1. 10-8 10-6 10-4 10-2 100 102 104
0
50
100
150
[KM409], µM
PercentageofhSERT-mediated3H-SerotoninUptake
WT
K490C
E493D
KM409 inhibits wild-type hSERT but has
an activating effect on hSERT E493D,
therefore KM409 display an intriguing
biphasic activity of stimulating a mutant
while inhibiting WT SERT.
KM409 inhibits wild-type hSERT and
different mutations of hSERT. KM409 has
an interesting effect on hSERT E493D, as
it is activating this mutant of hSERT.
Structural and functional characterization of novel allosteric SERT modulating compounds
Justin Pezick1, Derick Haas1, Sandhya Kortagere2, Ole V Mortensen1
1Department of Pharmacology & Physiology, Drexel University College of Medicine, Philadelphia, PA
2Department of Microbiology & Immunology, Drexel University College of Medicine, Philadelphia, PA
Serotonin transporters (SERTs) are monoamine transporter proteins
that can be found in the central nervous system (CNS). SERT works
by transporting serotonin from the synaptic cleft to the presynaptic
neuron. The actual transport by SERT terminates serotonin
neurotransmission and recycles serotonin through a sodium-
dependent process. Because of the actions of serotonin on the brain,
it is not surprising SERT is a target of several psychoactive drugs,
including psychostimulants and antidepressants. It is believed popular
antidepressants, such as, tricyclic antidepressants and selective
serotonin reuptake inhibitors achieves their therapeutic action by
specifically targeting and inhibiting SERT thereby elevating brain
serotonin levels.
Research performed previously by our lab identified a novel allosteric
binding pocket on human SERT (hSERT). This was achieved through
molecular dynamics simulations and comparative homology modeling
of human and the parasite Schistosoma mansoni monoamine
transporters. Site-directed mutagenesis has validated the functional
significance of this allosteric site and the hybrid structure-based (HSB)
method has identified molecules that target this allosteric site and
display unique transporter modulating activities.
In this study, we wanted to investigate further the structure of the
allosteric pocket. In order to accomplish this objective, we used two of
these allosteric small molecule modulators, KM409 and KM456 that
showed modulating activity of SERT function as determined in
previous experiments. We developed a biotinylation assay that labels
individual amino acids within SERT. This assay will be used to
examine the interaction of KM409 and KM456 with the allosteric
pocket and to study if additional amino acids within SERT interact with
the compounds. Functional serotonin uptake assays were also used
to determine the role of the specific residues in the action of the
compounds.
Development of novel transporter modulators could have therapeutic
potential for the treatment of several mental health disorders.
Developing a modulating compound could be beneficial to those who
do not respond to antidepressants or those who have untreatable
depression.
COS-7 cells and transfections
Wild-type hSERT or single point mutations cDNAs was transfected using COS-7 cells and
Plated on a 96-well plate and incubated for two days
Uptake Assays
Media was removed and cells washed using PBS containing 0.1 mM CaCl2, 1 mM MgCl2, 10 mM
clorgyline, 10 mM pargyline, 100 mM ascorbic acid (PBS-CM)
Compounds were added 10 minutes prior to addition of 5-HT.
3H-Serotonin at 50 nM was added and uptake allowed to proceed for 10 minutes at room temperature
For Michaelis-menten kinetics varying concentration of serotonin was added.
Uptake was terminated by washing with PBS-CM
Radiolabel was quantified using Wallac 1450 MicroBeta liquid scintillation counter.
Data was analyzed with GraphPad Prism and fitted with nonlinear regression
Biotinylation
HEK cells are transfected into a 6-well plate and incubated for 48 hours
Media is removed and cells are washed with PBS containing 0.1 mM CaCl2 and 1 mM MgCl2 (PBS-CM)
Cells are incubated with 1 mM MTSEA biotin in PBS-CM for 10 minutes
Biotin is removed and washed with PBS-CM containing 1 mM DTT for 15 minutes at 4℃
Cells are lysed with TNE lysis buffer (10 mM Tris, 150 mM NaCl, I mM EDTA, 1% Triton) containing
protease inhibitors for 90 minutes at 4℃
Lysed cells are collected and aliquoted into two divisions with one division receiving a 50% slurry of
NeutrAvidin agarose resin (Thermo Scientific)
Beads with cell lysate are left incubating on rotator at 4℃ overnight
The beads are washed with TNE lysis buffer
Sample is finally dissolved in denaturing sample buffer
Total and biotinylated SERT protein is visualized through immunoblotting
Results
A. B.
C.
Structure of a human SERT highlighting the salt bridge (green) forming the extracellular gate,
representative experimental compound (purple), and single point mutations (red) of interest
(A. top view; B. side view; C. labeled point mutations)
N112
I327
Q332
Y568
FIG 1. Serotonin transporter structure
FIG 2. KM456 noncompetitively inhibits wild-type hSERT
and displays partial inhibition compared to citalopram
FIG 5. Developing a biotinylation method to test the interaction of
hSERT modulating compounds KM456 and KM409 with hSERT
WT C109A I327C D328C Q332C Y568C
FIG 4. KM409 affects hSERT function in a biphasic manner
Conclusions & Future Directions
Ø KM456 and KM409 both are allosteric modulators of hSERT.
Ø KM456 is a partial non-competitive inhibitor of hSERT, suggesting
that it acts through an allosteric pocket of hSERT.
Ø KM409 affects hSERT function in a biphasic manner activating
uptake at low concentrations and inhibiting at higher concentrations.
Ø A mutant of the extracellular gate reveals a role of this gate in the
modulating activities of both KM456 and KM409.
Ø Successful development of the biotinylation assay allows for the
ability to test interaction of KM456 and KM409, with different hSERT
residues of interest.
Western blot blotted with a SERT antibody showing totals (T) and biotinylated precipitations (P) of four
different hSERT mutants. C109A is a negative control. No drug was used in this particular assay.
Saturation uptake kinetics
-6 -4 -2 0 2 4
0
50
100
150
log[Drug], µM
PercentageofhSERT-mediated3H-SerotoninUptake
Wild-Type + Citalopram
Wild-Type + KM456
KM456 Dose Response Curve
FIG 3. KM456 action is disrupted in hSERT E493D
compared to wild-type hSERT
KM456 has inhibitory actions on
several different mutations of hSERT.
However, E493D is the most
interesting mutation. KM456 loses its
inhibitory effect when this position is
mutated in hSERT. Suggesting that
this particular residue is important to
the structure of the allosteric binding
pocket.
Introduction
Methods & Materials
u MTSEA biotin labels an extracellular cysteine at residue 109 in wild-type hSERT (WT)
creating a signal
u Through mutagenesis we changed this cysteine into an alanine (C109A)
u Since there is no cysteine to label, we expect that there will be a diminished signal in C109A
u We confirmed the diminished signal in C109A, and we were able to use C109A as
background to create single point mutations with a substituted cysteine
u These single point mutations that were chosen are based on where we believe KM456 and
KM409 bind
u Therefore, we expect KM456 and KM409 should block the biotinylation signal at specific
single point mutations
WT C109A
T P T P T P T P TT PP
Amount of
hSERT
0 5 10 15 20 25
0
50
100
150
[5-HT], µM
PercentageofhSERT-mediated3H-SerotoninUptake
WT
WT + 30 µM KM456
WT: Vmax = 101.8% ± 1.97
Km = 0.63 µM ± 0.053
WT + 30 µM KM456: Vmax = 80.09% ± 3.30
Km = 1.13 µM ± 0.185