Reductases and Dehydrogenases
N‐Acyl and S‐Methyl Transferases
MEDCH527 – Re@e – 1/30/2013
ReducEon in Drug Metabolism • ReducEve drug metabolism is the least studied and, from an enzymological perspecEve, the most poorly characterized of the common drug metabolism processes.
• Testa’s treatment of the biochemistry of metabolic reducEon in Chem. Biodivers. Vol 4 (2007) is an invaluable guide to this complicated subject.
• ReducEon of carbonyls (aldehydes, ketones, quinones) is the most common metabolic reducEon reacEon that drugs, xenobioEcs and endogenous compounds undergo (compare with nitro reducEon, azo reducEon, etc).
Major Enzymes Catalyzing Carbonyl Reduc8on
and P450 reductase (CPR)
Adapted from Testa (2007)
Involved in causing cellular toxicity
and P450 reductase
CPR
Testa (2007) Reduc8on
CPR
CPR
Cofactors for Carbonyl ReducEon
Despite their molecular diversity, and the fact that some, but not all, catalyzes reversible reacEons, these enzymes are united by their use of NAD(H) and/or NADP(H) cofactors (see below).
Alcohol dehydrogenases (ADH) Zn‐containing, NAD(H)‐dependent
Aldo‐keto reductases (AKR) NAD(P)(H)‐dependent ‐ aldehyde/aldose reductases (AR) ‐ hydroxysteroid dehydrogenases (HSD) ‐ many dihydrodiol dehydrogenases /(DHD)
Carbonyl reductases (CR) NAD(P)(H)‐dependent
Quinone oxidoreductase (NQO) NADPH‐dependent
P450 reductase (CPR) NADPH‐dependent
Hydride transfer mechanism for carbonyl reducEon
Quinone ReducEon by NQO1 and CPR OH
OH
O
O
O
O
+ 1 e + 1 e
Quinone Semiquinone radical anion Hydroquinone
(+ 2H+)
• NADPH‐dependent quinone oxidoreductase (NQO1; DT‐Diaphorase) is an FAD‐containing, cytosolic enzyme with an exquisite sensiEvity towards the inhibitor, dicoumarol; Ki = 1 nM.
• NQO1 catalyzes the obligatory two electron reducEon of quinones.
• Cytochrome P450 reductase (CPR) catalyzes the one‐electron reducEon of quinones to a reacEve semiquinone radical intermediate.
Quinone toxicity: Fu8le cycling and protein aryla8on
• Quinones may react directly with thiols on proteins to cause toxicity • Quinones can react with molecular oxygen to form ROS aaer CPR‐catalyzed one‐electron reducEon to the semiquinone radical – ‘fuEle cycling’ • NQO1‐catalyzed two‐electron reducEon to the hydroquinone is a more benign process, since it bypasses the semiquinone radical
N‐acetyl transferases (NAT)
• NATs (NAT1 and NAT2) catalyze the transfer of an acetyl group from the cofactor, acetyl‐CoA, mainly to relaEvely lipophilic compounds that contain a primary amino group.
CoA SC
O
CH3
N-Acetyltransferase (NAT)
NAT Cys
C
CH3
O
C
O
CH3NH
R
RNH2 NAT
N‐Acetyltransferase Reac8ons
N‐acetylaEon is a major route of biotransformaEon for xenobioEcs containing a primary arylamine (R‐NH2) or a hydrazine group (R‐NH‐NH2).
Products are aromaEc amides (R‐NH‐COCH3) and hydrazides (R‐NH‐NH‐COCH3), respecEvely.
HNO
NH2
N
HN
NH2
N
N
HN
NH2
H2N
S
HN
O
O
N
N
H2N
CO2H N
NH
O
H2N
H2N NH2
NH2
PABA Sulfamethazine Procainamide
Hydralazine Isoniazid Phenelzine
Benzidine 2-Aminofluorene
N‐Acetyltransferase Reac8ons (cont’d)
• XenobioEcs containing primary aliphaEc amines are rarely substrates for N‐acetylaEon. The important excepEon being cysteine conjugates, which are formed from glutathione conjugates and converted to mercapturic acids by N‐acetylaEon in the kidney (see Atkins lecture on GSH).
• Some drugs are metabolized to primary amines before acetylaEon.
N
HN
O
S NH
O
O
N
N
N
O
HO
HO
O2N
Sulfasalazine Nitrazepam
Role of NAT1 and NAT2 in aroma8c amine metabolism
NAT1 appears to funcEon as both an O‐acetyltransferase (OAT) and an N,O‐acetyltransferase (N,O‐AT) when using acetyl coenzyme A or hydroxamic acids, respecEvely, as acetyl donors. NAT2 appears to act preferenEally as an OAT and NAT.
Decomposes to reac,ve arylnitrenium ion (DNA binding)
NATs: The Enzymes • N‐acetylaEon is carried out in mammals by NAT 1 and
NAT2, cytosolic enzymes of M.W. ~ 33‐34 kDa.
• NAT1 and NAT2 share 87% nucleoEde and 81% amino acid sequence idenEEes.
• Human NATs are encoded at 3 separate loci on chromosome 8. One of the loci contains a non‐expressed pseudogene – NAT3.
• NAT acEvity has been found in most organisms and all mammals, where there is high acEvity in the liver.
• There is ~50% overall sequence homology for all NATs with a conserved acEve site cysteine required for catalyEc acEvity as the acetylaEon site.
NAT1
• NAT1 is ubiquitously expressed. It catalyzes the acetylaEon of what are termed “monomorphic substrates”, such as p‐aminosalisylic acid (PASA) and p‐aminobenzoic acid (PABA).
• Although the gene has been tradiEonally known as the “monomorphic” acetyltransferase, many geneEc variants are now recognized. NAT1*4 represents the wild type gene.
NAT2
NAT2 is expressed primarily in the liver and intesEnal mucosa and catalyzes the acetylaEon of what has been termed “polymorphic” substrates, including sulfamethazine, isoniazid, dapsone, sulfamethoxazole, procainamide, hydralazine and caffeine.
The x‐ray crystal structures of the prokaryoEc NAT enzymes from S. typhimurium and M. smegma=s have been solved. The overall structure of the prokaryoEc NAT enzymes consists of three domains which are of approximately equal length. The first two N‐terminal domains are highly conserved in NATs throughout both the eukaryoEc and prokaryoEc kingdoms, and contain an acEve site cataly8c triad composed of Cys69‐His107‐Asp122 (numbering scheme from S. typhimurium).
NAT2*4 represents the wild type gene. Mutant alleles oaen generate protein proteins with decreased expression and/or stability.
NAT2 Polymorphisms: Caffeine Acetylator Status • Phenotyped in urine by the raEo of AFMU:1‐methylxanthine
NAT ‘slow acetylator’ Phenotype
• NAT polymorphism first idenEfied as the ‘slow acetylator’ phenotype
• Trimodal phenotype distribuEon – 55‐60% in Caucasians / Northern Europeans – 8‐10% Japanese – 20% Chinese – 90% North Africans
• SA due largely to polymorphisms in NAT2 • MutaEons in NAT1 discovered only in the last 15 years
NAT2 Ac8vity
• 2*4 is ‘wild‐type’, responsible for most ‘fast acetylator’ acEvity
• ‘Slow acetylator’ phenotype due largely to 2*5, 2*6 , 2*7, 2*14 alleles • Low acEvity due to:
– Poor expression/unstable protein (2*5) – Decreased catalyEc acEvity (2*6)
• Some studies demonstrate a much greater frequency of homozygous PMs (91%) in Caucasian children with documented skin allergies, than in disease‐free children (62%). SensiEzaEon may be mediated by increased formaEon of hydroxylamines or a slower clearance of histamine at the site of release. (Clin Pharmacol Ther 62:635‐42, 1997).
• Epiemiological studies on the role of NAT polymorphisms in cancer suscepEbility are extremely confusing and oaen contradictory.
S-Methyltransferases
• SAM is ‘Nature’s methyl iodide’” it serves commonly as the methyl group donor for both S‐methyltransferase (e.g. TPMT), O‐methyl transferase (e.g. COMT) and N‐methyltransferase enzymes.
CH2
CH2
CH
SH3C CH2 adenosine
CO2HH2N
CH2
CH2
CH
S
CH2 adenosine
CO2HH2N
R-X-H R-X-CH3
S-adenosyl methionine (SAM) S-adenosyl homocysteine
There are relaEvely few drugs that undergo S‐methylaEon, but the process is important for the detoxificaEon of xenobioEc thiol compounds, which tend to be toxic.
Microsomal TMT prefers to metabolize aliphaEc thioles, e.g. captopril.
Cytsosolic TPMT prefers to metabolize aromaEc or heteroaromaEc thiols.
S‐Methyltransferases: The Enzymes
N
N
HN
N
S
N
N
NO2
H3C
N
N
HN
N
SH
N
N
HN
N
S CH3
Azathioprine 6-Mercaptopurine 6-Thiomethyl mercaptopurine
S‐Methyltransferases: The Enzymes and Substrates
N
N
HN
N
S
N
N
NO2
H3C
N
N
HN
N
SH
N
N
HN
N
S CH3
Azathioprine 6-Mercaptopurine 6-Thiomethyl mercaptopurine
TPMT: thiopurine methyltransferase XO: xanthine oxidase HGPRT: hypoxanthine guanine phosphoribosyltransferase TIMP: 6‐thioinosine monophosphate MTMP: 6‐S‐methylthioinosine monophosphate TGN: 6‐thioguanine nucleoEdes 6‐MP: 6‐mercaptopurine MeMP: 6‐S‐methylmercaptopurine 6‐TU: 6‐thiouric acid
6‐MP TGN
MeMP
TPMT
6‐TU
XO
HGPRT DNA
TPMT
Bioac=va=on Pathway
Detoxifica=o
n Pa
thway
TIMP
MTMP
TPMT
(mulEple enzymaEc steps)
(Purine salvage)
6‐Mercaptopurine Disposi8on
Both TPMT and bioacEvaEon enzymes found in hematopoieEc cells; XO found only in the liver
Other products
Krynetski and Evans, Pharmacology 61:136‐46, 2000
Common Reduced Func8on TPMT Alleles
Although there are over 15 different mutant alleles, only a few account for the majority of PM acEvity throughout the world; *3A more common in Caucasians, *3C in Asians and Africans.
These mutaEons affect the stability of the enzyme (enhanced
proteasomal degradaEon), with
reduced steady‐state Vmax and Clint
Protein t1/2 ~ 18 hr
Protein t1/2 ~ 0.25 hr
6‐MP Dose Adjustment Based on TPMT Genotype
Krynetski and Evans, Pharmacology 61:136‐46, 2000
Strategy is to focus on the most common defecEve alleles and adjust 6‐MP dose downward for ~10% of paEents.
FDA Review: 6‐MP Product Labeling Changes • No mandatory requirement for geneEc tesEng
• GeneEc informaEon listed under Pharmacokine=cs, Warnings and Precau=ons secEons
• Dose and Administra=on ‐ “states tests are available”
Concerns with Mandatory TesEng: Extensive clinical experience High cure rates with”acceptable toxicity profile” Fear of under‐dosing and reduced cure rates (heterozygotes) No standardized therapy (requires center‐specific interpretaEon of
geneEc results) Delay in treatment iniEaEon Legal consequences
R. Padzur, Oncology Drug Products, FDA
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