medch527 reductases and transferases...
TRANSCRIPT
Reductases and Dehydrogenases N-‐Acetyl and S-‐Methyl Transferases
MEDCH527 1/26/2015
Collec2vely, these enzymes account for ~10% of metabolic reac2ons (Testa et al., Drug Discovery Today, 2012)
Reduc9on in Drug Metabolism • Reduc2ve drug metabolism is one of the least studied and, from an enzymological
perspec2ve, the most poorly characterized of the common drug metabolism processes. • Testa’s treatment of the biochemistry of metabolic reduc2on in Chem. Biodivers. Vol 4
(2007) is an invaluable guide to this complicated subject. • Reduc2on of carbonyls (aldehydes, ketones, quinones) is the most common metabolic
reduc2on that drugs, other xenobio2cs and endogenous compounds undergo.
O
O
CH3
Phytyl
OH
OH
CH3
Phytyl
Vitamin KH2
Vitamin K
Major Enzymes Catalyzing Carbonyl Reduc9on (Adapted from Testa, 2007)
and P450 reductase (CPR)!
CPR
Testa (2007) Reduc9on
CPR
CPR
Hydride transfer mechanism for carbonyl reduc2on
Cofactors for Carbonyl Reduc2on Despite their molecular diversity, and the fact that some, but not all, catalyzes reversible reac2ons, these enzymes are united by their use of NADH and/or NADPH cofactors (see below).
Alcohol dehydrogenases (ADH) Zn-‐containing, NADH-‐dependent
Aldo-‐keto reductases (AKR) NADPH-‐dependent -‐ aldehyde/aldose reductases (AR) -‐ hydroxysteroid dehydrogenases (HSD) -‐ many dihydrodiol dehydrogenases /(DHD)
Carbonyl reductases (CR) NADPH-‐dependent
Quinone oxidoreductase (NQO) NADPH-‐dependent
P450 reductase (CPR) NADPH-‐dependent
Quinone Reduc2on by NQO1 and CPR OH
OH
O
O
O
O
+ 1 e + 1 e
Quinone Semiquinone radical anion Hydroquinone
(+ 2H+)
• Microsomal NADPH-‐dependent cytochrome P450 reductase (CPR, POR) catalyzes the one-‐electron reduc2on of quinones to a reac2ve semiquinone radical intermediate. • NADPH-‐dependent quinone oxidoreductase (NQO1; DT-‐Diaphorase) is an FAD-‐containing, cytosolic enzyme with an exquisite sensi2vity towards the inhibitor, dicoumarol; Ki = 1 nM. • NQO1 catalyzes the obligatory two electron reduc2on of quinones, and so bypasses the semiquinone radical.
Quinone toxicity: ROS genera9on and protein aryla9on
• Quinones may react directly with thiols on proteins to cause toxicity • Quinones can react with molecular oxygen to form ROS • NQO1-‐catalyzed two-‐electron reduc2on to the hydroquinone is a more benign process, since it bypasses the semiquinone radical
Importantance of Quinones to Drug Toxicity [Testa et al., 2012; N=473 toxicity reac2ons]
Reduc2ve bioac2va2on of an2cancer pro-‐drugs • CPR-‐catalyzed one electron reduc2on forms radicals, which in normal cells react
with oxygen to forms superoxide anion that can be detoxified by SOD. • In solid tumors that are low in oxygen because of poor neo-‐vasculariza2on
radicals persist and can cause DNA strand breaks in tumor cells.
• NQO-‐catalyzed reduc2on to the E09 hydroquinone ac2vates the azaridinyl ring for nucleophilic afack by DNA. NQO1 is upregulated in many cancers.
N
CH2OH
CH3
O
O
R
N
N
CH2OH
CH3
O
OH
R
N
H
(+)XNQO
Azo-‐ and Nitro-‐reduc2on
• These reac2ons are typically catalyzed by intes2nal microflora in the anerobic environment of the lower GIT.
• However, under condi2ons of low oxygen tension, P450, NQO1 and aldehyde oxidase may also contribute.
Reduc2ve dehalogena2on and toxicity
• Reduc2ve dehalogena2on of CCl4 and the anesthe2c halothane generates carbon-‐centered radicals that ini2ate lipid peroxida9on. Downstream toxic metabolites include CO and phosgene.
• Oxida1ve dehalogena2on of halothane generates trifuuoroacetaldehyde that causes immune hepa99s following neo-‐an2gen forma2on.
N-‐Acetyl transferases (NAT)
• NATs (NAT1 and NAT2) catalyze the transfer of an acetyl group from the cofactor, acetyl-‐CoA, mainly to rela2vely lipophilic compounds that contain a primary amino group.
CoA SC
O
CH3
N-Acetyltransferase (NAT)
NAT CysC
CH3
O
C
O
CH3NH
R
RNH2 NAT
• X-‐ray crystal structures of prokaryo2c NAT enzymes from S. typhimurium and M. smegma1s have been solved demonstra2ng 3 domains.
• The first two N-‐terminal domains are highly conserved in NATs throughout both the eukaryo2c and prokaryo2c kingdoms, and contain an ac2ve site cataly9c triad composed of Cys69-‐His107-‐Asp122 (numbering scheme from S. typhimurium).
N-‐Acetyltransferase Reac9ons • N-‐acetyla2on is a major route of biotransforma2on for xenobio2cs
containing a primary arylamine (R-‐NH2) or a hydrazine group (R-‐NH-‐NH2).
• Products are aroma2c amides (R-‐NH-‐COCH3) and hydrazides (R-‐NH-‐NH-‐COCH3), respec2vely.
HNO
NH2
N
HNNH2
N
N
HN
NH2
H2N
S
HN
O
O
N
N
H2N
CO2H NNH
O
H2N
H2N NH2NH2
PABA Sulfamethazine Procainamide
Hydralazine Isoniazid Phenelzine
Benzidine 2-Aminofluorene
N-‐Acetyltransferase Reac9ons (cont’d)
• Xenobio2cs containing primary alipha2c amines are rarely substrates for N-‐acetyla2on. The important excep2on being cysteine conjugates, which are formed from glutathione conjugates and converted to mercapturic acids by N-‐acetyla2on in the kidney (see Atkins lecture on GSH).
• Some drugs are metabolized to primary amines before acetyla2on.
N
HN
O
S NH
O
O
N
N
N
O
HO
HO
O2N
Sulfasalazine Nitrazepam
NATs: The Enzymes • N-‐acetyla2on is carried out in mammals by NAT 1 and
NAT2, cytosolic enzymes of M.W. ~ 33-‐34 kDa. • NAT1 and NAT2 share 87% nucleo2de and 81% amino acid
sequence iden22es. • Human NATs are encoded at 3 separate loci on
chromosome 8. One of the loci contains a non-‐expressed pseudogene – NAT3.
• NAT ac2vity has been found in most organisms and all mammals, where there is high ac2vity in the liver.
• There is ~50% overall sequence homology for all NATs with a conserved ac2ve site cysteine required for cataly2c ac2vity as the acetyla2on site.
NAT1 is ubiquitously expressed. It catalyzes the acetyla2on of “monomorphic substrates”, such as sulfamethoxazole and p-‐aminosalisylic acid. NAT2 is expressed primarily in the liver and intes2nal mucosa and catalyzes the acetyla2on of what has been termed “polymorphic” substrates, including sulfamethazine, isoniazid, dapsone, sulfamethoxazole, procainamide, hydralazine and caffeine. .
NAT ‘slow acetylators’ • NAT polymorphism first iden2fied as the ‘slow acetylator’
phenotype in pa2ents using the hydrazine drug, isoniazid
• ~50% of pa2ents were observed to suffer ADRs, i.e. hepatotoxicity and peripheral neuropathy
• Slow acetylator frequencies: – 55-‐60% in Caucasians /Northern Europeans – 8-‐10% Japanese, 20% Chinese – 90% North Africans
• Slow acetylator status due largely to polymorphisms in NAT2
NAT2 Polymorphisms: Caffeine Acetylator Status
• Phenotyped in urine by the ra2o of AFMU:1-‐methylxanthine
NAT2-‐dependent slow acetyla9on
• NAT2*4 is ‘wild-‐type’, responsible for most ‘fast acetylator’ ac2vity • ‘Slow acetylator’ phenotype due largely to NAT2*5, NAT2*6 , NAT2*7,
NAT2*14 alleles • Low ac2vity due to:
– Poor expression/unstable protein (NAT2*5) – Decreased cataly2c ac2vity (NAT2*6)
• Some studies have demonstrated a much greater frequency of homozygous slow acetylators (91%) in Caucasian children with documented skin allergies, than in disease-‐free children (62%).
• Sensi2za2on may be mediated by increased forma2on of hydroxylamines, e.g. seen with sulfonamides.
• Epidemiological studies on the role of NAT polymorphisms in cancer suscep2bility and sulfonamide toxicity are quite confusing with oven contradictory findings.
Role of NATs in aroma9c amine genotoxicity
Reac%ve nitrenium ion; DNA binding
NR
OSO3H
NR
SULT
OSO3H
SAM and Methyltransferases • SAM -‐ ‘Nature’s methyl iodide’” – serves as the methyl group donor
for 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
SCH2 adenosine
CO2HH2N
R-X-H R-X-CH3
S-adenosyl methionine (SAM) S-adenosyl homocysteine
• Methyltransferase reac2ons differ from other conjuga2ons in that they typically decrease the water solubility of the resul2ng metabolites.
S-‐, O-‐ and N-‐Methyl Transferases
Main func2onal groups involved: • Thiols (TPMT/TMT) • Catechols (COMT) • Aroma2c and alipha2c amines (NMT) Mostly endogenous substrates, but some drugs are metabolized by these enzymes: • Captopril (TMT) • 6-‐Mercaptopurine (TPMT) • L-‐DOPA, Methyldopa (COMT) • Nico2ne (NMT)
S-‐Methyltransferases: 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
• At least two dis2nct enzymes (TMT and TPMT) each of which requires SAM. • Thiol methyl transferase (TMT) is a microsomal enzyme that catalyzes the
methyla2on of alipha2c thiols, e.g. captopril.
• Thiopurine methyl transferase (TPMT) is a 28 kDa cytosolic enzyme that catalyzes the methyla2on of aroma2c and heteroaroma2c substrates
N
O
SHHO2C
CH3
N
O
SHO2C
CH3
CH3
TPMT: thiopurine methyltransferase XO: xanthine oxidase HGPRT: hypoxanthine guanine phosphoribosyltransferase TIMP: 6-‐thioinosine monophosphate MTMP: 6-‐S-‐methylthioinosine monophosphate TGN: 6-‐thioguanine nucleo2des 6-‐MP: 6-‐mercaptopurine MeMP: 6-‐S-‐methylmercaptopurine 6-‐TU: 6-‐thiouric acid
6-‐MP TGN
MeMP
TPMT
6-‐TU
XO
HGPRT DNA
TPMT
Bioac1va1on Pathway
Detoxifica1
on Pathw
ay
TIMP
MTMP
TPMT
(mul2ple enzyma2c steps)
(Purine salvage)
6-‐Mercaptopurine Disposi9on
Other products
6-‐MP Dose Adjustment Based on TPMT Genotype
Krynetski and Evans, Pharmacology 61:136-‐46, 2000
Strategy is to focus on the most common defec2ve alleles and adjust 6-‐MP dose downward for ~10% of pa2ents.
Common alleles confer reduced enzyme stability
COMT
• Cytosolic (liver and kidney) and membrane-‐bound forms (brain)
• ~25 kDa enzymes requiring magnesium
• COMT inhibitors e.g. entacapone, used in Parkinsonism
• Metabolizes neurotransmifers, catechol estrogens and drugs that undergo metabolism to catechols.
• Methyla2on occurs at the meta posi2on.
NHCH3
O
O
CH3
NHCH3
HO
HO
CH3
NHCH3
HO
H3CO
CH3