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