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)