
A
- Caused by a deficiency in** **UGT
- UGT conjugates unconjugated bilirubin
- –> High concentration of unconj. Bilirubin
8
Q
Gray Baby Syndrome
A
- Caused by neonate inability to conjugate CHLORAMPHENICOL
- antibacterial that needs UGT to break it down
- UGT Deficiency
9
Q
UGT Polymorphism
UGT1A1*28 Varient
A
Express Less UGT1A1 Enzyme
- –> can lead to nonhemolytic unconjugated HYPERbilirubinemias
- –> Jaundice
- –> decreased enzyme activity effects Drug CLEARANCE
- IRINOTECAN (anticancer PRODrug)
- –> toxicity = diarrhea/neutropenia
- IRINOTECAN (anticancer PRODrug)
10
Q
UGT Enzyme INDUCER
A
Phenobarbital
11
Q
UGT Enzyme Inhibitor
A
Protease Inhibitors
Lopinavir/Ritonavir
Flavonoids (phenolic phytochemicals)
= competitive substrate / inhibitor of UGT
but will need a very high concentration (not seen)
12
Q
UGT Glucuronidation
Factors
A
Selective substrates / inibitors are LACKING
Low incidence of UGT Drug-Drug interactions
Important for detoxification reactions
inhibition –> reduce clearance of estradiol/bilirubin
13
Q
Irinotecan
A
Prodrug
SN38 Topoisomerase for HIV
- Protease Inhibitors & lopinavir/ritonavir
- Inhibit metabolization of SN38 by UGT
- –> INCREASED toxicity
- lopinavir also inhibits 3A4 metabolism
- –> increased toxicity
- Inhibit metabolization of SN38 by UGT
14
Q
How to make a UGT substrate more stable towards Metabolism?
A
UGT Mechanism is Sn2
to make optimize it (keep it from being metabolized):
+Add Rigidity (sterics)
+Change Stereochemistry
-Reduce Lipohilicity
15
Q
Why do Glucorinides have a relatively long
HALF LIFE?
A
Enterohepatic Recycling
- Glucornides (w/ MW>500)
- –> secreted into BILE
- –> Gallbladder
- –> B-glucoridase (bacterial)
- –> REABSORBED
- –> secreted into BILE
- Ultimately increases time in body and can interact w/ other drugs
- EX. CLORIPHENICOL
16
Q
Sulfate Conjugation
Enzymes & Cofactors
A
Detoxification & bioactivation Pathways
Conjugates activated Sulfate to:
–> Phenols
–> alcohols / aromatic amines / N-hydroxyl Groups
Enzyme = SULT1/2
Cofactor = PAPS
found Ubiquitous / LIVER / placenta / intestine
17
Q
Sulfate Conjugation
Mechanism
A
- Step 1: Activation of inorganic sulfate –> PAPS
- ATP-Sulfurylase / APS-Phosphokinase
- –> Active PAPS
- ATP-Sulfurylase / APS-Phosphokinase
- Step 2: Sn2 Displacement Reaction
- PAPS = Electrophile
- Drug/Substrate = Nucleophile
- Enzyme = ST (sulfotransferase)
- –> Sulfonated Product + PAP
- enhanced water solubility –> excretion
18
Q
Sulfate Conjugation
of Estradiol
A
Estradiol + PAPS/ST –> Sulfate Metabolite
- Sulfate Metabolite = Water soluble –> Blood
- In target tissues Sulfatase removes the sulfate
- –> reactivated estradiol in tissue
- “acts as a carrier”
- so drug can travel
- In target tissues Sulfatase removes the sulfate
19
Q
Sulfate Conjugation
DMBA Bioactivation
A
DMBA + ST/PAPS –> Sulfate metabolite
- Sulfate –> Sn1 Rxn = Carbocation
- carbocation is highly reactive can form TUMORS
- DNA Adducts
- Carbocation can also –> GSH –> detoxifcation
20
Q
PAPS / ST
SUBSTRATES
A
Primarily with PHENOLS
alcohols
aromatic amines
N-hydroxyl groups
Drug = Nucleophile
PAPS = Electrophile
21
Q
Glutathione Conjugation
Enzyme/Cofactors
A
Conjugates reactive/soft electrophiles such as
–> epoxides / RX / quinones
–> a/b-unsaturated carbonyls / quinone imines
Enzyme = GST = nu-
alpha/mu/pi-class of GST’s
Cofactor = Glutathione (nu-)
found in basicaly all mammalian tissues
22
Q
GST Inducers
A
Phenobarbital
3-methylcholanthrene
23
Q
Glutathione Conjugation
Mechanism
A
Can be conjugated w/o prior activation
Detoxification –> Inactive /nontoxic metabolites
- Sn2 Reaction “INVERTED SN2”
- GST = Nucleophile
- Drug/substrate = electrophile
- Product = Glutathione Conjugate(-GS)
- REDUCES pKa
- Can further be metabolized
- –>Mercapturic Acid Derivative
24
Q
Glutathione Conjugation
SUBSTRATES
A
Elimination of ELECTROPHILES
Epoxides
R-X (alkyl halides)
Quinones (QI’s / QM’s)
alpha/beta -unsaturated carbonyls
GSH = Nucleophile
25
**GSH**
**Detoxification of Aflatoxins**
* **Epoxide** (Aflatoxin)
* **strong electrophile --\> _attack nu- proteins_**
* **__--\>** protein modification
* --\> DNA adducts
* **Carcinogenic**
* ******Epoxide + GST/GSH --\> Glutathione Product**
* **Can be excreted, DETOXIFIED**
* **\****depletion of GST ---\> more epoxide as a carcinogen*
26
**N-Acetyltransferase (NAT)**
Reaction
Transfer of **Acetyl group** to **nitrogen atom** of:
--\> **aromatic Amine**
**--\> Hydrazine**
Cofactor = **Acetyl CoA**
Enzyme = **NAT**
_NAT1 found in urothelium / colon epithelial cells_
_NAT2 found in LIVER / intestine_
27
**N-acetyltransferase (NAT)**
**Mechanism**
* **Step 1) Transfer of Acetyl CoA --\> NAT**
* **Step 2) Transfer of Acetyl --\> Substrate**
* ****substrate is aromatic amine or hydrazine
* Produces **Mercapturic Acid Derivative**
* ****as third step after **GST** metabolism
28
**NAT**
Polymorphisms
**Isoniazid (hydrazine group) = Substrate of NAT**
* **Polymorphism in NAT2** (liver/intestine)
* ***SLOW Acetylators***
* ***--Accumalate Isoniazid --\> Neuropathy***
* ******found in many Caucasians /egyptians/moroccans
* ****RAPID acetylators**
* ****Isoniazid --\> more hydrolysis into **acetyl radicals**
* --\> **Hepatic Damage**
29
**Mercapturic Acid Pathway**
* **After GST Conjugation**:
* Product has **Glutamine / Cysteine / Glycine**
* 1) Y-glutamine Amide Hydrolysis
* 2) Glycine Amide Hydrolysis
* 3) **Cys-N-Acytylation**
* ******NAT + AcetylCoA**
* **PRODUCE MERCAPTURIC ACID**
* ****excreted in urine
30
**Mercapturic Acid Products**
**Tridiphane = Herbicide**
**ACETAMINOPHEN**
~only 2% of metabolism but is still found as a product
31
Chemical Toxicology
## Footnote
**Drug ITSELF is Toxic**
* Exagerated **on-target pharmacology**
* Off-Target Pharmacology
* **Trigger Immune response**
* **--\> inflammation******
* Structure Alerts from toxicity:
* **Sulfate**
* **NO2 (nitrates)**
* **Aziridiniums**
* **Epoxides**
32
**Fenfluramine**
Anti-suppresant drug
**Found to cause Rare Valvular Disease**
**when in combo with Phenteramine**
--\> potentiation of serotonin effects
**_DRUG ITSELF WAS TOXIC_**
33
**Vioxx**
COX-2 Inhibitor (similar to Celecoxib)
**Increased Risk of CV Problems**
COX2--\> in endothelial cells --\> vessel constriction
**DRUG ITSELF WAS TOXIC**
34
**DES**
| (diethylstilbestrol)
**Teratogenic Drug**
**_DRUG ITSELF WAS TOXIC_**
used to prevent miscarriage --\> led to increase in CERVICAL CANCER in DES daughters
**Endocrine disrupting chemical**
**--\> DNA methylation**
35
**Thalidomide**
**Teratogenic Drug**
**_DRUG ITSELF WAS TOXIC_**
Used for sleep aid / morning sickness
**caused severe birth defects**
_now used to treat leprosy & considered for HIV / multiple myeloma (cancer)_
36
**Metabolite Toxicity**
**Metabolite of the drug is the one that is TOXIC**
**Ex. ACETAMINOPHEN**
37
Metabolite Toxicity
## Footnote
**3 Major Mechanisms**
* **Adducted Protein/DNA**
* ******MAJOR one**
* Covalent bonding to cellular components
* enzymes / regulatory proteins /DNA
* **ROS production**
* non-covalent interactions
* **Covalent Protein Adducts --\> IMMUNOLOGICALLY mediated toxicity**
* ****
38
Metabolite Toxicity
## Footnote
**Sulfamethazole**
Leads to **Hypersensitivity reactions**
Specifically for **HIV Patients**
--\> Protein Adducts --\> Sn2 --\> **_More Immune Supression_**
39
Metabolite Toxicity
## Footnote
**Electrophile Reactivity/Selectivity Principle**
**The MORE REACTIVE the electropile**
**The *LESS Selective***
***=*MORE SIDE EFFECTS**
* ****Electrophile --\> Water (nu-)
* Most common reaction, b/c A LOT OF WATER
* E+ --\> **GSH/GST**
* *Toxic Reactions:*
* **E+ --\> Protein
* **Cytotoxic**
* E+ --\> DNA
* only a small amount reaches here but still **Genotoxic**
40
Carcinogens
**Direct Acting Genotoxic Agents**
**Electrophiles**
(usually w/o additional bioactivation)
**_Nitrogen Mustards_**
--\> **Aziridinium Ion --\> DNA ALKYLATION**
**=**apoptosis
41
Carcinogens
## Footnote
**Inderect Acting Genotoxic Agents**
**Need to be BIOACTIVATED to be toxic**
**= Ultimate Carcinogen/Toxin**
**_Benzo(a)pyrane_**
42
Carcinogens
## Footnote
**NON-Genotoxic / Epigenetic Agents**
--\> Proteins but do not change genetically
Sustained Cytotoxity / Receptor Mediated
ER / AhR
**Increase of Oxidative Stress**
**DNA Methylation**
43
_Carcinogens:_
Enzymes involved in
**Bioactivation Reactions**
**Mainly p450** (phase 1)
Mainly Class 1 (1A1 / 1A2 / 1B1)
**SULT**
**AKR**
**NAT**
FMO / COX
44
Enzymes Involved in
## Footnote
**Detoxification Reactions**
**Mainly _GST / UGT_**
**AKR**
**P450**
Little SULT / NAT / COMT / EH
45
**Types of Reactive Intermediates**
**Electrophiles (E+)**
**Free Radicals**
**_Nucleophiles (Nu-)_**
46
**Nitrenium Ions**
**Carbacations**
**Aziridinium Ion**
**Positively Charged Electrophiles (E+)**
**MOST REACTIVE** = Least selective
Can react with water
47
**Epoxide**
**Arene Oxide**
**Acyl Halide**
**Alkyl Halide**
**Neutral Electrophiles (E+)**
Reactive intermediates
48
**Quinone**
**Quinone Imine**
**Quinone Methide**
**Neutral Electrophiles (+)**
Reactive intermediates
MOST COMMON TOXIC METABOLITES
**Quinone Methide = Not as bad as other quinones**
**does NOT undergo Redox Cycling --\> ROS**
49
Reactive Intermediates
**Epoxides**
**Aflatoxin**
**Metabolicly Activated**
Mycotoxin / **Aflatoxin B1**
* Haptic carcinogen / acute toxin
* from mold Aspergillus flavus
* **Apurinic Sietes on DNA**
* **Mutagenic Lesions**
* **GST/GSH or EH**--\> detoxify the epoxide
* **_Oltipraz_** _= Chemopreventative for aflatoxin liver cancer_
50
**Oltipraz**
**_Induces GST & Quinone Reductase_**
***INHIBITS 1A2 / 3A4***
= CHEMOPREVENTATIVE for **Aflatoxin Epoxide**
* Oltipraz --\> More GST/GSH
* More Detoxification of Aflatoxin Epoxide
* Oltipraz -/-\> *inhibits P450*
* *Less formation of epoxide*
51
**Pulegone**
From _pennyroyal oil_
used as a **Abortifacient**
**P450 Oxidation --\> Epoxide Electrophile (-)**
**= Liver Damage / Death @ high doses**
52
**Benzo[a]pyrene**
Found in _Cigarettes_
**Potent Mutagen**
**1A1 metabolism --\> Epoxide = DNA Adducts**
also *EH metabolism --\> Diol --\> **AKR --\>** DNA Dmg*
53
**Chloroform**
Chloroform + 2E1 --\> **Phosgene** (**_ACYL Halide_**)
**_Neutral Electrophile+_**
**= Lung Toxin / rxns w/ amines of proteins**
54
**Dichloroethylene**
Found in _dry cleaning solvents_
**Causes Lung Toxicity**
* 3 Bioactivation Pathways 2E1:
* **Chloroacetyl Chloride (most reactive electrophile)**
* DCE Epoxide
* DCaldehyde
55
**Safrole**
**Tamoxifen**
**DMBA**
Bioactivated into **Genotoxic _Carbocation_**
(Positively Charged Electrophile)
* P450 --\> **_PAPS/ST_ --\> Sn1 Displacement**
* **--\> Carbocation**
* ****Attacks DNA = Forms DNA Adducts
* Bulky adducts are stable
56
**Nitrenium Ion**
**Positively Charged Electrophile+**
* Amide --\> 1A2 --\> **_PAPS/ST --\> Sn1 Displacement_**
* __= **Nitrenium Ion**
* DNA Binding / Tumor Formations
57
**Aristolochic Acid**
**Form Nitrenium Ion** (ultimate carcinogen)
Positively Charged Electrophile+
_Dietary supplement for weight los_
* Nitro + Reductase --\> Amine --\> Amide --\> P450
* --\> **_PAPS/ST --\> Sn1 Displacement_**
* __= **Nitrenium Ion**
* DNA Binding / Tumor Formations
* **Neprotoxicity Kidney**
* **Many patients had End-Stage Grenal Failure**
* **& Urothelial Cancer**
* *Misidentified chinese name that caused issues*
58
**Quinoids**
**Neutral Electrophiles**
_Most Common Functional Group in Drugs_
* **BENZENE + P450 + OXYGEN**
* **--\> Quinones**
* **Can undergo REDOX RECYLING**
* **--\> ROS**
59
**Phenytoin Bioactivation**
**Just Oxygen + P450**
**= Quinone (**Electrophile)
**Also undergoes Redox Cycle**
**--\> ROS's**
60
**Endogenous Estrogen Bioactivation**
* Estrogen + p450 --\> **Quinones**
* **Redox recycling --\> ROS**
* **Depurinating Adducts = GENOTOXICITY**
* * ******COMT = Catecholamine METHYLTRANSFERASE**
* Also metablize estrogen and form
* both **Genotoxic Biomarkers**
* ***Non-Toxic Biomarkers***
61
**Bioactivation of Equilenin**
Form **Redox Active / Electrophilic Quinones**
## Footnote
**+ Superoxide**
**= Carcinogen/Toxic**
62
**HRT**
**Timing Hypothesis**
* Thought that using **HRT PRE-MONOPAUSE**
* More benifits vs risks
* *stroke/cancer risk*
* Average age of HRT was \>60 = POST-menopause
* More risks vs benefits
* *heart disease / cognition / cancer / stroke risk*
63
**Black Cohosh / Red Clover**
P2 Clinical Trial proved that
## Footnote
***They DO NOT* reduce hotflashes in postmenopausal women**
64
**Tamoxifen Bioactivation Pathway(s)**
**PAPS/ST --\> CARBOCATION**
**P450 + Oxygen --\> QUNONE /** Quinone Methide
*quinones can undergo **GSH** conjugation --\> excreted*
65
**STAR**
**Study of Tamoxifen & Raloxifene**
(SERMS)
**Both form Quinoids** (electrophiles)
*But can be **GSH** conjugated **--\> -GS excretion***
* ******Found that 3rd/4th generation **SERMS** (selective estrogen receptor modulators)
* are **ALSO CONVERTED TO QUINOIDS**
* ex. _DMA / Acolbifene_
66
**Etoposide**
--\> **QUINONE PRODUCTS**
as a side effect
* A **Anticancer agen**t from podophyllotoxin
* --\> _Small cell lung / testicular cancer_
* __**INHIBITS Topoisomerase**
* ****--\> unwinds supercoiled DNA
* Inhibits tumor replication
67
**Kava Kava**
**Bioactivated to QUINONES**
* Methysticin / 7-8Dihydromethysican
* --\> **Quinones --\> Liver Toxicity**
* ****hepatitis / cirrhosis / liver failure
* _Marked as a SEDATIVE dietary supplement_
68
**Acetaminophen/Diclofenac Bioactivation**
**APAP --\> O/2E1 --\> NAPQI**
NAP- **Quinone Imine**
GI / Liver Toxicity
69
**Eugenol / BHT**
Bioactivation
**Eugenol** = _cloves/cigarettes / food / flavor agent_
**BHT =** _antioxidant / dried foods --\> prevent lipid oxidation_
**Both metablolized to _Quinone Methide_**
Quinone methide = tumor promotor
*DOES NOT undergo rodox cyling*
70
**Redox Cycling**
Mechanism of toxicity for
**QUINONES & QUINONE IMINES**
*not for quinone methides*
* **Quinone + P450 Reductase --\> Semiquinone Radical**
* **O2 radical --\> O2**
* **ROS** --\> RADICAL DNA DMG
71
**Doxorubin**
**Undergo Redox Cycling**
Quinone --\> Semiquinone
forms **ROS**
* ROS = O2 radical
72
**Consequences of ROS**
* **ROS = O2 Radical**
* O2 + 2H+ **--\> _SOD (Superoxide Dismutase)_**
* **= H2O2 (hydrogen peroxide)**
* ****H2O2 + **Fe2+** (**FENTON REACTION)**
* ****= HO- **+ HO RADICAL**
* **HO Radical** can cause:
* **Lipid Peroxidation**
* **Protein Oxidation**
* ****disulfide bond formation
* **DNA Oxidation**
* --\> Mutagenic Lesion
* Form **(8-oxo-dG) = Biomarker**
73
**Fenton Reaction**
* **Converts Hydrogen Peroxide with Fe2+******
* = HO- + **HO RADICAL**
* HO Radical can cause:
* Lipid Peroxidation
* Protein Oxidation
* DNA Oxidation
74
**Superoxide Dismutase**
**SOD**
**_Converts ROS --\> Hydrogen Peroxide_**
O2 Radical --\> H2O2
--\> fenton reaction
75
**Counterbalancing ROS**
* **OH Radical** formed from Fenton Rxn
* can be **counterbalanced by OXIDATIVE DEFENSE**
* Enzymatically
* Non Enzymatically
* **Gutathione**
* **2GSH + 2OH Radicals --\> GS-SG**
* --\> excreted
76
**Lipid Peroxidation**
**Causes Many Diseases**
End Product = **Lipid Aldehyde (Biomarker)**
* Occur as a result from **ROS** --\> **Fenton RXN**
* **OH RADICAL**
* **Radicals KEEP CYCLING**
* until hit by **GSH or another detoxifyer**
77
**Vitamin C**
**Flavonoids** (quercetin)
**Antioxidants that SCAVANGE _OH Radicals_**
* Take **OH Radicals** --\> Form **WATER** TWICE
* --\> forms a quinone, not as bad as the OH radicals
78
**Enzyme Catalyzed**
**ROS Scavanger**
**Scavange O2 Radicals**
* 2O2 + **SOD** --\> Hydrogen peroxide
* _H2O2 can undergo two detoxification reactions_
* **Glutathione Peroxidase**
* +2GSH --\> 2x Water
* **Catalase**
* --\> 2x Water
79
**Non-Enzymatic**
**ROS Scavenger**
* **PHENOL****S**(from food) +**OH Radical**
* abstracts the radical --\> Water
* **BHT** + **OH Radical**
* Abstracts radical --\> WATER
* --\> forms **Quinone Methide**
* *not as bad, does not go through REDOX RECLYING*
80
What metabolizes
**Michael Acceptors**
**a/b -unsaturated carbonyls**
**GST**
GSH = nucleophile -\> Carbonyl (michael acceptor)
--\> **-GS product**
81
**Bioactivation of Carbocation**
**P450** --\> -OH group
**PAPS / ST** --\> Sulfonated product
**Sn1 Rxn** --\> **CARBOCATION+**
genotoxic molecule --\> mutations on DNA
82
**Glutathione Peroxidase**
**Ezyme catalyzed**
**Scavenger of ROS**
H2O2 + 2GSH --\> **2x water + GSSG**
83
**Catalase**
**Enzyme catalyzed**
**Scavenger of ROS**
H2O2 + Catalase --\> **2x Water + o2**
84
**BHT**
**Phenols in food**
**Non-Enzyme Scavenger of ROS**
Abstracts -OH Radicals
--\> form **Quinone Methide**
*does not go through redox recycling*
85
**P450 1A1**
**Induced by POLYCYCLIC HYDROCARBONS**
1-methyphenanthrene
**If it has a lot of benzene rings it is likely oxidized by 1a1**
86
**Redox Active**
**Means it can UNDERGO REDOX RECYCLING**
**Quinones**
*Epoxides are NOT redox active*
87
**Aromatic Amines can be Metabolized by what?**
**NAT --\>** Acetyl group on the amine
**PAPS/ST --\>** Sulfonated amine
**UGT/UDPGA** --\> N-glucuronide
88
**What can react with Aromatic Nitro Groups**
**-NO2**
**AKA NITROSO**
**GST**
glutathione s- transferase
89
MedChem Final
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