Identification and characterization of novel NMDA receptor positive allosteric modulators (PAMs)
David H Hackos Dept. of Neuroscience Genentech, Inc.
Schizophrenia and NMDA receptors
Schizophrenia
• A common disorder - about 1% of general population
• Mechanism and cause is poorly understood
• Antipsychotics treat some symptoms, but show limited efficacy in negative and cognitive symptoms
• NMDA receptor blockers (such as PCP) induce schizophrenia-like symptoms in normal individuals
• Animal models of NMDAR hypofunction exhibit schizophrenia-like phenotypes
• Previous generation NMDAR enhancers, such as GlyT inhibitors, indirectly potentiate NMDARs.
The structure and function of NMDA receptors
Gouaux et al Nature 2014, 511, 191
uHTS screen for NMDA receptor PAMs
GluN1/GluN2A-expressing 293 cells
Culture in the presence of 1mM ketamine
Induce with Dox
Wash cells to remove ketamine
Plate cells in acidic media (pH 5.5)
Hamamatzu FDSS
Load cells with calcium indicator dye
uHTS screen for NMDA receptor PAMs
Assay started when neutralization buffer is added (saturating Gly and EC30 Glu)
uHTS screen for NMDA receptor PAMs
uHTS screen for NMDA receptor PAMs
2.5 million compounds screened 7 verified hits
GNE-3476
Medicinal chemistry efforts led to drug-like compounds
GNE-3476 GNE-3419
GNE-7728 GNE-6901
GNE-6901
Example PAM discovered by the Genentech NR2A PAM project team
GNE-6901 slightly shifts Glu potency with no effect on Gly potency
Different impacts of related NR2A PAMs
GNE-8324 slows deactivation by increasing glutamate potency
GNE-8016
GluN2A 1.2 uM
GNE-9178
GluN2A 0.44 uM
GNE-4208
GluN2A 0.09 uM
[GNE-8016] = 10 uM
[GNE-9178] = 10 uM [GNE-4208] = 10 uM
Phenyl Pyrazole Aniline Ether
GNE-6901 GluN2A 0.38 μM
Deactivation increases with substitution on the right-hand side
[GNE-6192] = 100 uM
[Glu] = 100 uM
[Gly] = 50 uM
GluN2A GluN1
Structure of the binding site within the ligand-binding domain (LBD)
GluN2A
GluN1
Compounds of this series are highly selective for GluN2A
A single residue accounts for the NR2A selectivity of GNE-6901
H780
L780
P527P527
P532P532T759
T758
V 783
Y535Y535
E530E530
R755
I514
K531
V526
F528
F
1 3 10 30
Glu:
GN E-6901:
WT GluN2A
GluN2A V783F
WT GluN2B
GluN2B F784V
GluN2A WT
GluN2A V783F
GluN2B WT
GluN2B F784V
0
100
200
300
400
500
600
700
800
900
0.1 1 10 100
% 3
00 G
lu o
nly
resp
on
se
GNE-6901 (mM)
G H
H780
L780
P527P527
P532P532T759
T758
V 783
Y535Y535
E530E530
R755
I514
K531
V526
F528
F
1 3 10 30
Glu:
GN E-6901:
WT GluN2A
GluN2A V783F
WT GluN2B
GluN2B F784V
GluN2A WT
GluN2A V783F
GluN2B WT
GluN2B F784V
0
100
200
300
400
500
600
700
800
900
0.1 1 10 100
% 3
00 G
lu o
nly
resp
on
se
GNE-6901 (mM)
G H
GluN2A V783 = GluN2B F784 (larger side chain)
Do these GluN2A selective PAMs potentiate native NMDA receptors?
GNE-6901 potentiates NR2A-containing NMDA receptors in hippocampal slices
0
50
100
150
200
250
0 5 10 15 20 25
WT
GluN2A KO
0
50
100
150
200
250
0 5 10 15 20 25
GNE-6901 GNE-6901
time (min)
EP
SC
pe
ak (
% b
aselin
e)
EP
SC
are
a (
% b
aselin
e)
WT
GluN2A KO
time (min)
Do PAMs with differential effects on NR2A have differential effects on physiology?
NMDA receptor dependent long term potentiation
HFS HFS
Time (min)
100
120
140
160
180
200
220
0 10 20 30 40 50 60 70
GNE-8324
Intact inhibition
EP
SP
slo
pe
(%
ba
se
line
)
0 10 20 30 40 50 60 70
EP
SP
slo
pe
(%
ba
se
line
)
0 10 20 30 40 50 60 70
Time (min)
EP
SP
slo
pe
(%
ba
se
line
)
DMSOGNE-6901
DMSO
GNE-6901GNE-8324
100
120
140
160
180
200
220
100
120
140
160
180
200
220
GNE-6901 enhances LTP whereas GNE-8324 inhibits LTP
Jesse Hanson, David Hackos
Acknowledgements
Structural Biology
Patrick Lupardus
Heidi Ackerly Wallweber
Christopher Koth
Protein Production
Baculovirus Expression
Group
Legal
Shannon Chi
Robert Hall
Neurobiology
Jesse Hanson
Yelin Chen
Qiang Zhou
Kimberly Scearce-Levie
Morgan Sheng
Biochemical and Cellular
Pharmacology
James Herrington
Paul Reynen
Saundra Clausen
Amy Gustafson
Peter Thana
Yichin Liu
Chemistry
Matt Volgraf
Cuong Ly
Elisia Villemure
Richard Pastor
Allen Jiang (PH)
Po-wai Yuen (PH)
Mingcui Liu (PH)
Xifeng Luo (PH)
Compchem
Ben Sellers
Guosheng Wu (PH)
Aijun Lu (PH)
IBENS, France
Teddy Grand
Pierre Paoletti