EnPlex: a high-throughput method for family- wide profiling of acvity

Daniel A. Bachovchin November 5, 2014 Potency typically drives drug discovery, selecvity follows

Traditonal drug discovery representatve compound ? ? ? ? ? ? ? ? ? ? High-throughput compound library ? ? ? ? ? ? selectvity profiling? potency selectvity evaluate potency evaluate potency against target against many targets

hits ? ? ? ? ?

choose selectve hit, optmize potency and optmize potency and (some) selectvity Beter selectvity selectvity profiling methods lead ? are needed optmize potency, (some) selectvity, and PK/PD more efficient process? clinical candidate ? more selectve inhibitors? Selecvity determinaon is difficult due to the number and similarity of serine

Major Human Enzyme Serine hydrolases Families

700

600

500

400 300 • Involved in clong, neural 200 signaling, inflammaon, cancer

Number of Members 100 0 • Currently 8 are targeted by 14 drugs

• Majority are uncharacterized

The ideal soluon? A high-throughput, mulplexed enzyme acvity assay

1. Selecvity profiling of drugs and chemical probes

2. Inhibitor discovery based on both potency and selecvity

Acvity-based protein profiling (ABPP): a general method to assay enzyme acvity

activity-based probe fluorophosphonate (FP)-biotin

O O P reactive F O NH tag OEt group O S N H HN NH O

B: O H O P P O F O OEt OEt Tag Tag

active serine probe-labeled serine hydrolase

Evans & Crava, Chem Rev, 2006 Compeve ABPP can be used for inhibitor discovery

Inhibitor ABPP probe kDa 75- < SDS-PAGE 50-

37- <

< Proteome Inhibitor-treated Probe-labeled 25- proteome proteome In-gel fluorescence scanning

Avidin

N m/z C N N C N C LC-MS/MS N On-Bead C C m/z

Identification Peptides and Quantification Evans & Crava, Chem Rev, 2006 The advantages and limitaons of ABPP for inhibitor discovery

Advantages • Does not require a specific substrate assay • Can simultaneously evaluate mulple enzymes

Limitaons • Typically assays only a small number of enzymes • Laborious detecon methods severely limit throughput

Comparing ABPP screening plaorms (for serine hydrolases)

100,000 fluorescence polarizaon 10,000 new 1,000 technology?

100 SDS-PAGE

10

number,of,compounds mass spectrometry 1 0 50 100 150 200 250 number,of,enzymes human serine hydrolase family

Is a possible to simultaneously screen many enzymes in high-throughput? EnPlex: A mulplexed, bead-based plaorm for screening

Bachovchin et al. Nat Chem Biol, 2014 EnPlex is sensive and robust assay for inhibitor discovery

Extraordinary Compability with Works in a sensivity: many enzymes: compeve format:

1.0 PME1 (625 pM) y t i PME1 (125 pM) v i t c A

1

E 0.5 M

P IC50 = 12 nM

0.0 1 10 100 1000 10000 [ABL127] nM

Requires 8,000-fold Catalycally dead Accurate IC50 less protein than enzymes give no determinaon fluopol-ABPP signal Assembly of a diverse 97 human serine hydrolase panel DPP4 Acetylcholinesterase

Purified, acve enzymes

Thrombin Uses of a high-throughput, mulplexed enzyme acvity assay

1. Selecvity profiling of drugs and chemical probes

2. Inhibitor discovery based on both potency and selecvity

Representave EnPlex data of known inhibitors

Enzymes

IC50 (nM) IC values < 10 50 10-100 100-1,000 1,000-10,000 10,000-33,300 >33,300 First family-wide selecvity profiling of commonly used inhibitors

Thrombin inhibitors

HCV protease DPP4 inhibitors inhibitors FAAH/MGLL inhibitors CELA1 and PRCP are previously unknown off- targets of telaprevir

CELA1 PRCP . Boc Tel.

O H N O O O O NH O O N H NH2 N N H H H HN N N N N H O O O N O H Telaprevir Boceprevir (Incivek) (Victrelis) Telaprevir is associated with a severe rash that is not currently understood

• Telaprevir received a Black Box Warning from the FDA in December 2012

• PRCP and CELA1 possible mediators?

-CELA1 is only expressed in skin, funcon is poorly understood

-PRCP globally expressed, regulates several bioacve pepdes

Roujeau et al. JAMA Dermatol, 2013 Telaprevir-PRCP interacon underscores the value of family-wide profiling PRCP: α/β hydrolase fold, cleaves aer proline

NS3 protease and CELAs: -like folds, cleave aer hydrophobic residues Bortezomib highlights comprehensiveness of EnPlex compared to exisng methods

Bortezomib, first-in-class proteasome inhibitor approved for mulple myeloma

% Inhibition

0 50 100 n D 1 1 2 3 n 2 B 5 B B a 1 0 1 S11 1 bi 7 2 1 n 1 2 2 1 2 1 2 4 4 6 14 1 2 4 S H1 H1 1 1 1 A 1 G B A 9 D E m 8 1 L A H B L 1 H P E G T 4 7 8 9 C H 2 5 A E 4 L 1 2 5 LA LA 1 2 H T 1 I a P U o P a E A E C HD G r FA FA M 1 N F A D E II R BP L SS S A a P D L G LA h TS F T P P A A B AM108B L M NL V MPR R RC YP YP A P I ABHD1 ABHD1 AB ABHD ABHD ACH ACOT ACOT ACOT AFMI AOA APE BCH C1 C1 CAT CE CELA CES CES CES4 CES C CMA Chymotrypsi CPV C DDHD DPP DPP DPP DPP ELAN ES FAA FAM108A F F F Fx FXI GZM GZ H HTRA HTRA HTRA IAH KLK KLK KLKB LACT LC LIP LIP L LIP LIP L L L LYPLAL MGL O OVCA P P PCSK PLA2 PLA2G4 PLA2G P PLA PL P PNLIPRP PNPL P PPT PPT P PRE PREP PRO PRSS2 P QRSL R SCPEP SIA S T T Trypsi bortezomib

• Previous studies discovered 6 targets • EnPlex found those 6, plus 7 addional targets Uses of a high-throughput, mulplexed enzyme acvity assay

1. Selecvity profiling of drugs and chemical probes

2. Inhibitor discovery based on both potency and selecvity

Inhibitor discovery from a boronic acid library

O Y OH Boronic acids drive potency, X N B H but achieving selecvity is OH challenging ~600 boronic acids DPP4 RBBP9 APEH all other enzymes 33 µM 3.3 µM 100 100 n n o o i i t t i i b b i i nh nh I I t t

n 50 n 50 ce ce r r e e P P Inhibion (%) Inhibion (%)

0 0 0 100 200 300 0 100 200 300 compounds compounds Examples of lead inhibitors: APEH and RBBP9

APEH RBBP9 5 10 O 9 43 132 40 N 4 NH 8 22 s s t t 7

e B 3 N O OH e 33 244

g 6 g 107 OH 5 ar 2 ar 99 51

T 4 89 Py(D)Ala-boroPro T # # 3 280 217 selectivity 1 226 2 60 36 1 0 0 25 50 75 100 50 75 100 % APEH Inhibition % RBBP9 inhibition

potency n D 2 3 n A 11 2 B B 5 B B e rypsi a 1 0 1 t 1 bi 7 2 1 n 1 2 2 1 2 1 2 4 4 6 1 2 4 SS 1 H1 H1 1 1 1 G B A A 9 D E A A 8 L 108 A H B L R H P E G T 4 7 8 9 C H 2 5 A 4 E L T 1 2 5 L L 2 H T 1 I a P U P a A E RA RA RA C G N F A S D E R L S A a P D L G LA T T T T F L E AM108 YP YP P ABHD1 ABHD1 ABHD14 ABHD ABHD ACH ACOT ACOT ACOT AFMI AOA APE BCH C1 C1 CAT C CEL1 CES CES CES4 CES C Chymas Chymo CPV C DDHD DPP DPP DPP DPP ELAN ES FAA FAM F FA FVII Fx FXI GZM GZM HP H H H IAH KLK KLK KLKB LAC LC LIP LIP LIP LIP LIP L L L LYPLAL MGL O OVCA PAFA PAFA PCSK PLA2 PLA2G4 PLA2G P PLA PL PME- PNL PNLIPRP PNPLA PPT PRC PRE PREP PRO PRSS2 PRSS QRSL RBBP SCPEP SIA ST1 Throm TMP Trypsi 226 Glu-boroSar thioxamide (ARI-2408): A remarkably selecve DPP4 inhibitor

5 HO O 4 s t

e 3 OH g

ar 2 N B T H N OH

# 2 1 S 0 Glu-boroSar thioxoamide 90 95 100 ARI-2408 % DPP4 Inhibition

% Inhibition

0 50 100 n D 2 3 n A 11 2 B B 5 B B rypsi 1 0 1 t 1 bi 4A 7 2 A A 1 n 1 2 B 2 1 2 1 2 4 4 6 2 4 SS H1 H1 1 1 2 1 G B G A A 9 D E A A m 8 1 L 108 A H B L 1 R H P E G T 4 7 8 9 C H 2 5 2 A 4 E L T 1 2 5 L L 2 H T 1 I a P U o P a A E RA RA C G r N F A S D E R A L SS S A a P D L G LA T T h T F L P L AM108 R RC YP YP A P I ABHD1 ABHD1 ABHD14 ABHD ABHD ACH ACOT ACOT ACOT AFMI AOA APE BCH C1 C1 CAT CE CEL1 CELA2 CELA3 CELA3 CES CES CES4 CES C CMA Chymo CPV C DDHD DPP DPP DPP DPP ELAN ES FAA FAM F F FVII Fx FXI GZM GZM HP H H IAH KLK KLK KLKB LAC LC LIP LIP L LIP LIP L L L LYPLAL MGL O OVCA PAFA PAFA PCSK PCSK PLA2 P PLA2G P PLA PL PME PNL PNLIPRP PNPLA PPT P PRE PREP PRO PRSS2 P QRSL RBBP SCPEP SIA ST1 T TMP Trypsi Glu-boroSar thioxoamide

Demonstrates boronic acids can be selecve inhibitors ARI-2408 vs. ARI-2243: Similar in efficacy, but dramacally different in safety

5 HO O HO OH 4 S B s t

e 3 OH N g ar 2 N B T H N OH

# 2 NH2 1 S Ala-boroPro thioxoamide 0 Glu-boroSar thioxoamide ARI-2243 90 95 100 ARI-2408 % DPP4 Inhibition

Similar efficacy in OGTT: ARI-2408 safe, ARI-2243 toxic:

ARI-2408 ARI-2243 Animals(with(adverse(events((( ( ( (e.g.,(skin(lesions,(edema,(or(death)! Dose! ! ! ! (mg/kg)! ARI92408( ARI92243( Sitagliptin! 0.3! 6! 5/12! ! 1.0! 6! 11/12! 6! 3.0! 6! 9/12! 6!

% Change 100.0! 0/4! 6! 0/6! 300.0! 0/6! 6! 6! Blood Glucose AUC Blood Glucose

Summary of the EnPlex technology and its applicaons EnPlex: a mulplexed, high-throughput enzyme acvity assay that can profile 100s of enzymes against 1000s of compounds Applicaons: • Aid lead selecon and opmizaon in drug discovery (DPP4, APEH, RBBP9)

• Profile exisng drugs for selecvity (sitaglipn, bortezomib)

• Discover off-target interacons mediang toxicity (telaprevir)

• Repurpose drugs and probes

• Mechanisc insights into enigmac biology Acknowledgements

Todd Golub Benjamin Crava

Golub Lab: Crava Lab: Luke Koblan Katsunori Tsuboi Jason Barne Shasha Ji Jadwiga Grabarek Jon Long David Peck Channing Yu Tus Medical School: John Davis Jack Lai Wengen Wu NIH/NIAID: Sarah Poplawski Robert Munford William Bachovchin David Sanford

Current Acvity-Based Probe Repertoire

• Directed approaches - Cysteine proteases (Bogyo et al) - Metalloproteases (Crava et al, Yao et al) - Kinases, Phosphatases (Acvx, Taunton, Zhang) BHAcATP probe - Histone deacetylases (Crava et al) (Kinases) - Cytochrome P450s (Crava et al) - Protein Arginine Deiminases (Thompson et al) - Protein deubiquinases (Kessler et al)

• Non-directed approaches (Crava et al) - Alcohol/aldehyde dehydrogenases - Enoyl hydratases - Epoxide hydrolases - Glutathione S- Phenyl sulfonate probe - (acvated cysteines; GSTO1) - Transglutaminases