Application of Chemical Biology in the Discovery
of Fatty Acid Amide Hydrolase Inhibitors and
-Secretase Modulator Mechanism of Action
Proteinase 2013
Douglas S. Johnson
Pfizer Worldwide Research and Development
April 15, 2013
Outline
•
Introduction
•
Chemical proteomic technologies for target ID and selectivity profiling
•
Integration of clickable chemical probes with bioorthogonal conjugation of
reporter groups
•
Fatty acid amide hydrolase (FAAH) inhibitors
•
FAAH inhibition to enhance action of endogenous cannabinoids
•
Discovery of PF-04457845: a highly potent and irreversible urea FAAH
inhibitor as a clinical candidate for pain and other Neuroscience indications
•
Clickable analogs to profile selectivity of urea and carbamate FAAH inhibitors
•
Notch-sparing -secretase inhibitors (GSIs) and modulators (GSMs)
•
Aβ42-lowering therapy for the treatment of AD
•
Target and MOA was not known
•
Clickable photoaffinity probes to identify the target(s) and probe binding site
Chemical Proteomic Technologies for Target ID and
Selectivity Profiling
•
Affinity chromatography – noncovalent capture of targets with immobilized
compounds
O
HN
H
N
H
S
O
H
N
N
N
N
Activity-based protein profiling (ABPP) – covalent capture of targets with active
site-directed reactive probes
Cysteine protease activity-based probe
(Nat. Struct. Mol. Biol. 2012, 19, 9)
Serine hydrolase activity-based probe
(Annu. Rev. Biochem. 2008, 77, 383)
•
H
N
N
Biotin-imatinib to pull down GSAP
(Nature, 2010, 467, 95)
Thalidomide linked to FG beads to
identify target of teratogenicity
(Science, 2010, 327, 1345)
•
N
O
NH H
Photoaffinity labeling – covalent capture of targets with photoreactive probes
O
O
HN
H
N
O
OH
H
N
O
O
N
H
H
N
O
H
H
N
O
H
NH
H
S
L646, gamma-secretase photoaffinity probe
(Nature, 2000, 405, 689)
Geoghegan and Johnson, Ann. Rep. Med. Chem. 2010, 45, 345
Clickable Probes and
Bioorthogonal Conjugation Reactions
•
Direct incorporation of bulky reporter groups (i.e. biotin) can often cause a
significant decrease in the affinity with the target or influence the cell penetration
and distribution of the compound
•
Alternatively one can apply chemical proteomic methods with clickable probes (of
covalent inhibitors or photoaffinity probes) to capture protein targets followed by
bioorthogonal conjugation of reporter groups with click chemistry
•
•
Design probe with small clickable handle (usually alkyne or azide)
Introduction of tag by Cu-catalyzed azide alkyne cycloaddition (CuAAC) or Cu-free
strain-promoted azide alkyne cycloaddition (SPAAC)
Probe-labeled protein
CuAAC
(Huisgen, Sharpless & Meldal)
SPAAC
(Bertozzi)
Example of
Dendrogram Showing the ~240 Predicted Human
Serine Hydrolases
The metabolic serine
hydrolases are in blue
The remaining enzymes are
serine proteases:
• chymotrypsin-like enzymes
are in grey
• subtilisin-like enzymes are in
red
• other smaller serine protease
classes are in green
Nat. Rev. Drug Disc. 2012, 11, 52
FAAH Inhibition Results in Elevated Levels of FAAs Including
the Endocannabinoid Anandamide
• The balance between eCB synthesis/release and inactivation determine the extent of eCB accumulation
• FAAH is the principal enzyme responsible for the hydrolysis/inactivation of anandamide
Phospholipids
Biosynthesis
(Ca2+ )
Arachidonic Acid
Hydrolysis
(FAAH)
Cravatt,
Nature, 1996
Anandamide (eCB)
Devane, Science, 1992
(Decreased release of excitatory neurotransmitters
from nociceptive neurons)
membrane-bound intracellular
serine hydrolase of the
amidase signature class
• Pharmacological effects without apparent sideeffects of direct CB1 agonists
• Inhibition of FAAH activates CB receptors
indirectly by boosting the levels of their
endogenous agonists
• Enhances endocannabinoid signal only in
tissues and cells with ongoing synthesis and
degradation of endocannabinoids
The Path from HTS Hit to Clinical Candidate
PF-00103080 (HTS hit)
HTMS IC50 = 434 nM
hFAAH: IC50 (60 min) = 382 nM
rFAAH: IC50 (60 min) = 7020 nM
PF-03604750
hFAAH: kinact/Ki = 598 M-1s-1
rFAAH: kinact/Ki = 99 M-1s-1
HLM t1/2 = 25 min
%F (rat) = 67%
Hot Plate: MED = 100 mg/kg, po
PF-04323845
hFAAH kinact/Ki = 12,600 M-1s-1
rFAAH kinact/Ki = 3,900 M-1s-1
HLM t1/2 = 35 min
clogP = 4.9
CFA: MED = 3-10 mg/kg
PF-04394864
hFAAH kinact/Ki = 21,600 M-1s-1
rFAAH kinact/Ki = 51,100 M-1s-1
HLM t1/2 = 68 min
clogP = 4.6
CFA: MED = 1 mg/kg, po
PF-04161001
hFAAH: kinact/Ki = 9,230
rFAAH: kinact/Ki = 2,140
HLM t1/2 = 53 min
clogP = 6.2
DDI: CYP2D6 IC50 = 290 nM
CFA: MED = 1 mg/kg, po
PF-04457845 (Clinical candidate)
hFAAH kinact/Ki = 40,200 M-1s-1
rFAAH kinact/Ki = 32,300 M-1s-1
HLM t1/2 = 105 min
clogP = 3.9
CFA: MED = 0.1 mg/kg, po
Achieved significant potency improvement while lowering clogP
Johnson, et al, ACS Med. Chem. Lett. 2011, 2, 91
PF-04457845 is an Irreversible FAAH Inhibitor
Overlay of PF-04457845-h/rFAAH
and MAP-rFAAH Crystal Structures
PF-04457845
h kinact/Ki = 40,200 M-1s-1
r kinact/Ki = 32,300 M-1s-1
clogP = 3.9; PSA = 80; MW = 455.4
MDCK/MDR1 Papp: AB = 15.2, BA = 20.1 x 10-6
%F (rat) = 88% (parent, crystalline)
CL = 3.0 mL/min/kg; Vdss = 3.2 L/kg; t1/2 = 14 h
CFA: MED = 0.1 mg/kg, po
Evidence for irreversible inhibition:
• Apparent potency increased with pre-incubation time
• FAAH preincubated with the urea inhibitor did not
recover activity in a rapid dilution experiment
• The mass of the active site peptide containing the
Ser241 following treatment with PF-04457845 and
tryptic digestion corresponded to the benzylidene
piperidine carbamylated adduct
Biochemistry, 2007, 46, 13019
PF-04457845 is covalently attached to the
S241 through a carbamate linkage and binds
in the same region as the arachidonyl chain
of MAP in the acyl-chain binding pocket
MAFP
(methyl arachidonyl fluorophosphonate)
Chemistry & Biology, 2009, 16, 411
Efficacy and Biomarker Modulation for PF-04457845
(FAAH Activity and Anandamide Levels)
N
2
20
1
[PF-04457845] (mg/kg, po)
en
3000
2000
1000
0
Ve
hi
cl
e
0.
00
3
0.
01
0.
03
0.
1
0.
3
1
ro 0
xe
n
3
N
ap
1
0
Ve
hi
cl
0. e
00
3
0.
01
0.
03
0.
1
0.
3
0
4000
Brain
Plasma
JPET, 2011, 338, 114
[PF-04457845] (mg/kg, po)
10
40
5000
3
3
Plasma AEA (pmol/ml)
Brain AEA (pmol/g)
D. PF‐04457845 Exposure
[PF-04457845] (ng/g or ng/ml)
C. Anandamide Levels (mech biomarker)
Brain
Plasma
1
[PF-04457845] (mg/kg, po)
[PF-04457845] (mg/kg, po)
60
ro
x
le
0.
00
3
0.
01
0.
03
0
Ve
hi
c
3
ap 1
ro 0
xe
n
1
0
ap
5
50
N
10
1
* * * *
>90% FAAH
inhibition
appears to
be necessary
for efficacy
100
3
15
* *
Blood Leukocytes
0.
20
Brain
150
1
anti-hyperalgesic effects in CFA
(reduction of mechanical allodynia)
0.
25
B. Residual FAAH Activity (target biomarker)
FAAH Activity (% of Vehicle)
Paw Withdrawal Threshhold (g)
N
a
Ve ive
hi
c
0 . le
00
3
0.
01
0.
03
0.
1
0.
3
A. Efficacy in Rat CFA Inflammatory Pain Model
FAAH Mechanism:
Pros and Cons of Irreversible Inhibitors
• Inhibition of FAAH leads to elevated levels of endogenous N-acyl ethanolamine (NAE)
substrates by up to 10-20-fold
• >90% inhibition of FAAH is necessary to achieve efficacy in animal models of inflammatory
pain
Pros of Irreversible Inhibitors
 Increased biochemical efficiency - Nonequilibrium binding limits the competition with
high endogenous substrate/ligand concentrations
 Potential longer duration of action dependent on the synthesis of new enzyme
 Most efficient strategy when complete inactivation of target is required
Potential Cons
 Potential immunogenicity of protein-adduct leading to an allergic response or drug
hypersensitivity reaction (idiosyncratic) – derisk with low dose compound
 Higher risk if covalent inhibitor lacks specificity (nonspecific covalent binding should be
avoided) – use chemical proteomics to profile proteome-wide selectivity
To minimize the risk associated with developing a covalent inhibitor, we needed to demonstrate:
• high potency to achieve as low a dose as possible
• high selectivity to avoid off target toxicity
D. C. Swinney, Nat. Rev. Drug Disc. 2004, 3, 801
Johnson, Weerapana & Cravatt, Future Med. Chem. 2010, 2, 949
Characterization of the In Vivo Selectivity of
Covalent Inhibitors using CC-ABPP
CC-ABPP
probe
• Mice are administered an alkyne-modified covalent inhibitor at increasing doses.
• The tissue is isolated, homogenized and reacted with an azide-modified reporter tag
(i.e. rhodamine azide for detection).
• Labeled proteins are separated by SDS-PAGE and visualized by in-gel fluorescence.
Alexander and Cravatt, Chemistry & Biology, 2005, 12, 1179
Johnson, Weerapana and Cravatt, Future Med. Chem. 2010, 2, 949
Click Chemistry Probe to Evaluate Selectivity of Ureas
In Vivo
CC-ABPP to directly analyze
the protein targets in vivo
PF-04457845
PF-04457845yne
PF-04457845yne
hFAAH kinact/Ki = 11,900 M-1s-1
Convert to clickable activity-based probe by incorporating an alkyne into the
structure which serves as a bioorthogonal handle to attach a reporter group via CC
JPET, 2011, 338, 114
FAAH
In Vivo Selectivity of Urea vs Carbamate FAAH Inhibitors
by CC-ABPP
PF-04457845yne
SA-57yne
JP104
10 mg/kg
10 mg/kg
FAAH
mouse brain proteome
ACS Chem. Neurosci. 2012, 3, 418
mouse brain proteome
Micah Niphakis and Ben Cravatt (Scripps)
Eric Ballard (Pfizer)
Chemistry & Biology, 2005, 12, 1179
PF-04457845 Human Phase I Data
1 mg dose gives >24 hours of sustained biomarker modulation
8
100
97% FAAH inhibition
95
90
pk1mg
AEA1mg
FAAH1mg
6
85
5
80
4
75
AEA Emax
70
3
65
2
60
1
PK: Cmin
0
55
50
0
24
48
72
Post Dose (hrs)
Br. J. Clin. Pharmacol. 2011, 73, 706
96
120
144
% FAAH Inhibition
Plasma PK or AEA (ng/mL)
7
PF-04457845 Phase II Clinical Trials
www.clinicaltrials.gov
4 mg tablet once daily
Pain, 2012, 153, 1837
PF-04457845 is currently being investigated for other neuroscience indications
Outline
•
Introduction
•
Chemical proteomic technologies for target ID and selectivity profiling
•
Integration of clickable chemical probes with bioorthogonal conjugation of
reporter groups
•
Fatty acid amide hydrolase (FAAH) inhibitors
•
FAAH inhibition to enhance action of endogenous cannabinoids
•
Discovery of PF-04457845: a highly potent and irreversible urea FAAH
inhibitor as a clinical candidate for pain and other Neuroscience indications
•
Clickable analogs to profile selectivity of urea vs. carbamate FAAH inhibitors
•
Notch-sparing -secretase inhibitors (GSIs) and modulators (GSMs)
•
Aβ42-lowering therapy for the treatment of AD
•
Target and MOA not known
•
Clickable photoaffinity probes to identify the target(s) and probe binding site
Class Distribution of Human Proteases
The 16 intramembrane proteases are found in the membranes on the cell
surface, endoplasmic reticulum and mitochondria and are involved in
regulated intramembrane proteolysis
Nat. Rev. Mol. Cell Biol. 2007, 8, 245
Amyloid Hypothesis of Alzheimer’s Disease
sAPP
Fibril assembly
sAPP
Amyloid Plaque
A oligomers
A40
A42
α-CTF
β-CTF
AICD
Amyloid hypothesis
• States that Aβ accumulation is the primary event in AD pathogenesis
• A42 is the earliest and most predominant form deposited
• FAD mutations in APP increase generation of A including A42 (i.e., APP V717I,
V717F)
• FAD mutations in presenilin increase the A42/A40 ratio
• Convergence of genetic and pathological studies has provided support for the amyloid
hypothesis of AD
Hardy and Selkoe, Science, 2002, 297, 353
-Secretase as a Target to Inhibit A Production
-Secretase
A
• Transmembrane aspartyl protease
comprising at least 4 subunits
C99
AICD
AICD
• PS-1 or PS-2 (presenilin)
• Nicastrin (NCT)
• Pen 2 (presenilin enhancer)
• Aph 1a (L or S) or 1b (anterior
pharynx defective)
• Endoproteolysis of PS to NTF and
CTF yields active enzyme (active-site
directed GSI photoprobes played an
important role in characterization)
GSI photoaffinity probe labels PS1-NTF
• Success finding potent brain penetrant inhibitors
• Multiple compounds have been in clinical trials (Lilly, Wyeth, Elan, BMS)
• But safety is an issue
• Many GS substrates emerging, leading to potential for mechanism-based
toxicity (i.e., Notch).
• GS inhibition results in accumulation of the β-CTF of APP
Ann. Neurol. 2009, 66, 48
LY-450139 (semagacestat)
Aβ IC50 = 3-15 nM
Notch1 IC50 = 29 nM
Notch1/Aβ = 2-10
GSIs in Clinical Trials
Ann. Neurol. 2009, 66, 48
LY-450139 (semagacestat)
Aβ IC50 = 3-15 nM
Notch1 IC50 = 29 nM
Notch1/Aβ = 2-10
CF3
Phase III Termination of LY-450139:
• Semagacestat was associated with worsening of
clinical measures of cognition and the ability to perform
activities of daily living to a statistically significantly
greater degree than those treated with placebo.
• It was associated with an increased risk of skin
cancer compared with those who received placebo
Cl
O
H2N
N
O
F
S
O
N
N O
ACS Med. Chem. Lett. 2010, 1, 120
BMS-708163 (phase II)
Aβ IC50 = 0.3 nM
Notch1 IC50 = 58 nM
Notch1/Aβ = 193
• Oral administration of BMS-708163 significantly reduced Aβ40
levels for sustained periods in brain, plasma, and CSF in rats
and dogs
• Good plasma and CSF Aβ lowering reported in human
• 193-fold selectivity against Notch1
• What is the mechanism of these “Notch-sparing” GSIs?
Design photoaffinity probes to interrogate the binding site and
mechanism of action of these “Notch-sparing” GSIs
Clickable Photoprobe Design
CF3
Cl
Cl
O
H2N
O
O
S
N
O
H2N
N
O
S
O
N
F
O
N
BMS-708,163
O
163-BPyne
• Photoreactive group
• Incorporate benzophenone, phenylazide or diazirine photoreactive group to convert the
non-covalent small molecule-protein interaction into a covalent adduct upon UV irradiation
• Clickable handle
• The alkyne provides a click chemistry handle for conjugation of an azide-linked reporter
group (i.e., TAMRA for fluorescence detection or biotin to pull down targets)
• Direct incorporation of bulky reporter groups (i.e. biotin) can often cause a significant
decrease in the affinity with the target or decrease cell permeability
Clickable Photoaffinity Probes to Determine Target of
GSIs and GSMs
BMS-708,163 targets Presenilin and lacks
Notch-sparing activity
BMS-708,163 has been reported to be 193-fold selective for APP over Notch cleavage using a
reporter-based assay that relies on NICD-mediated activation of CBF1, however …
Photolabeling with 163-BPyne
PS1-NTF
Competition:
BMS-708,163
163-BPyne
GSIs
In vitro and cell based assays directly
measuring Aβ and NICD showed only
3-7 fold selectivity for APP over Notch
GSMs
163-BPyne labeling was blocked by the
allosteric and active site-directed GSIs,
but was not effected by GSMs
Cell-free in vitro assay (IC50, nM)
Compound
BMS-708,163
IC50
Aβ40
0.26
IC50
Aβ42
0.35
IC50
NICD
0.84
NICD/
Aβ40
H
N
F
O
3
F
163-BPyne
0.20
0.40
0.61
L458
3
Collaboration with Yueming Li at Sloan-Kettering
(Biochemistry, 2012, 51, 7209)
LY-450139
E2012
O
O
N
N
H
N
Cmpd E
GSM-1
Photophore Walking to Interrogate -Secretase Active Site
Asp
Asp
O
O
H
H
N
O
-O
O
P1’
P3’
H
O
O
H
N
O
L646 (P2)
O
N
H
NH2
JC-8 (P1’)
O
P2
P1
L-685,458
GY-4 (P1)
CS-1
L646
P1
JC‐8
GY‐4
P2
L505
Labeling of PS1-NTF
PNAS, 2009, 106, 20228
L‐685,458 DMSO
L‐685,458 Proposed MOA:
Binding of CS-1 to an allosteric site in
-secretase alters the shape of the
active site such that L646 and GY-4
can not label S1 and S2 pockets.
DMSO
L505 (P3’)
CS-1
BMS-708,163 Exhibits Characteristics of Nonselective GSIs
O
R=
*
BP
R1 = BP
L646
R2 = BP
GY4
R3 = BP
JC8
Photolabeling with
CF3
R4 = BP
L505
L646
GY4
JC8
L505
Cl
PS1-NTF
O
H2N
N
O
S
O
Competition: BMS-163
+
-
+
-
+
-
+
-
N
F
O
N
BMS-708,163
All four TS GSI photoprobes were completely inhibited by
BMS-708,163, a characteristic of non-selective pan-GSIs
Consistent with this profile, doses of BMS-708,163 at 100 mg or above were associated with
higher discontinuation rates due to gastrointestinal adverse events as well as skin-related
adverse events including non-melanoma skin cancer in a phase II clinical trial.
(Arch. Neurol. 2012, 69, 1430)
Biochemistry, 2012, 51, 7209
Attention Shifting to -Secretase Modulators (GSMs) for
“Selective” Inhibition of A42 Production
O
N
N
N
F
O
Imidazole Series
E2012 (Eisai)
Selectively lowers A 42 and A 40
while increasing A 38 and A 37
•
GSMs were discovered when select NSAIDs were found to selectively lower A42 in cell culture
and transgenic mouse models
•
Shift cleavage of APP from longer (A42/ A40) to shorter (i.e. A38) species without changing
overall amount of -CTF cleaved
•
In contrast to GSIs, GSMs do not result in an accumulation of APP C-terminal fragments and do
not broadly inhibit the cleavage of other -secretase substrates that are critical for normal cellular
signaling such as Notch
•
There have been conflicting reports as to target ID and MOA (both APP and -secretase complex
are proposed to contain binding sites for GSMs)
Design photoaffinity probes to determine the target of GSMs and
gain a better understanding of the molecular determinants for GSM action.
Biotinylated Photoprobe of NSAID GSM Flurbiprofen
CO2H
F
Me
CO2H
F
Photoreactive
biotin-tagged
probe
O
NH
Nature, 2008, 453, 925
O
O
Flurizan (Myriad)
IC50 Aβ42 = 134 μM
HN
O
H
N
O
O
•
•
•
NH
O
N
H
2
S
Flurbiprofen photoprobes did not label the core proteins of the -secretase complex, but
instead labeled the substrate APP.
Caution: these are low-affinity probes so nonspecific labeling could be a problem – difficult
to do competition studies at such high concentrations.
In fact, it was recently demonstrated that these NSAID-based GSMs form aggregates at
concentrations >50 μM and bind nonspecifically to Aβ (see Biochemistry, 2011, 50, 10328).
Design of Clickable Photoaffinity Probes for Acid
GSMs
CO2H
Photoreactive
“clickable”
probe
F
N
F
CF3
F
N3
F
GSM-1
A42 IC50 = 211 nM (CHO-APP)
183 nM (He La membranes)
GSM-4
A42 IC50 = 3.8 uM (CHO-APP)
358 nM (HeLa membranes)
GSM-5
A42 IC50 = 672 (CHO-APP)
268 nM (HeLa membranes)
Photoaffinity Labeling with Acid GSM Photoprobe
CO2H
Photoreactive
“clickable”
probe
F
N
F
CF3
F
N3
F
GSM-1
GSM-5
CC with TAMRA-azide and in gel fluorescence
Fluorescence
-
+
-
CC with biotin-azide and PS1-NTF Western blot
250
GSM-5
+
+
-
+
+
GSM-616
-
GSM-1
37
PS1-NTF
DMSO
50
1 µM
GSM-616
75
0.5 µM
GSM-1
+
DMSO
GSM-1 (50 μM)
Coomassie Blue
25
20
PS1-NTF is labeled by GSM-1693 in HeLa membranes
and is competed by 50 µM GSM-1 and GSM-616.
15
ACS Chem. Neurosci. 2011, 2, 705
Proposed Model for the Mechanism of Action of Acid GSMs
Photophore walking approach to
interrogate -secretase active site
S1’
S3’
S2
S1’

M
M
4
D
-1
-1
0
L505
P3’
4 µM
GSM
Allosteric Site
S1
-
S3’
S2
100
SO
JC8
P1’
S1
200

M
GY4
P1
GSM
binding
300
12
L646
P2’
Densitomery (%of DMSO)
GY4 Labeled PS1-NTF
GY4
Active site
shape change
GSM-1
HeLa membranes were labeled with 20 nM of GSI
photoprobe L646, GY4, JC8 or L505 in the presence or
absence of 4 µM GSM-1, followed by streptavidin pull
down and western blot analysis with PS1-NTF antibody.
ACS Chem. Neurosci. 2011, 2, 705
Enhanced
GY4 labeling
Modulation of
Aβ cleavage
GSM binding to PS1 allosterically influences the
S1 subsite within the active site, leading to an
alteration of -secretase cleavage specificity, and
an observed increase in GY4 labeling.
Design of Clickable Photoaffinity Probe of E2012
Photoreactive
“clickable” probe
E2012
A42 IC50 = 106 nM (CHO-APP)
74 nM (HeLa membranes)
E2012-BPyne
A42 IC50 = 374 nM (CHO-APP)
96 nM (HeLa membranes)
Specific Photolabeling of PS1-NTF with E2012-BPyne
E2012
E2012-BPyne
E2012-BPyne labeling in HeLa membranes
(CC with biotin-azide
and Western blot)
E2012-BPyne
E2012-BPyne
Nicastrin
PS1-NTF
E2012
(5 M)
(200 nM)
(200 nM)
+
+
-
2
20
2
50
-
PS1-CTF
PS1-NTF
PS1- E9
APH-1a
E2012-BPyne (µM)
E2012 (µM)
50
37
2
25
1
-
1
10
1
25
E2
01
2
D
M
SO
in
pu
t
PEN-2
Fluorescence
Coomassie
J. Biol. Chem. 2013, 288, 9710
Cross Competition Studies of GSM Photoprobes with
Various GSMs and GSIs to Probe Binding Sites
CO2H
F
N
F
CF3
F
N3
F
1
2
M- MS 458
01
S
2
B
G
E
L
CF3
H2N
O
Cl
O
S
N
O
N
F
E2012
O
N
GSM-1
BMS-708,163
L458
Acid and imidazole GSM photoprobes bind at distinct allosteric sites on PS1-NTF
E2012-BPyne Labeling is Enhanced in the Presence of
the Active-site GSI L458
E2012-BPyne
E2012-BPyne (2 M)
-
E2012
50
L458
10
1.0
10-1
10-2
L679
10-3
10-4
10
M
L458
PS1-NTF
HeLa cell membranes
L679
Dose response of E2012-BPyne
Cooperativity exists between the
-secretase active site and the
E2012 GSM binding site
Nick Pozdnyakov and Heather Murrey
E2012-BPyne Photolabeling in HeLa Cells and Neurons
PS1-NTF
E2012-BPyne
L458
•
•
•
•
Clickable photoprobe E2012-BPyne is cell permeable
Photoaffinity labeling in live cells, then lyse and perform click chemistry
E2012-BPyne labels PS1-NTF in native environment
Labeling is enhanced by L458 in both HeLa cells and cortical neurons
Heather Murrey
E2012-BPyne Preferentially Labels Active PS1-NTF
E2012-BPyne
L4
58
E2
01
2
L4
58
80
in 0 n
pu g
t
L458-BPyne
Full-length PS1
PS1-NTF
•
•
ANP24 cells overexpress PS1, Aph1 and nicastrin but not Pen2 resulting in the
accumulation of full-length PS1
Imidazole GSM E2012 preferentially binds active PS1-NTF over inactive full-length PS1
Chemical Biology Reveals that GSMs and GSIs have
Distinct Binding Sites on Presenilin
Presenilin
E2012
(Imidazole GSM)
L-458
(TSA)
GSM-1
(Acid GSM)
BMS-708,163
(Allosteric GSIs)
Summary
•
Application of click chemistry in the context of existing chemical proteomic
technologies can be very powerful
•
Clickable covalent inhibitors to assess in vitro and in vivo selectivity
•
Discovered piperidine ureas as covalent irreversible inhibitors of FAAH
•
Covalent inhibitors can be readily modified with clickable tags resulting in
activity-based probes (CC-ABPP) that can be used to inventory their on- and
off-targets in complex biological systems
•
The urea FAAH inhibitor PF-04457845 is exquisitely selective for FAAH
•
Clickable GSI and GSM photoaffinity probes for target ID and MOA studies
•
Clickable photoprobe based on BMS-708,163 targets PS1-NTF and has
characteristics of a nonselective pan-GSI
•
Acid and imidazole GSM photoaffinity probes specifically labels PS1-NTF
and have distinct binding sites
•
Active site-directed inhibitor L458 influences the conformation of the E2012
GSM binding site
•
Photophore walking to interrogate the effect of allosteric GSIs and GSMs on
-secretase active site
•
Taken together our chemical biology tools further strengthen our ability to
differentiate GSMs from GSIs
Acknowledgements
FAAH
Pfizer FAAH project team
Cory Stiff
Kay Ahn
Eric Ballard
Ben Cravatt (Scripps)
Eranthie Weerapana (Scripps)
Micah Niphakis (Scripps)
Gamma-secretase
Nick Pozdnyakov
Kelly Bales
Heather Murrey
Eric Ballard
Chris amEnde
Martin Pettersson
Ben Fish
Yueming Li (Sloan-Kettering)
Christina Crump (Sloan-Kettering)
39