Department of Pharmacology (A.C.A., Y.-F.X., M.K.), Southern
Illinois University School of Medicine, Springfield, Illinois; and
Departments of Psychiatry (R.G., G.L.) and Chemistry (D.P., R.C.),
University of California, Irvine, California
 |
Introduction |
AMPA-type
glutamate receptors are abundant throughout the brain and account for
much of the transmission occurring at excitatory synapses. Ito et al.
(1990)
made the seminal observation that the current through these
receptors can be enhanced by the nootropic compound aniracetam. The
drug did not influence other types of glutamate receptors, and it had
no evident effect on AMPA receptors in the absence of glutamate. Other
compounds were subsequently discovered that "up-modulate" or
"potentiate" AMPA receptor function in a similar manner, including
diazoxide (Yamada and Rothman, 1992
), cyclothiazide (Yamada and Tang,
1993
), IDRA-21 (Bertolino et al., 1993
), and PEPA (Sekiguchi et
al., 1997
). Using the structural leads given by these compounds,
several laboratories have developed entire families of potent AMPA
receptor modulators beginning with the ampakines (Arai et al., 1994
,
1996b
,c
, 2000
; Staubli et al., 1994a
,b
) and including the
pyridothiadiazines (Pirotte et al., 1998
) and the
biarylpropylsulfonamides (Ornstein et al., 2000
).
The interest in these compounds has been fostered in part
by the possibility that some neurological disorders, such as
age-related memory impairment, schizophrenia, and perhaps depression,
may be associated with lower than normal excitatory transmission in some brain regions (Masliah et al., 1993
; Tamminga, 1998
). If so,
up-modulation of AMPA receptors could potentially be of therapeutic value. Experimental evidence to support this has been obtained in
behavioral studies with ampakines (e.g., Granger et al., 1993
, 1996
;
Staubli et al., 1994b
; Larson et al., 1996
) and recently also with
other modulators (Zivkovic et al., 1995
; Lebrun et al., 2000
; Li et
al., 2001
). Several of these compounds have been shown to cross the
blood-brain barrier to increase excitatory responses in vivo (Staubli
et al., 1994a
; Vandergriff et al., 2001
), and their ability to
facilitate long-term synaptic potentiation (Arai and Lynch, 1992
,
1996a
; Staubli et al., 1994a
) has been suggested to account for the
observed improvements in various memory tests. The drugs may have
similar actions in humans according to preliminary clinical tests
(Lynch et al., 1996
; Goff et al., 2001
).
Most current AMPA receptor modulators belong to one of two
structural families, referred to as benzamides (aniracetam and ampakines) and benzothiadiazides (BTDs). PEPA and the more recent biarylpropylsulfonamides fall outside these two categories but share
elements with the latter. Behavioral tests have largely involved the
benzamides because the first modulators in the BTD family (diazoxide
and cyclothiazide) have clinically important peripheral effects and
only weakly affect field EPSPs in hippocampal slices, even at
concentrations far above their affinities for the AMPA receptor (Larson
et al., 1994
; Arai and Lynch, 1998
; Hjelmstad et al., 1999
). Some
modulators have substantial effects on extracellular synaptic responses
that align well with their affinities for AMPA receptors, whereas
others do not; this is thought to be related to how the compounds
affect receptor kinetics. Diverse experiments indicate that
cyclothiazide acts mainly on desensitization (Johansen et al., 1995
;
Partin et al., 1996
), whereas ampakines also have a strong, even
primary, influence on channel gating (Arai and Lynch, 1998
). This would
explain the observed differences in the effects of the modulators if,
as has been argued, the latter process plays a larger role than the
former in shaping the size and waveform of undisturbed synaptic responses.
IDRA-21, which does not have the peripheral side effects associated
with diazoxide and cyclothiazide and unlike the latter rapidly
increases synaptic potentials (Arai et al., 1996a
), was the first
benzothiadiazide examined in behavioral tests (Zivkovic et al., 1995
).
However, IDRA-21 has modest potency, with concentrations above 200 µM
typically needed to enhance excitatory transmission in hippocampal
slices (Arai et al., 1996a
). Our efforts as well as those of others
(Desos et al., 1996
; Pirotte et al., 1998
) have therefore been directed
at finding analogs that have higher potency yet maintain effect
profiles that are suitable for behavioral applications. In the present
project, we systematically modified substituents at various locations
around the benzothiadiazide core of IDRA-21 and measured the effects on
various aspects of AMPA receptor operation. Some of the modifications
resulted in compounds that have much greater potency than IDRA-21 and
have effects on AMPA receptor kinetics that differ radically from those of cyclothiazide. A comprehensive description of the compounds that
were synthesized and examined in this study is given elsewhere (Phillips et al., 2002
), and some data have been presented in abstract
form (Arai et al., 1999
).
 |
Materials and Methods |
AMPA Receptor Currents in Excised Patches.
Patch-clamp
studies were carried out with outside-out patches excised from
pyramidal neurons in field CA1 of organotypic hippocampal slices (Arai
et al., 1996c
, 2000
). The slice cultures were prepared from 13 to
14-day-old Sprague-Dawley rats and grown for 2 weeks on cellulose
membrane inserts (Millipore CM; Millipore Corp., Bedford, MA). Patches
were excised in a medium containing 125 mM NaCl, 2.5 mM KCl, 1.25 mM
KH2PO4, 2 mM
CaCl2, 1 mM MgCl2, 5 mM
NaHCO3, 25 mM D-glucose, and 20 mM
HEPES, pH 7.3, and relocated to a chamber perfused with recording
medium containing 130 mM NaCl, 3.5 mM KCl, 20 mM HEPES, 0.01 mM
dizocilpine maleate, and 0.05 mM
D-2-amino-5-phosphonopentanoic acid. Patch pipettes had a
resistance of 3 to 8 M
and were filled with a solution of 65 mM CsF,
65 mM CsCl, 10 mM EGTA, 2 mM MgCl2, 2 mM ATP
disodium salt, and 10 mM HEPES, pH 7.3. A piezo device was employed to switch solutions applied to the patch within a fraction of a
millisecond (Arai et al., 1996b
). In brief, background medium and
agonist containing medium were flowing continuously through two lines of a double pipette that was moved by a piezo device across a distance
of 50 µm in 0.4 ms (Arai et al., 1996b
,c
). Data were acquired with a
patch amplifier (AxoPatch-1D; Axon Instruments, Inc., Foster City, CA)
at a filter frequency of 5 kHz and digitized at 10 kHz with
PClamp/Digidata 1200 (Axon Instruments, Inc.). The holding potential
was
50 mV. The drugs were applied at the same concentration in both
background and glutamate lines, and background flow lines were switched
at least 15 s before applying the first glutamate pulse.
Typically, five responses were collected and averaged for each
condition. Measurement with each patch was alternated repeatedly
between control (A: glutamate alone) and test conditions (B: glutamate + drug). For data analysis, response B was compared with the average of
the responses A taken before and after response B, and peak and
steady-state currents recorded in the presence of drug were normalized
to those without drug. Deactivation rates were determined by fitting
the decay phase of the response to a 1-ms glutamate pulse (10 mM) with
a single or double exponential function. Drug solutions were prepared
from 1000-fold stock solutions in dimethyl sulfoxide (DMSO); the same final concentrations of DMSO (maximum, 0.1%) were included in all drug
and control solutions.
Whole-Cell Recording from Pyramidal Cells in Field CA1 of
Hippocampal Slices.
Male Sprague-Dawley rats of postnatal day 15 to 21 (Harlan, Indianapolis, IN) were decapitated under anesthesia
following National Institutes of Health guidelines and an
institutionally approved protocol. Transverse hippocampal slices (400 µm) were prepared using a vibratome (Leica Microsystems, Deerfield,
IL). The slices were submerged in oxygenated artificial cerebrospinal fluid (ACSF) infused at 0.5 ml/min. The experiments were carried out at
ambient temperature. The ACSF contained 124 mM NaCl, 3 mM KCl, 1.25 mM
NaH2PO4, 2 mM
CaCl2, 1 mM MgSO4, 5 mM
NaHCO3, 10 mM glucose, and 10 mM HEPES, pH 7.4. The intrapipette solution contained 130 mM CsF, 10 mM EGTA/K, 2 mM ATP
disodium salt, 2 mM MgCl2, and 10 mM HEPES, pH
7.4. Pyramidal cells were visualized with an infrared microscope
(BXI50; Olympus, Tokyo, Japan) with differential interference contrast
configuration. Synaptic responses were recorded using borosilicate
glass electrodes (2-5 M
) in response to activation of
Schaffer-commissural fibers stimulated by a bipolar nichrome electrode
in stratum radiatum. After establishing a stable baseline, the
perfusion line was switched to one containing the drug; solution
exchange in the recording chamber was complete within 3 min. EPSCs were
recorded with AxoPatch 200B and digitized at 10 kHz with
Digidata1200/PClamp 7. The holding potential was
70 mV, and the
signals were filtered at 5 kHz. Recordings were discarded if the input
resistance varied by greater than 10% over the course of the experiment.
Whole-Cell Recordings from HEK 293 Cells.
Patch-clamp
recordings were carried out in whole-cell configuration from human
embryonic kidney (HEK) 293 cells that stably express homomeric AMPA
receptors consisting of GluR3 flop subunits (see Hennegriff et
al., 1997
). Recordings were made at room temperature in serum-free
minimal essential medium (Invitrogen, Carlsbad, CA). Patch
pipettes had a resistance of 3 to 7 M
and were filled with 130 mM
CsF, 10 mM EGTA, 2 mM MgCl2, 2 mM ATP disodium
salt, and 10 mM HEPES (pH 7.4). The holding potential typically was
100 mV. Agonist was applied with a fast solution switch system in
which cells are exposed to a constant flow of the background solution
that is momentarily interrupted during application of glutamate. The
drugs were included in both background and agonist lines.
Extracellular Recording in Hippocampal Slices.
Transverse
hippocampal slices (400 µm) were prepared as described elsewhere
(Arai et al., 1996c
) and placed in an interface chamber, which was
perfused at 0.5 ml/min with oxygenated ACSF containing 124 mM NaCl, 3 mM KCl, 1.25 mM KH2PO4, 3.4 mM CaCl2, 2.5 mM MgSO4, 26 mM NaHCO3, and 10 mM D-glucose and
exposed to humidified 95% O2/5%
CO2. Field EPSPs were recorded from the stratum radiatum in response to activation of Schaffer-commissural fibers in
the same stratum. The input-output relation of the synaptic response
was first established to determine the maximum EPSP amplitude without
spike component, and the stimulation intensity was adjusted to 50% of
the maximum EPSP amplitude. After establishing a stable baseline, the
perfusion line was switched to one containing the drug.
Binding Assays.
Binding tests were carried out with
membranes from rat brain and from HEK 293 cells that express one of the
AMPA receptor subunits. Membranes from rat brain were prepared
according to conventional procedures (Kessler et al., 1996
) involving
homogenization in an isotonic sucrose solution and differential
centrifugation to obtain a P2 pellet fraction,
followed by an osmotic lysis and repeated washing by centrifugation and
resuspending in the binding assay buffer (100 mM HEPES/Tris and 50 µM
EGTA, pH 7.4). Aliquots were frozen at
80°C; after thawing, the
membranes were sonicated and washed twice by centrifugation. For tests
with recombinant receptors, HEK 293 cells were collected into
Tris-buffered saline (150 mM NaCl and 10 mM Tris/HCl, pH 7.4)
containing in addition 2 mM EGTA and a protease inhibitor cocktail
(Sigma-Aldrich, St. Louis, MO). The cells were centrifuged at
least four times (2000g for 10 min) and resuspended in the
Tris-buffered saline without the additions; after the first
centrifugation, 0.1% saponin was included in the medium to
permeabilize the membranes, and the cell suspension was left at 25°C
for 5 min. All other steps were carried out at 0-4°C. The cells were
stored on ice and washed before use.
Binding tests with rat brain membranes were conducted with the
centrifugation method, generally at 25°C. Aliquots of the membrane suspension containing 50 µg of protein were incubated for 45 min with
the radioligand and appropriate additions in a final volume of 100 µl. Sets of 24 samples were then centrifuged for 15 min in a Beckman
Coulter JA 18.1 rotor (Fullerton, CA) kept at the incubation
temperature. The supernatant was aspirated, and the pellet was quickly
rinsed with ice-cold buffered saline containing 50 mM KSCN (wash
buffer). The pellets were dissolved in 20 µl of issue solubilizer
(Beckman Coulter, Inc.) and counted after addition of acidified
scintillation fluid. Binding incubations with HEK 293 cells were
terminated by filtration through Whatman GF/C filters (Fisher
Scientific, Pittsburgh, PA) after diluting the sample in 5 ml of
ice-cold wash buffer; the filters were rapidly washed with three
additional volumes of wash buffer. Drugs were dissolved in DMSO and
diluted into the assay buffer at twice the highest final drug
concentration; the assay buffer was heated to 60°C for the dilution
and then quickly cooled to ambient temperature. Further dilutions were
made with assay buffer containing the equivalent concentration of DMSO
(0.1-1%; DMSO concentrations in this range did not have a significant
effect on drug potency). Background values ("nonspecific binding")
were measured by inclusion of 5 mM L-glutamate and
subtracted from total binding; separate background values were
determined for incubations with and without drug. Protein content was
determined according to the Bradford method with the reagent available
from Bio-Rad (Hercules, CA) and bovine serum albumin as standard.
Binding curves were fitted to the data points with nonlinear regression
(GraphPad Prism; GraphPad Software, San Diego, CA).
(R,S)-[3H]AMPA and
[3H]CNQX were purchased from PerkinElmer Life
Sciences (Boston, MA), and
[3H]fluorowillardiine and
(S)-[3H]AMPA were obtained from
Tocris Cookson Inc. (Ballwin, MO). Other reagents were from the usual
commercial sources.
 |
Results |
Two of the more closely examined compounds of this study are shown
in Fig. 1 along with three widely studied
BTDs. The new compounds are derived from IDRA-21 and differ from it
mainly by an alkyl substitution at the 5' position. A comprehensive
description of syntheses and structure-activity relations is given in
Phillips et al. (2002)
, and drug names accord with those used therein.

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Fig. 1.
Structure of benzothiadiazide compounds. Three
conventionally used compounds of this drug family are shown in the
upper row. The second row shows the structure of the two most
extensively tested alkyl-BTDs. The benzothiadiazide core structure and
the numbering of substituent locations are indicated on the right.
|
|
AMPA Receptor Currents in Excised Patches.
Figure
2A shows the effect of the
5'-ethyl-benzothiadiazide D1 on currents induced by 800-ms application
of 1 mM glutamate in patches excised from hippocampal pyramidal cells.
In the absence of drug, the agonist-induced current declined rapidly to
a steady-state level that was 5 to 10% of the peak current. Increasing
concentrations of D1 progressively raised the steady-state current and
essentially blocked desensitization at concentrations above 100 µM.
The increases in steady-state values occurred without changing the rate
at which those values were reached, suggesting that D1 completely
blocked desensitization in receptors that have bound the drug and that increasing the drug concentration in essence shifted the balance between drug-free and drug-bound receptors. Fitting a four-point logistic equation to the dose-effect data gives an
EC50 estimate of 36 µM with a Hill coefficient
of 1.6; the threshold concentration was around 2 µM (Fig. 2, inset).

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Fig. 2.
Effects of D1 on AMPA receptor currents in patches
from hippocampal pyramidal neurons. A, application of 1 mM glutamate
for 800 ms to measure the effect of the drug on receptor
desensitization. The upper graph shows a representative experiment from
a single patch with responses recorded in the absence of drug and in
the presence of the D1 concentrations shown above the graph. The
horizontal bar indicates the duration of glutamate application. D1 was
present before and during the glutamate pulse; the background medium
was switched to one containing the desired drug concentrations at least
20 s before applying glutamate. The bottom graph shows the
concentration-effect relation for the steady-state current expressed as
percentage of the peak current (means and S.E.M. from 4-6 patches).
Data points were fitted with a four-point logistic equation. The
EC50 value and the Hill coefficient obtained from this
curve fit are shown in the graph. Traces in the absence of drug and at
2 µM D1 are shown as insets. B, application of fast (1-ms) pulses of
glutamate (10 mM) to mimic receptor activation during synaptic
transmission. The upper graph shows a representative experiment from a
single patch; the drug was equilibrated with the patch before glutamate
application as described under A. The return of the response to
baseline after glutamate offset is called deactivation. An approximate
time constant for this deactivation phase was determined by fitting a
two-exponential function to the data points and forming a weighted
average of the two time constants by multiplying them with the
fractional amplitude of each component. Its values are plotted in the
lower graph (means and S.E.M. from 3-8 patches). Raising the drug
concentration increased the time constant of the fast component (from
3.4 ms at 15 µM D1 to 9.5 ms at 240 µM), the slow component (from
15.5 to 60.8 ms, respectively), and the fractional contribution of the
slow component (from 5 to 75%, respectively). C, two 1-ms pulses of 10 mM glutamate were applied at an interval of 40 ms. The upper graph
shows representative records in the absence of drug and in the presence
of 200 µM D1. In the absence of drug, the second response was reduced
due to receptor desensitization. In the graph underneath, the
percentage reduction in the amplitude of the second response relative
to that of the first response was plotted against the concentration of
D1. D, responses to prolonged application of glutamate in the absence
of drug and in the presence of IDRA-21, D1, and the 5'-methyl analog
16g, each at 200 µM. Experimental procedures were the same as in A. Averaged values (mean and S.E.M.) for the steady-state/peak ratios are
shown in the bar graph. The calibration marked by an asterisk refers to
compound 16g. E, responses to 1-ms application of glutamate in the
absence and presence of 200 µM D1 and 16g. Experimental procedures
were as in B. The deactivation phase of the responses was fitted with
single- and double-exponential equations. Control responses were in
both cases fit best with the single-exponential function. Curve fits to
responses in the presence of drug were superior according to F-tests
when using two-exponential functions (D1: F = 2618, P <0.0001; 16g: F = 653, P <0.001; analysis by GraphPad Prism). Averaged time
constants for the fast (f) and slow (s) component are shown in the bar
graph (mean and S.E.M.; n = 5 for D1,
n = 4 for 16g). The calibration marked by refers to compound 16g.
|
|
Figure 2B summarizes data from experiments in which glutamate (10 mM)
was applied for only 1 ms to measure the rate at which responses
deactivate after glutamate offset. In the absence of drug, the response
returned to baseline with a time constant of 3.4 ± 0.4 ms. In the
presence of D1, the responses were greatly prolonged (Fig. 2, B and E).
This effect is not the result of blocking desensitization, because no
significant prolongation was observed with a near-saturating
concentration of cyclothiazide in some of the same patches (12 ± 6%, n = 4) and in previous studies (Arai and Lynch,
1998
; Arai et al., 2000
). Deactivation in the presence of drug
generally revealed two exponential components, as illustrated in Fig.
2E for the case of 200 µM D1. These components may reflect distinct
kinetic processes and/or the presence of receptors with different
degrees of drug occupancy at subsaturating drug concentrations. The
dose-effect relation shown in Fig. 2B was constructed using a weighted
average of the two time constants. Its value increased from 3.4 ± 0.4 ms in the absence of drug to 58.4 ± 8.1 ms at 240 µM D1, or
by a factor of 17, providing an EC50 value of 77 µM with a Hill slope near 3. In Fig. 2C, two 1-ms pulses were applied
in rapid succession (40-ms interpulse interval). Under these
conditions, the second response is typically reduced by about 40%, as
expected if a sizable proportion of the receptors activated by the
first glutamate pulse desensitize and are not available to respond to
the second pulse. As seen in Fig. 2, this reduction was eliminated in
the presence of 200 µM D1.
Effects similar to those of D1 were obtained with other alkyl-BTDs. The
5'-methyl analog 16g (see Fig. 5 for structure) had a lower potency
than D1 (see Fig. 4, below) but was similarly efficacious in blocking
desensitization (Fig. 2D). By comparison, IDRA-21 at 200 µM produced
only a 2- to 3-fold increase in the steady-state current, which is in
agreement with the data shown by Bertolino et al. (1993)
, and it slowed
the rate at which the steady state was reached, suggesting that
desensitization is attenuated but not completely blocked by this drug.
Response deactivation was slowed by all active alkyl-BTDs, but the
magnitude of the effect was generally smaller with 5'-methyl analogs
than with the 5'-ethyl derivative D1. Thus, compound 16g at 200 µM
increased the slow component of the deactivation phase to only 29 ± 13 ms (n = 4), and the weighted average at that
concentration was 14.0 ms (Fig. 2E).
Effects on Agonist Binding.
By changing receptor kinetics,
most AMPA receptor modulators also enhance or reduce the binding of
receptor agonists, such as [3H]AMPA and
[3H]fluorowillardiine. The magnitude and
direction of the binding effect depend to some extent on assay
variables, such as temperature and buffer composition, but
EC50 values for drug effects correlate well with
those obtained from physiological recordings (e.g., Hall et al., 1993
;
Kessler et al., 1996
; Arai et al., 1996b
, 2000
). Binding tests were
therefore used together with excised patch recordings to screen and
characterize the compounds developed in this project (Phillips et al.,
2002
). Figure 3 illustrates the typical
effects of D1 on the binding of
[3H]fluorowillardiine (FW) (A) and
[3H]AMPA (B) under several commonly employed
assay conditions. In all cases, D1 produced a robust increase in the
binding of the agonist. At an assay temperature of 0°C, the
EC50 value was 5.5 µM, which indicates a
potency comparable with or higher than that of cyclothiazide (~30
µM; Hall et al., 1993
). All other tests with brain membranes were
conducted at ambient temperature to facilitate comparison with
physiological measures. The EC50 at this
temperature was 17 µM; the increase in [3H]FW
binding was 3.6 fold and thus considerably larger than that produced by
any other modulator under those assay conditions (Arai et al., 2000
).
Drug potency depended only weakly on the choice of the agonist (AMPA
versus fluorowillardiine) and was not affected by the chaotropic anion
SCN
, which is often used to augment the
affinity for [3H]AMPA (Fig. 3B). Thus, binding
tests provide a consistent measure for the potency of D1 that matches
within a factor of two the EC50 values obtained
in patch experiments.

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Fig. 3.
Allosteric modulation of agonist binding by D1. A,
effect of D1 on the binding of [3H]FW (20 nM) to rat
brain membranes at 0 and 23°C. Incubations were terminated by
centrifugation. Control binding without drug was measured in
quadruplicate; binding in the presence of drug was determined in
duplicate or triplicate and normalized to that in the absence of drug.
Data from typical experiments are shown as means and S.E.M.; the
absence of an error bar indicates an error smaller than the size of the
symbol. The data points were fitted with four-point logistic equations
forced through 100% as the lower asymptote (GraphPad Prism). Binding
in the absence of drug was 0.62 pmol/mg protein at 23°C and 0.98 pmol/mg protein at 0°C. Average values for drug potency
(EC50) and the Hill slope
(nHill) obtained from these curve fits are
shown as insets. Average values at 23°C were EC50,
17.4 ± 1.8 µM; nHill, 1.8 ± 0.1; and maximal increase, 360 ± 15% (n = 9). Average values at 0°C were EC50, 5.5 ± 1.1 µM; nHill, 1.7 ± 0.1; and maximal
increase, 127 ± 4% (n = 2). B, effect of D1
on the binding of
(R,S)-[3H]AMPA (50 nM) in
the absence and presence of the chaotropic ion SCN (50 mM; potassium salt). Binding was measured at 23°C. Average binding
parameters in the presence of SCN were EC50,
26.6 ± 1.8 µM; nHill, 2.2 ± 0.1; and maximal increase, 269 ± 6% (n = 11). Average values in the absence of SCN were
EC50, 25.4 ± 0.3 µM;
nHill, 1.6 ± 0.1; and maximal
increase, 673 ± 28% (n = 2). C, displacement
of [3H]CNQX binding (40 nM) by the glutamate
concentrations indicated on the x-axis, measured in the
absence and presence of 200 µM D1. Fitting of a sigmoidal curve
(nHill = 1) gives an IC50
of 53 µM, which, after Cheng-Prusoff correction, provides an affinity
estimate for glutamate of 47 µM. In the presence of D1, the
IC50 was lowered to 4.2 µM (affinity estimate, 3.7 µM).
A similar, approximately 10-fold increase in affinity was observed in
two additional experiments.
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The enhanced binding seen in these assays could be either due to an
increase in affinity or an increase in the
Bmax. This was examined more closely
in Fig. 3C in which the affinity for glutamate was determined by
measuring the degree to which the agonist displaces the high-affinity
antagonist [3H]CNQX. The latter radioligand was
used because it has similar affinity at high- and low-affinity variants
of the AMPA receptor and the different subunits and because its binding
can be reliably measured without additions such as thiocyanate (see
also Kessler et al., 1996
). The affinity of glutamate was increased
from 47 µM without modulator to less than 4 µM in the presence of
saturating D1. Thus, [3H]fluorowillardiine
binding apparently underestimates the magnitude of the affinity
increase for the endogenous ligand glutamate. [3H]CNQX binding itself was slightly
increased by D1; this stands in contrast to the 2-fold binding
reduction observed with cyclothiazide (Kessler et al., 1996
).
Figure 4 shows the effects on binding of
other 5'-alkyl derivatives and of the parent compound IDRA-21. The
isopropyl homolog 16c (which, like D1, extends a distance of two
carbons from the ring) was similar to D1, but the longer chain propyl
homolog 16e was much less potent with an EC50 of
170 µM. However, the maximum binding increase was larger than that
produced by D1 for both drugs. In contrast, shortening the alkyl length
reduced both drug potency and the maximal effect on binding. Thus the
methyl-BTD 16g increased FW binding less than 2-fold, and IDRA-21
produced at most a 30% enhancement. Few of the earlier studies on
IDRA-21 provided a potency estimate for this compound. Yamada et al.
(1998)
found an EC50 of 150 µM in cultured
neurons, but extrapolation of the patch data in Bertolino et al. (1993)
suggests an EC50 value on the order of 0.5 to 1 mM, and EPSP measurements in adult hippocampal slices (Arai et al.,
1996a
) and other physiological tests (Puia et al., 2000
) are more
consistent with the latter value. A similar EC50
estimate of about 1 mM was obtained here for the effect of IDRA-2 on FW
binding. Thus, the ethyl substituent in D1 conferred an approximately
50-fold gain in potency over the parent compound IDRA-21. The potency
of the methyl-compound 16g was intermediate between those of IDRA-21
and D1 with an EC50 of 100 µM. The differences
between methyl and ethyl compounds extend to other compounds developed
in this study. As shown in Fig. 5, the
maximum increase in binding was always less than 2-fold for 5' methyl
substituents as opposed to the 260 to 430% seen with larger alkyl
groups. Secondly, none of the 5'-methyl compounds had an
EC50 lower than 90 µM, which confirms a
progressive gain in potency with extension of the 5'-alkyl group. A
further noteworthy aspect is that Hill coefficients of many compounds
were in the vicinity of 2 and that all of them were significantly
larger than unity.

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Fig. 4.
Relation between 5'-alkyl length and effect on
[3H]fluorowillardiine binding. Binding was measured as in
Fig. 3A with 20 nM [3H]FW in the absence of
SCN . The incubation temperature was 23-25°C. Data are
from representative experiments. The nature of the alkyl group and
average EC50 values are shown next to the drug name.
Complete drug structures and additional binding parameters are compiled
in Fig. 5. In the case of IDRA-21, data for each drug concentration
were averaged from three experiments; limits in drug solubility
prevented testing at concentrations above 1 mM, and thus the
EC50 of 0.9 mM obtained from curve fitting is an
approximation.
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Fig. 5.
Summary of structure-activity relations for
5'-alkyl-benzothiadiazides. Fifteen of about 50 compounds that were
tested are shown together with their effects on agonist binding.
Binding was measured as in Fig. 3A at 23-25°C with 20 nM
[3H]fluorowillardiine (without SCN ) to
construct dose-response relations. Data were fitted with four-point
logistic equations; average values for the EC50, Hill
coefficient, and maximal increase in binding are shown next to the drug
structure. Frames connect compounds with close structural relations.
The left column shows compounds with progressively larger alkyl side
chains at the 5' position; the right column shows variations in the
alkyl substituent at the 3' position. The upper horizontal frame shows
5' methyl compounds that differ only in their 7' substituent.
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Additional observations from binding experiments are illustrated in
Fig. 6. GYKI 52466 is a selective
downmodulator that effectively blocks AMPA receptor responses, probably
by slowing channel opening (Arai, 2001
). GYKI was previously reported
to have no detectable impact on agonist binding, and it did not seem to
interfere with the influence of cyclothiazide on
[3H]AMPA binding (Kessler et al., 1996
). The
effect of D1 was greatly altered, however, when GYKI was present (Fig.
6, A and B). In the presence of thiocyanate,
[3H]AMPA binding was reduced rather than
increased, and in its absence, the binding increase caused by D1 was
less extensive than without GYKI. The EC50 for
GYKI in either case was around 20 µM (Fig. 6C), which is in the range
commonly reported from physiological tests (Zorumski et al., 1993
).
More importantly, the EC50 for D1 changed by a
factor of 2 or less, and the Hill coefficient remained virtually
unaltered, indicating that the interaction between D1 and GYKI is not
competitive.

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Fig. 6.
D1 interaction with GYKI 52466 and D1 effect on
[3H]kainate binding. A, binding of
(S)-[3H]AMPA (50 nM) was measured at
25°C in the presence of the D1 concentrations indicated on the
x-axis at 30, 20, or 300 µM GYKI 52466;
all assays contained in addition 50 mM KSCN. Data for 0 and 300 µM
GYKI are averages from three experiments each, EC50 and
Hill slope were determined for the averaged data, and the curve for 20 µM GYKI is from a representative experiment. B, same as in A except
that binding was measured in the absence of thiocyanate. Data points
are averages of three separate experiments. C, dose-effect curves for
GYKI 52466; binding of [3H]AMPA was measured at a fixed
concentration of D1 (100 µM) and the GYKI concentrations indicated on
the x-axis. Binding at 0 µM GYKI was in this case set
as 100%. Data shown in the graph are from a set of representative
experiments with and without thiocyanate; nearly identical curves were
obtained in a second set of experiments. Averaged binding constants
were 17.2 ± 3.6 µM (S.D.),
nHill = 1.05 ± 0.10 in the
absence of thiocyanate and 20.2 ± 0.1 µM,
nHill = 1.03 ± 0.04 in the
presence of thiocyanate. D, effect of D1 on [3H]kainate
binding to rat brain membranes. Binding was measured at 25°C with 40 nM [3H]kainate and the D1 concentrations shown on the
x-axis. Some incubations contained in addition 200 µM
GYKI 52466. Similar results were obtained in two additional
experiments. The percentage increase in binding produced by D1 was
lower at a [3H]kainate concentration of 4 nM (data not
shown).
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Positive and negative allosteric modulators of the AMPA receptor in
general seem to be very selective, with no detectable action at kainate
receptors (Ito et al., 1990
; Paternain et al., 1995
). This was examined
again for the case of D1 by testing whether the compound influences
agonist binding to kainate receptors (Fig. 6, right). Unexpectedly,
binding of [3H]kainate was increased by 30%.
However, the EC50 for this effect (27 µM) and
the Hill slope (2.4) are close to those seen with FW and AMPA binding,
and the effect was partially reversed by GYKI 52466, which does not act
on kainate receptors (Paternain et al., 1995
). It is thus likely that
some of the [3H]kainate binding occurs at AMPA
receptors and that the observed enhancement by D1 is mediated entirely
through this component. This accords with our earlier observation that
kainate binds with micromolar affinity to subpopulations of AMPA
receptors (Hall et al., 1994
).
Effects of Alkyl-BTDs on Synaptic Responses.
Effects on
synaptic responses were examined using field recording in area CA1 of
hippocampal slices (Fig. 7) and
whole-cell recording from pyramidal neurons of this region (Fig.
8). As shown at the top of Fig. 7, the
methyl-BTD 16h and its ethyl analog 16a (the 7'-fluoro version of D1)
significantly enhanced synaptic responses at concentrations of 50 and
20 µM, respectively. Onset of the effect was fast, and responses
returned to baseline within 30 min of drug washout. Interestingly,
response amplitude and response duration were affected in different
ways by the two compounds. The 5'-methyl-BTD 16h produced a large
increase in the amplitude with very small effects on the half width of
the response, whereas the ethyl analog 16a caused a marked widening
with modest amplitude effect. A similar differentiation was observed
with other drugs with either methyl or longer chain alkyls, some of
which are included in the diagram at the bottom of Fig. 7. As shown
there, methyl-BTDs in all of 20 experiments enhanced the amplitude more
than the response half width; in the 12 experiments involving compound 16h, the average ratio between the two effects was 4.6. Effects produced by the ethyl derivative 16a showed a nearly inverse pattern; in 9 of 13 experiments, the response duration was significantly increased, with little or no effect on amplitude, and only two experiments showed a preferred action on the amplitude.

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Fig. 7.
Effects of 5'-methyl-BTDs and 5'-ethyl-BTDs on field
EPSPs in CA1 of hippocampal slices. Top, representative experiments.
Recording and stimulation electrodes were placed in stratum radiatum of
the field CA1 of a hippocampal slice. After establishing a stable
baseline, 50 µM of compound 16h or 20 µM of 16a was added to the
perfusion line for the duration indicated by the horizontal bar.
Amplitude and half width of the EPSP were normalized to those of the
baseline responses and plotted against time. Traces taken before and
during drug infusion are shown superimposed. Bottom, summary of drug
effects. Each point represents one perfusion experiment. Percentage
change in EPSP amplitude at the peak of the drug effect was plotted on
the x-axis, and percentage change in response half width
was plotted on the y-axis. Experiments at different drug
concentrations are combined; the concentrations were 16h, 50 to 200 µM; 22a, 100 to 400 µM; 24, 50 to 100 µM; and 16a, 10-50 µM;
only experiments in which responses returned to baseline were selected.
Drug structures are shown in Fig. 5.
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Fig. 8.
Effects of 5'-methyl-BTDs and 5'-ethyl-BTDs on EPSCs
recorded from hippocampal CA1 pyramidal neurons. Whole-cell recordings
were made from stratum pyramidale of field CA1. Synaptic responses were
evoked by stimulation of the Schaffer-commissural fibers. Compounds 16g
and 16h were infused at a concentration of 100 µM, and D1 and 16a
were infused at a concentration of 50 µM. Representative traces taken
before and during drug application are shown at the top. The graphs for
the 5'-methyl-BTDs also show the responses with drug normalized to the
amplitude of the control responses. Holding potential: 70 mV.
Calibration: 100 pA/200 ms. The hatched and clear columns in the bar
graphs show the mean percentage increase (± S.E.M.) in the amplitude
and half width over the predrug response (n = 9 for
D1; n = 6 for all other compounds). The filled bars
show the average preference for amplitude or half-width effect across
all trials with either methyl- or ethyl-BTDs (experiments in which both
amplitude and half width increased by less than 20% were excluded from
this analysis). For this purpose, the ratios
RA (amplitude with drug)/(amplitude before
drug) and RHW (half width with drug)/(half
width before drug) were calculated for each trial and then divided by
each other to obtain a preference ratio
RP = RA/RHW, where
RP = 1 indicates equal increases in
both amplitude and half width. The RP values
from all trials were then averaged and are shown as mean and S.E.M.
(n = 10 for methyl-BTDs, n = 14 for ethyl-BTDs).   , P < 0.001 for the
comparison between the two RP values
(t test).
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The effects of the drugs on synaptic transmission were also examined in
whole-cell recording with voltage-clamp configuration (Fig. 8). EPSCs
recorded from CA1 pyramidal cells revealed a double dissociation
between the drug effects similar to that seen with extracellular
recording. The methyl compound 16h (100 µM) increased response
amplitude by 83% (±36%, n = 6), with only 37%
(±23%) increase in the response half width, and a similar amplitude
preference was observed for compound 16g (97 ± 27%,
n = 6 for amplitude versus 51 ± 20% for half
width). Conversely, the ethyl derivatives D1 and 16a (50 µM) mainly
increased response half width (83 ± 23%, n = 9 for D1 and 178 ± 63%, n = 6 for compound 16a)
with less pronounced effects on amplitude (12 ± 21% and 53 ± 24%, respectively). The difference in the preference ratio
(amplitude-effect/half width-effect) was highly significant between the
two drug groups (Fig. 8, bottom). Thus, according to these
observations, changes in the size of the 5' substituent have
consequences not only for drug potency but also for the way in which
receptor kinetics is modulated by the drug.
Effects on Homomeric Receptors.
The effects of D1 on
recombinant homomeric AMPA receptors were examined in HEK 293 cells
that stably express individual receptor subunits (Hennegriff et al.,
1997
). Measurements of whole-cell currents in such cells typically
reveal small basal responses to glutamate because receptor
desensitization proceeds faster than solution exchange. D1 entirely
blocked this desensitization in GluR3 flop receptors (Fig.
9, left) and those made from other AMPA
receptor subunits (data not shown) and thereby greatly increased the
response. The compound was considerably more effective in this regard
than cyclothiazide, which causes at most a slowing of desensitization
in receptors of the flop variant (Partin et al., 1994
; Arai et al.,
2000
). Dose-response relations for this effect yielded an
EC50 of 38 µM, which is about the same as that obtained with native receptors (Fig. 2), and were characterized by a
steep Hill slope of 3. Whether D1 has a preference among AMPA receptor
subunits was examined in binding tests. Dose-effect curves were
constructed at 0°C at a fixed concentration of
(S)-[3H]AMPA in the absence of
thiocyanate. Under these conditions, D1 increased agonist binding to
all subunits by 50 to 200% (Table 1).
EC50 values were on the order of 5 µM (which
corresponds to the value obtained with brain membranes at 0°C) with
the exception of GluR4 flip, which exhibited an
EC50 of 0.64 µM. It is also apparent, however,
that D1 had a general preference for flip variants with selectivity
ratios between 1.6 (GluR2) and 6 to 8 (GluR4). Most Hill coefficients
were again between 1.5 and 2. Complete dose-effect curves are shown for
GluR4 flop and flip in Fig. 9B. IDRA-21 exhibited a similar flip
preference as did the 5'-methyl-BTD, which lacked the methyl group
normally present at the 3' position (compound 20d; Table 1). IDRA-21
enhanced [3H]AMPA binding to GluR4 flip with an
EC50 of 116 µM, indicating again two orders of
magnitude difference in potencies between it and D1, but the Hill slope
was the same for both compounds. This suggests that flip preference and
a high value of the Hill coefficient are general properties of
benzothiadiazides that contain the IDRA core structure.

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Fig. 9.
Effect of D1 on homomeric recombinant AMPA receptors.
Tests were carried out with HEK 293 cells that stably express homomeric
AMPA receptors made of one of the subunits GluR1 through 4 in either
the "flop" or the "flip" splice variant (Hennegriff et al.,
1997 ). Left, whole-cell current mediated by GluR3 flop receptors during
application of 10 mM glutamate. The drug was present at the indicated
concentration in both glutamate containing solution and background flow
solution; background flow lines were switched at least 30 s before
the first glutamate pulse. The peak current at each drug concentration
was normalized to that measured in the presence of 100 µM D1. Data
(means and S.E.M.) were collected from a total of nine patches; the
number of patches tested at each concentration is shown in parentheses.
The data points were fitted with a four-point logistic equation. The
inset shows traces from a typical experiment in which 3, 10, 100, and
300 µM D1 were applied alternatingly to the same patch; the duration
of glutamate infusion is indicated by the horizontal bar. Right,
effects on [3H]AMPA binding to GluR4 flop and flip
receptors. Binding was measured at 0°C without KSCN at an
(S)-[3H]AMPA concentration of 10 nM in the
case of GluR4 flop and at 50 nM in the case of GluR4 flip; the
(S)-AMPA concentrations were selected to give about 10%
receptor occupancy. Binding at each drug concentration was normalized
to that in the absence of drug, and data from four (D1) and three
experiments (IDRA-21) were averaged (means and S.E.M.). The data were
fitted with four-point logistic equations. Data for other subunits are
summarized in Table 1.
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TABLE 1
Binding affinities for homomeric AMPA receptors (0°C)
The selectivity for AMPA receptor subunits was determined as described
in Fig. 9 by measuring the effect of the drug on the binding of
(S)-[3H]AMPA. Binding incubations were
conducted at 0°C with a fixed concentration of
(S)-[3H]AMPA in the absence of KSCN. They
were terminated by filtration through GF/C glass fiber filters.
(S)-AMPA concentrations were selected between 10 and 50 nM
to obtain approximately 10 to 20% receptor occupancy in the absence of
drug. In each experiment, binding was measured at seven to nine drug
concentrations and fitted with a four-point logistic equation. Binding
constants from the indicated number of experiments were then averaged
(means and S.E.M.). To obtain the binding parameters for the effect of
IDRA-21, binding data from three experiments were first averaged and
then fitted; its effect on GluR4 flop was fitted with an
nHill = 1 because of the smallness of the
binding change.
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Discussion |
Cyclothiazide has been much more effective than IDRA-21 with
regard to AMPA receptor currents in excised patches but not with regard
to enhancing AMPA receptor-mediated field EPSPs. There is also evidence
that the latter compound is behaviorally active while lacking the
antidiuretic and antihypertensive qualities of the former. The goal of
this study has therefore been to develop more potent and functionally
distinct derivatives of IDRA-21 by modifying substituents around its
bicyclic core. The most important findings have been 1) that an alkyl
group at the 5' position greatly potentiates drug-receptor interaction,
whereas modifications at other positions generally were silent or
disabling; 2) that D1 and cyclothiazide differ radically in their
effect on AMPA receptor kinetics despite many commonalities in
structure, potency, and subunit preferences; and 3) that the length of
the 5' substituent not only controls potency but also the manner in
which the compounds affect the waveform of synaptic responses.
Potency estimates for IDRA-21 vary, but its effects on AMPA receptors
in adult brain generally indicate an EC50 of
about 1 mM. Introducing an ethyl group at the 5' position increased the potency by at least one order of magnitude and resulted in a compound with a potency comparable with cyclothiazide. Binding tests and patch-clamp experiments with long glutamate applications produced similar EC50 values for D1 of about 20 to 40 µM. The EC50 obtained with 1-ms glutamate
responses was somewhat higher and probably reflects a lower potency of
the drug at receptors that are not equilibrated with the agonist,
suggesting that drug and agonists mutually increase their affinity.
Insights into structure-activity relations for the alkyl-BTDs are
discussed in greater detail elsewhere (Phillips et al., 2002
). As shown
there, the large gain in potency conferred by the ethyl group in D1 is
close to the theoretical maximum for a hydrophobic substituent of this
size, pointing to the conclusion that a major part of its surface is in
contact with the receptor. This, and the abrupt loss in potency upon
extending the alkyl group further, suggests that the 5'-ethyl group
fits into a pocket of the receptor. Space around the
R3 substituent also seems to be confined insofar
as extension beyond the methyl group at that position or introducing a
second methyl substituent again reduced binding affinity. Larger
substituents were however readily accommodated at the
R2 position. This suggests that the compounds
described here bind to the receptor in such a way that their 3'-5' edge
is in close contact with the surface of the receptor, perhaps facing a groove.
Given that D1 and cyclothiazide belong to the same class of compounds,
it was surprising that their effects on AMPA receptors differ in
several fundamental ways. Deactivation of fast responses to millisecond
glutamate pulses was slowed nearly 20-fold by D1, a value substantially
greater than that reported for most other AMPA receptor modulators
(Arai et al., 1996b
, 2000
), whereas cyclothiazide had almost no effect
under the same test conditions (Arai and Lynch, 1998
). Presumably
related to this, the alkyl-BTDs markedly increased extracellular
synaptic responses in CA1, whereas cyclothiazide is among the least
effective of the AMPA receptor modulators in this measure. Furthermore,
D1 caused an unprecedented increase in the equilibrium binding of
agonists, even in assays containing the chaotropic ion thiocyanate
(SCN
) in which cyclothiazide reliably causes a
10-fold reduction in agonist binding (Hall et al., 1993
; Kessler et
al., 1996
). Lastly, the effects of cyclothiazide in those tests could
be adequately fitted with curves that had Hill slopes near 1, whereas
D1 exhibited a large degree of cooperativity.
These observations suggest that D1 and cyclothiazide have very distinct
effects on AMPA receptor kinetics, but it remains to be determined
which aspects are preferentially targeted. Because we have previously
shown that desensitization contributes minimally to response
deactivation in hippocampal AMPA receptors (Arai and Lynch, 1998
), the
prolongation of fast glutamate responses by D1 indicates that this
drug, unlike cyclothiazide, is also highly effective in slowing channel
closing and/or dissociation of the agonist. Slowing of channel closing
does by itself lead to a reduction in the macroscopic response
desensitization, as seen during long glutamate applications
(Ambros-Ingerson and Lynch, 1993
). However, this is not likely to
account for the complete blocking of desensitization caused by the
alkyl-BTDs, because the methyl-BTDs were as effective as D1 in this
regard despite lower efficacy in prolonging response deactivation.
Also, the loss in paired-pulse inhibition in Fig. 2C indicates that the
alkyl-BTDs, like cyclothiazide, directly block transition into the
desensitized receptor state. In all, it seems that the alkyl-BTDs
differ from cyclothiazide in being able to influence a much broader
range of kinetic properties that encompasses both deactivation and desensitization.
The binding data support the above arguments. Equilibrium binding is
mainly governed by the desensitized receptor states, but the
dissociation constant KD remains a
mathematical function of all kinetic rate constants, including those
for channel gating, as explicitly shown for the five-state receptor
model by Ambros-Ingerson and Lynch (1993)
. Calculations with their
equation indicate that slowing response deactivation will, under most
conditions, increase equilibrium binding affinity, whereas blocking
desensitization will have the opposite effect. Consistent with this,
modulators that markedly slow deactivation have been found to produce
larger increases in agonist binding. Thus, the methyl-BTDs were
moderately effective in slowing the decay of fast responses and
increased binding by less than 100%, whereas D1 had a very strong
effect and produced the largest increases in binding affinity so far obtained with AMPA receptor modulators. Conversely, cyclothiazide has
barely detectable effects on deactivation and generally reduces agonist binding.
It is also clear, however, that a simple scaling of drug effects with
regard to effects on deactivation does not predict the full range of
drug actions on synaptic responses. For example, the balance of effects
on response amplitude versus duration differed substantially between
methyl- and ethyl-BTDs in a manner than was not related in any obvious
way to deactivation or desensitization. Thus, methyl-BTDs were more
effective in enhancing the response amplitude despite having a lesser
influence on response duration. Although it can not be ruled out from
the present data that the drugs influenced other aspects of synaptic
physiology, it seems more likely that the waveform of synaptic
transmission is governed by subtle aspects of AMPA receptor kinetic
that are not yet fully appreciated in their importance but happen to be
affected differentially by minimal variations in drug structure.
Whatever the underlying mechanism, the amplitude-versus-duration
distinction could prove to be of practical significance, because the
compounds predominantly affecting the former variable seemed to be less
likely to cause epileptiform discharges. If so, then methyl-BTDs
may have advantages over their ethyl counterparts in behavioral applications.
A consistent and unusual feature of the drugs described here is the
size of the Hill coefficient. Binding effects of cyclothiazide, ampakines, and other agents are generally governed by Hill slopes near
1 (Arai et al., 1996c
, 2000
). By contrast, D1 effects consistently exhibited Hill coefficients larger than 1, usually on the order of 2, in binding tests and in recordings involving native and recombinant
receptors. Hill slopes significantly higher than unity were found for
all analogs with the possible inclusion of IDRA-21; the significance of
this observation remains unclear. Because AMPA receptors
probably are tetrameric proteins, each functional unit contains four
homologous drug sites in addition to the same number of agonist sites.
It is thus possible that alkyl-BTDs, but not other modulators, invoke
cooperativity between homologous sites on adjacent subunits.
Alternatively, the subunits may have multiple modulatory sites, as
indicated by competition studies with cyclothiazide and ampakines (Arai
et al., 2000
). Possibly, then, those alkyl-BTDs with Hill slopes near 2 bind to two sites with similar affinity, whereas those with smaller
coefficients have various degrees of preference for one of the sites.
The dramatic reduction in the effect of D1 on binding when GYKI was
present raised the possibility of a common site, but further analysis showed that the D1 effect in the presence of saturating GYKI was still
governed by a Hill slope larger than unity. Interactions with GYKI were
also found in an earlier study with ampakines (Arai et al., 2000
), and
it was suggested there that the effect is due to opposing actions at
the level of receptor kinetics rather than competition for a shared
modulatory site.
The functional distinctions discussed above emphasize the importance of
the structural differences between the alkyl-BTDs and cyclothiazide. It
is of interest in this regard that IDRA-type compounds and
cyclothiazide cannot be "connected" through conservative substitutions in the sense that compounds with intermediary structural features have little potency at AMPA receptors. Thus, introducing a
norbornyl group into alkyl-BTDs at R3
(Phillips et al., 2002
) or replacing chloride at
R7 with the sulfonamide group present in
cyclothiazide (Bertolino et al., 1993
) caused a loss in potency. Similarly, several cyclothiazide analogs in which the norbornenyl group
had been removed or replaced by other substituents were inactive at
AMPA receptors (Yamada and Tang, 1993
). It thus seems likely that the
topology of drug-receptor association and the contact points
responsible for bonding are different for IDRA-type compounds and cyclothiazide.
Another interesting aspect of comparison between D1 and cyclothiazide
concerns the selectivity for the AMPA receptor subunits and their
splice variants. Cyclothiazide has a large preference for the flip
variant of all four subunits (Partin et al., 1994
) that critically
depends, however, on the norbornenyl moiety that is present in
cyclothiazide but not other benzothiadiazides. In fact, replacing the
norbornenyl substituent conservatively with a cyclohexyl ring was
sufficient to switch the flip preference to flop at most subunits
(Kessler et al., 2000
). It was therefore quite unanticipated that
IDRA-21 and D1 were again associated with a marked flip preference, at
least at GluR4 subunits, and it suggests that this may be a rather
widespread feature of this class of drugs despite the possibility of
different docking modes mentioned above.
In conclusion, the drugs described here possess properties that differ
in many aspects from those of other AMPA receptor modulators, including
the ampakines, and thus provide new tools to study receptor function.
Other compounds have recently been described that also exhibit greatly
improved potency for the AMPA receptor. Some of them are structurally
very similar to the compounds described here, such as S18986
(Desos et al., 1996
) and the pyridothiadiazines (Pirotte et al., 1998
).
Others do not contain the characteristic bicyclic core but have a
sulfonamide embedded in an elongated molecular structure, such as PEPA
(Sekiguchi et al., 1997
) and LY395153 and related compounds (Ornstein
et al., 2000
), the latter of which exhibited submicromolar
EC50 values in various physiological tests. It
will be of interest to compare these compounds with the alkyl-BTDs in
the paradigms used here, because they have not yet been fully
characterized regarding their effects on the synaptic waveform,
receptor kinetics, or agonist binding.
This work was supported by grants from the Central Research
Committee of Southern Illinois University (201-08; to A.C.A.), the Air
Force Office of Scientific Research (98-1-03317; to G.L.), and
the National Institutes of Health (NS41020 to A.C.A. and NS27600 to
R.C.), and by a Graduate Research Fellowship from DuPont
Pharmaceuticals (to D.P.).