Biomedical Engineering Reference
In-Depth Information
target for cocaine's stimulatory action. DAT knockout mice are insensitive to the administration of
cocaine and, moreover, knockin mice expressing a DAT mutant incapable of binding cocaine shows
insensitivity to cocaine administration. It is, therefore, the current view that the rapid increase in
extracellular dopamine concentration elicited by cocaine inhibition of DAT produces the psycho-
motor stimulant and reinforcing effect that underlie cocaine abuse.
Some closely related cocaine analogues possess higher potency toward the biogenic amine
transporters and, thus, have been more suitable than cocaine itself in experimental setups (e.g., radi-
oligand binding assays) directed toward understanding the pharmacological properties of the trans-
porters. Important examples include CFT (2
β
-carbomethoxy-3
β
-(4-l uorophenyl)tropane or WIN
35,428) and RTI-55 ((-)-2
-CIT) (Figure 14.4).
Both compounds display nanomolar afi nity for the biogenic amine transporters; however, while
CFT shows selectivity for DAT over NET and SERT, RTI-55 shows selectivity for SERT and DAT
over NET.
For compounds of the tropane class, the tropane ring and the 2
β
-carbomethoxy-3
β
-(4-iodophenyl)tropane or
β
-carbomethoxy group are crucial
for their afi nity. An exception for this rule is the benztropine class. This group of tropanes lack
the 2
β
-carbomethoxy
group, the benztropines contain a diphenylmethoxy moiety. Recently, there has been increasing
focus on benztropine analogues. Several of these compounds posses similar or even higher afi n-
ity and greater selectivity for the DAT than cocaine. The compounds tested so far readily cross
the blood-brain barrier and produce increases in extracellular levels of dopamine for even longer
durations than cocaine. Nonetheless, several of these DAT inhibitors are less effective than cocaine
as behavioral stimulants. Furthermore, one benztropine analogue, JHW 007, has been found to
potently antagonize the behavioral effects of cocaine (Figure 14.4). Assuming a correlation between
behavioral effects of cocaine in laboratory animals and abuse potential in humans, these i ndings
suggest JHW 007 as a potential lead for development of cocaine abuse pharmacotherapeutics. The
reason for this discrepancy in the stimulating effect between cocaine and the benztropines has been
suggested at least in part to be related to different pharmacodynamic properties of the compounds.
Interestingly, recent studies suggest that while cocaine and cocaine analogues bind and stabilize an
outward facing conformation of the transporter, benztropine analogues bind and stabilize a more
closed conformation of the transporter. It is possible that binding to the open and likely more prev-
alent outward facing conformation of DAT conformation results in a faster on-rate, which may
facilitate faster inhibition of DAT function and thereby a more rapid rise in extracellular dopamine
concentration. In contrast, binding to a more closed and predicted less prevalent conformation of the
transporter may result in a slower on-rate of the compound and thereby a slower rise in dopamine
levels and a less stimulatory effect.
The molecular mode of interaction of cocaine and analogues with DAT has long been the subject
of speculation. In particular, it has been debated whether or not the cocaine-binding site in DAT
overlaps with that of dopamine. If inhibition of dopamine uptake by cocaine is the result of an
allosteric mechanism, it would be possible, at least in theory, to generate a cocaine antagonist for
treatment of cocaine addiction that might block cocaine binding without affecting dopamine trans-
port. An experimentally validated molecular model of the cocaine-binding site in the DAT has
been reported (Figure 14.5). The DAT model was generated on the basis of the LeuT
Aa
structure
followed by molecular docking of dopamine, cocaine, and other inhibitors into the model. The
docking procedure revealed a binding site for cocaine and cocaine analogues that was deeply buried
between TM1, 3, 6, and 8, and overlapped with the binding site for dopamine. There were, however,
also signii cant differences between the binding modes: cocaine and cocaine analogues displayed a
unique interaction with Asn157 and, moreover, the binding mode of cocaine/cocaine analogues dis-
torted the conformation of the binding (Tyr156) that (as described earlier) plays a key role in closing
the dopamine-binding pocket to the extracellular environment through formation of a stabilizing
hydrogen bond with Asp79 in TM1 (Figure 14.5). In the cocaine-binding model, Tyr156 is pushed
away by the 2
β
-carbomethoxy group but still bind DAT with high afi nity. Instead of the 2
β
-methylester substituent of cocaine resulting in disruption of the hydrogen bond and
a conformation of the binding pocket that is more open to the outside. This is in agreement with
β
