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Application of Chemical Biology in the Discovery of Fatty Acid Amide Hydrolase Inhibitors and-Secretase Modulator Mechanism of Action
Proteinase 2013
Douglas S. JohnsonPfizer 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
• Activity-based protein profiling (ABPP) – covalent capture of targets with active site-directed reactive probes
• Photoaffinity labeling – covalent capture of targets with photoreactive probes
HN
HN
NH
HN
O
O
OH
O
O
O
HN
OS
NHHN
O
HH
H
L646, gamma-secretase photoaffinity probe(Nature, 2000, 405, 689)
Serine hydrolase activity-based probe(Annu. Rev. Biochem. 2008, 77, 383)
Thalidomide linked to FG beads to identify target of teratogenicity
(Science, 2010, 327, 1345)
N
N
N
HN
HN
O
NN
NH
O
S
NH
HN
OH
H
Biotin-imatinib to pull down GSAP(Nature, 2010, 467, 95)
Geoghegan and Johnson, Ann. Rep. Med. Chem. 2010, 45, 345
Cysteine protease activity-based probe(Nat. Struct. Mol. Biol. 2012, 19, 9)
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 Example of
(Bertozzi)
(Huisgen, Sharpless & Meldal)
SPAAC
CuAAC
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
Anandamide (eCB)
Biosynthesis(Ca2+)
Hydrolysis(FAAH)
Phospholipids Arachidonic Acid
• The balance between eCB synthesis/release and inactivation determine the extent of eCB accumulation
• Pharmacological effects without apparent side-effects 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
(Decreased release of excitatory neurotransmitters from nociceptive neurons)
membrane-bound intracellular serine hydrolase of the amidase signature class
• FAAH is the principal enzyme responsible for the hydrolysis/inactivation of anandamide
Cravatt, Nature, 1996
Devane, Science, 1992
The Path from HTS Hit to Clinical Candidate
PF-03604750hFAAH: 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-00103080 (HTS hit)HTMS IC50 = 434 nMhFAAH: IC50 (60 min) = 382 nMrFAAH: IC50 (60 min) = 7020 nM
PF-04457845 (Clinical candidate)hFAAH kinact/Ki = 40,200 M-1s-1
rFAAH kinact/Ki = 32,300 M-1s-1
HLM t1/2 = 105 minclogP = 3.9CFA: MED = 0.1 mg/kg, po
PF-04161001hFAAH: kinact/Ki = 9,230 rFAAH: kinact/Ki = 2,140 HLM t1/2 = 53 minclogP = 6.2DDI: CYP2D6 IC50 = 290 nMCFA: MED = 1 mg/kg, po
PF-04323845hFAAH kinact/Ki = 12,600 M-1s-1
rFAAH kinact/Ki = 3,900 M-1s-1
HLM t1/2 = 35 minclogP = 4.9 CFA: MED = 3-10 mg/kg
PF-04394864hFAAH kinact/Ki = 21,600 M-1s-1
rFAAH kinact/Ki = 51,100 M-1s-1
HLM t1/2 = 68 minclogP = 4.6CFA: MED = 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
PF-04457845h 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 hCFA: MED = 0.1 mg/kg, po
Evidence for irreversible inhibition:
MAFP(methyl arachidonyl fluorophosphonate)
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
Overlay of PF-04457845-h/rFAAH and MAP-rFAAH Crystal Structures
• 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
Chemistry & Biology, 2009, 16, 411Biochemistry, 2007, 46, 13019
Efficacy and Biomarker Modulation for PF-04457845(FAAH Activity and Anandamide Levels)
Vehicl
e0.0
03 0.01
0.03 0.1 0.3 1
Napro
xen
0
50
100
150Blood LeukocytesBrain
[PF-04457845] (mg/kg, po)
FAAH
Act
ivity
(% o
f Veh
icle
)
B. Residual FAAH Activity (target biomarker)
Vehicl
e0.0
03 0.01
0.03 0.1 0.3 1 3 10
Napro
xen
0
20
40
60
0
1
2
3PlasmaBrain
[PF-04457845] (mg/kg, po)
Bra
in A
EA (p
mol
/g)
Plasma AEA (pm
ol/ml)
Vehicl
e0.0
03 0.01
0.03 0.1 0.3 1 3 10
0
1000
2000
3000
4000
5000
PlasmaBrain
[PF-04457845] (mg/kg, po)
[PF-
0445
7845
] (ng
/g o
r ng
/ml)
C. Anandamide Levels (mech biomarker) D. PF‐04457845 Exposure
A. Efficacy in Rat CFA Inflammatory Pain Model
Naive
Vehicl
e0.0
03 0.01
0.03 0.1 0.3 1 3 10
Napro
xen
0
5
10
15
20
25
[PF-04457845] (mg/kg, po)
***** *
Paw
With
draw
al T
hres
hhol
d (g
)
>90% FAAH inhibition appears to be necessary for efficacy
JPET, 2011, 338, 114
anti-hyperalgesic effects in CFA (reduction of mechanical allodynia)
• 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
FAAH Mechanism: Pros and Cons of Irreversible Inhibitors
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
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
D. C. Swinney, Nat. Rev. Drug Disc. 2004, 3, 801Johnson, Weerapana & Cravatt, Future Med. Chem. 2010, 2, 949
Characterization of the In Vivo Selectivity of Covalent Inhibitors using CC-ABPP
• 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, 1179Johnson, Weerapana and Cravatt, Future Med. Chem. 2010, 2, 949
CC-ABPP probe
Click Chemistry Probe to Evaluate Selectivity of Ureas In Vivo
PF-04457845 PF-04457845ynehFAAH kinact/Ki = 11,900 M-1s-1
FAAH
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
CC-ABPP to directly analyze the protein targets in vivo
JPET, 2011, 338, 114
PF-04457845yne
In Vivo Selectivity of Urea vs Carbamate FAAH Inhibitors by CC-ABPP
PF-04457845yne JP104SA-57yne
10 mg/kg
FAAH
10 mg/kg
Micah Niphakis and Ben Cravatt (Scripps)Eric Ballard (Pfizer)
mouse brain proteome mouse brain proteome
ACS Chem. Neurosci. 2012, 3, 418 Chemistry & Biology, 2005, 12, 1179
PF-04457845 Human Phase I Data
Br. J. Clin. Pharmacol. 2011, 73, 706
0
1
2
3
4
5
6
7
8
0 24 48 72 96 120 144
Post Dose (hrs)
Plas
ma
PK o
r AEA
(ng/
mL)
50
55
60
65
70
75
80
85
90
95
100
% F
AA
H In
hibi
tion
pk1mgAEA1mgFAAH1mg
97% FAAH inhibition
AEA Emax
PK: Cmin
1 mg dose gives >24 hours of sustained biomarker modulation 1 mg dose gives >24 hours of sustained biomarker modulation
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
Hardy and Selkoe, Science, 2002, 297, 353
A40A42
Fibril assembly
Amyloid PlaqueA oligomers
sAPPsAPP
β-CTFα-CTFAICD
Amyloid hypothesis• States that Aβ accumulation is the primary event in AD pathogenesis • A42 is the earliest and most predominant form deposited• FAD mutations in APP increase generation of A including A42 (i.e., APP V717I,
V717F)• FAD mutations in presenilin increase the A42/A40 ratio• Convergence of genetic and pathological studies has provided support for the amyloid
hypothesis of AD
-Secretase as a Target to Inhibit A Production
AICD
A
AICD
C99
LY-450139 (semagacestat) Aβ IC50 = 3-15 nM
Notch1 IC50 = 29 nMNotch1/Aβ = 2-10
Ann. Neurol. 2009, 66, 48
• 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
• Transmembrane aspartyl protease comprising at least 4 subunits
• 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)
-Secretase
GSI photoaffinity probe labels PS1-NTF
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
LY-450139 (semagacestat) Aβ IC50 = 3-15 nMNotch1 IC50 = 29 nMNotch1/Aβ = 2-10
Ann. Neurol. 2009, 66, 48
GSIs in Clinical Trials
BMS-708163 (phase II)Aβ IC50 = 0.3 nMNotch1 IC50 = 58 nMNotch1/Aβ = 193
ACS Med. Chem. Lett. 2010, 1, 120
H2N
ON
CF3
SO
OCl
FN O
N
• 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
H2N
ON
SO
OCl
O
163-BPyneBMS-708,163
H2N
ON
CF3
SO
OCl
NO
NF
• 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
163-BPyneBMS-708,163
Compound IC50Aβ40
IC50Aβ42
IC50NICD
NICD/Aβ40
BMS-708,163 0.26 0.35 0.84 3
163-BPyne 0.20 0.40 0.61 3
Cell-free in vitro assay (IC50, nM)
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 …
In vitro and cell based assays directly measuring Aβ and NICD showed only 3-7 fold selectivity for APP over Notch
Collaboration with Yueming Li at Sloan-Kettering(Biochemistry, 2012, 51, 7209)
PS1-NTF
Photolabeling with 163-BPyne
Competition:
GSIs GSMs163-BPyne labeling was blocked by the allosteric and active site-directed GSIs, but was not effected by GSMs
GSM-1E2012
L458
LY-450139
Cmpd E
HN
NH
NO
ONO
F
F
Photophore Walking to Interrogate -Secretase Active Site
DMSO
L‐68
5,45
8 L646
GY‐4
P1
P2
CS-1
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.
Labeling of PS1-NTFPNAS, 2009, 106, 20228
GY-4 (P1)
HN
HN
NH
NH2O
O
O
O
O
O
H
OOH
O
AspAsp
O-
L-685,458
P1’ P3’
P1
P2
L646 (P2)
JC-8 (P1’)
L505 (P3’)
L505
JC‐8
DMSO
L‐68
5,45
8
CS-1
BMS-708,163 Exhibits Characteristics of Nonselective GSIs
All four TS GSI photoprobes were completely inhibited by BMS-708,163, a characteristic of non-selective pan-GSIs
H2N
ON
CF3
SO
O
Cl
NO
NF
BMS-708,163
PS1-NTF
L646 L505GY4 JC8
+ - + -+ - + -
Photolabeling with
Competition: BMS-163
O
*R3 = BPJC8
R =
R1 = BPL646
R2 = BPGY4
R4 = BPL505
BP
Biochemistry, 2012, 51, 7209
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)
NO
N
N
O
F
Imidazole SeriesE2012 (Eisai)
Selectively lowers A 42 and A 40while increasing A 38 and A 37
• GSMs were discovered when select NSAIDs were found to selectively lower A42 in cell culture and transgenic mouse models
• Shift cleavage of APP from longer (A42/ A40) to shorter (i.e. A38) 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.
Attention Shifting to -Secretase Modulators (GSMs) for “Selective” Inhibition of A42 Production
Biotinylated Photoprobe of NSAID GSM Flurbiprofen
• 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).
CO2H
F
NHO
O
O
OHN
ONH
OHN NH
S
O
2
F
Me CO2H
Flurizan (Myriad)IC50 Aβ42 = 134 μM
Photoreactive biotin-tagged
probe Nature, 2008, 453, 925
Design of Clickable Photoaffinity Probes for Acid GSMs
GSM-1A42 IC50 = 211 nM (CHO-APP)183 nM (He La membranes)
GSM-4A42 IC50 = 3.8 uM (CHO-APP)358 nM (HeLa membranes)
N
CO2H
CF3
FF
FF
N3
GSM-5A42 IC50 = 672 (CHO-APP)268 nM (HeLa membranes)
Photoreactive “clickable”
probe
Photoaffinity Labeling with Acid GSM Photoprobe
GSM-1
Photoreactive “clickable”
probe N
CO2H
CF3
FF
FF
N3
GSM-5
Fluorescence Coomassie Blue
25
37
50
75
20
15
250
+ - + -CC with TAMRA-azide and in gel fluorescence
GSM-1 (50 μM)
PS1-NTF is labeled by GSM-1693 in HeLa membranes and is competed by 50 µM GSM-1 and GSM-616.
- + + - + +
GSM
-1
GSM
-616
DM
SO
GSM
-1
GSM
-616
DM
SO
0.5 µM 1 µM
PS1-NTF
GSM-5
CC with biotin-azide and PS1-NTF Western blot
ACS Chem. Neurosci. 2011, 2, 705
Proposed Model for the Mechanism of Action of Acid GSMs
DMSO
M - 1
4
M - 1
12
0
1 0 0
2 0 0
3 0 0G
Y4
Labe
led
PS1
-NTF
Den
sito
mer
y (%
of D
MSO
)
JC8P1’
L646 P2’
L505 P3’
GY4 P1
4 µM - GSM-1
GSMAllosteric Site
GSMAllosteric Site
S1
S2
S1’ S3’
S1
S2
S1’ S3’
GSM binding
Enhanced GY4 labeling
Active siteshape change
Modulation of Aβ cleavageHeLa 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.
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.
ACS Chem. Neurosci. 2011, 2, 705
Photophore walking approach to interrogate -secretase active site
GY4
Design of Clickable Photoaffinity Probe of E2012
E2012A42 IC50 = 106 nM (CHO-APP)
74 nM (HeLa membranes) E2012-BPyneA42 IC50 = 374 nM (CHO-APP)
96 nM (HeLa membranes)
Photoreactive “clickable” probe
Specific Photolabeling of PS1-NTF with E2012-BPyne
J. Biol. Chem. 2013, 288, 9710
E2012 E2012-BPyne
25
3750
PS1- E9
Fluorescence Coomassie
+ -
E2012-BPyne(200 nM)
E2012(5 M)
+ -
E2012-BPyne(200 nM)
Nicastrin
PS1-NTF
PS1-CTF
APH-1a
PEN-2
input
DMSOE20
12
2 2 2 1 1 1 - 20 50 - 10 25
PS1-NTF
E2012-BPyne labeling in HeLa membranes(CC with biotin-azide and Western blot)
E2012-BPyne (µM)
E2012 (µM)
Cross Competition Studies of GSM Photoprobes with Various GSMs and GSIs to Probe Binding Sites
L458
H2N
ON
CF3
SO
O
Cl
NO
NF
BMS-708,163
Acid and imidazole GSM photoprobes bind at distinct allosteric sites on PS1-NTF
N
CO2H
CF3
FF
FF
N3
GSM-1E2012
L458E2012
GSM-1BMS
E2012-BPyne Labeling is Enhanced in the Presence of the Active-site GSI L458
L458
HeLa cell membranes
E2012-BPyne (2 M)
E2012 L458
10 1.0 10-1 10-2 10-3 10 M50
L679
10-4
-
PS1-NTF
E2012-BPyne
Nick Pozdnyakov and Heather Murrey
Cooperativity exists between the -secretase active site and the E2012 GSM binding site
Dose response of E2012-BPyne
L679
E2012-BPyne Photolabeling in HeLa Cells and Neurons
Heather Murrey
• 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
PS1-NTF
L458E2012-BPyne
800 ng
input
E2012
L458
L458
E2012-BPyne Preferentially Labels Active 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
E2012-BPyne
Full-length PS1
PS1-NTF
L458-BPyne
Chemical Biology Reveals that GSMs and GSIs have Distinct Binding Sites on Presenilin
E2012(Imidazole GSM)
GSM-1(Acid GSM)
BMS-708,163(Allosteric GSIs)
L-458 (TSA)
Presenilin
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
39
FAAHPfizer FAAH project teamCory StiffKay AhnEric Ballard
Ben Cravatt (Scripps)Eranthie Weerapana (Scripps)Micah Niphakis (Scripps)
Gamma-secretaseNick PozdnyakovKelly Bales Heather MurreyEric BallardChris amEndeMartin PetterssonBen Fish
Yueming Li (Sloan-Kettering)Christina Crump (Sloan-Kettering)