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