Biology Reference
In-Depth Information
membrane proteins has four members, designated
hENT1-4 (158). hENT1, hENT2, and hENT3 are the
best characterized of this family and display similar
selectivity for transporting for purine and pyrimidine
nucleosides.
158
In addition to transporting nucleosides,
hENT2 and hENT3 can also transport nucleobases
whereas hENT1 cannot.
159
hENT4 is unique in trans-
porting adenosine as well as various organic cations.
160
hCNTs differ from hENTs by three important
features. First, nucleoside transport via hCNTs is via
aNa
þ
-dependent process.
161
Secondly, most hCNT
family members display nucleoside specificity in which
hCNT1 prefers to transport pyrimidine nucleosides
while hCNT2 prefers purines.
162
hCNT3 is the lone
exception as this transporter shows broad nucleoside
specificity.
162
Finally, hCNT family members bind nucle-
osides with ~10-fold higher affinity than hENT family
members.
163
Once inside the cell, most purine nucleosides accu-
mulate mainly as the corresponding 5
0
-triphosphate. In
the case of F-ara-A, the rate-limiting step in the forma-
tion of triphosphate is conversion of F-ara-A to its mono-
phosphate which is catalyzed by deoxycytidine
kinase.
164
Although F-ara-A is a poor substrate for this
pyrimidine kinase, the high specific activity of this
enzyme in lymphoid tissues results in overall efficient
phosphorylation. The monophosphate is converted to
the diphosphate by AMP kinase, and then to the corre-
sponding triphosphate by nucleoside diphosphate
kinase.
165
Adenosine deaminase is an important enzyme
involved in purine metabolism by catalyzing the irre-
versible deamination of adenosine and deoxyadenosine
to inosine and deoxyadenosine, respectively. Mutations
in the gene for adenosine deaminase can lead to a defi-
ciency in this enzyme to cause a syndrome referred to
as severe combined immunodeficiency (SCID).
166
It
was recognized that a deficiency in adenosine deami-
nase leads to an accumulation of deoxyribonucleotides
in lymphocytes to cause lymphocytopenia.
167
Based
upon this, it was proposed that inhibiting the activity
of adenosine deaminase by nucleoside analogs could
also generate a similar beneficial effect against various
forms of leukemia.
168
Indeed, nucleoside analogs such
as fludarabine, cladribine, and pentostatin are resistant
to deamination by this enzyme and often function to
inhibit its activity to produce anticancer effects.
169
Fludarabine
1
F-ara-A
2
3
4
F-ara-AMP
F-ara-A
F-ara-ADP
8
5
F-ara-ATP
6
7
RNA-dFdCMP
DNA-dFdCMP
FIGURE 5.11
Key pharmacokinetic features associated with the
metabolism of fludarabine. Step 1 is dephosphorylation of fludarabine
(F-ara-AMP) by 5'-nucleotidase to form F-ara-A which can then be
transported into cells by various nucleoside transporters (step 2). Step
3 is the initial phosphorylation step converting F-ara-A to F-ara-AMP
and is catalyzed by dCK. Steps 4 and 5 are subsequent phosphory-
lation events to form F-ara-ADP and F-ara-ATP, respectively. Step 6
is incorporation of F-ara-ATP into DNA while step 7 is incorporation
of F-ara-ATP into RNA. Step 8 is dephosphorylation of F-ara-AMP by
5'-nucleotidase to F-ara-A.
conversion to the corresponding nucleoside triphos-
phate. The metabolism of fludarabine is provided as an
example for key steps used in the anabolism and catab-
olism of most purine nucleoside analogs. Fludarabine is
a prodrug that must first be converted from the mono-
phosphate form to the free nucleoside, F-ara-A, prior
to gaining cellular entry. This conversion is catalyzed
by 5-nucleotidase and occurs readily in plasma.
156
After
dephosphorylation, the free nucleoside must enter the
cells via the action of one of several nucleoside trans-
porters. In general, nucleosides are rather hydrophilic
and typically show negligible permeability across
hydrophobic cellular membranes. To facilitate uptake,
cells use specific proteins that translocate natural and
modified nucleosides from the extracellular milieu into
the cytosol. There are two types of cellular nucleoside
transport, an equilibrative transport mechanism and
a concentrative transport mechanism (reviewed else-
where
157
). The equilibrative transporters show broad
specificity but relatively low affinity for natural nucleo-
sides and various nucleoside analogs. In contrast,
concentrative transporters are Na
þ
-dependent and
display higher affinity for nucleosides compared to equi-
librative transporters. These two types of nucleoside
transport processes are catalyzed by classes of
membrane-bound proteins designated as
e
quilibrative
n
ucleoside
t
ransporters (ENT) or
c
oncentrative
n
ucleo-
side
t
ransporters (CNT). The
h
uman ENT family of
Biochemical Mechanism of Action
The primary cytotoxic mechanism for most nucleo-
side analogs is incorporation of their corresponding
nucleoside triphosphates into DNA, causing chain
termination of DNA synthesis and the activation of
apoptosis. The mechanism for the incorporation of
