EnPlex: a high-throughput method for family- wide profiling of enzyme ac vity
Daniel A. Bachovchin November 5, 2014 Potency typically drives drug discovery, selec vity 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? Selec vity determina on is difficult due to the number and similarity of serine hydrolases
Major Human Enzyme Serine hydrolases Families
700
600
500
400 300 • Involved in clo ng, neural 200 signaling, inflamma on, cancer
Number of Members 100 0 • Currently 8 enzymes are targeted by 14 drugs
• Majority are uncharacterized
The ideal solu on? A high-throughput, mul plexed enzyme ac vity assay
1. Selec vity profiling of drugs and chemical probes
2. Inhibitor discovery based on both potency and selec vity
Ac vity-based protein profiling (ABPP): a general method to assay enzyme ac vity
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 hydrolase probe-labeled serine hydrolase
Evans & Crava , Chem Rev, 2006 Compe ve 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 limita ons of ABPP for inhibitor discovery
Advantages • Does not require a specific substrate assay • Can simultaneously evaluate mul ple enzymes
Limita ons • Typically assays only a small number of enzymes • Laborious detec on methods severely limit throughput
Comparing ABPP screening pla orms (for serine hydrolases)
100,000 fluorescence polariza on 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 mul plexed, bead-based pla orm for enzyme inhibitor screening
Bachovchin et al. Nat Chem Biol, 2014 EnPlex is sensi ve and robust assay for inhibitor discovery
Extraordinary Compa bility with Works in a sensi vity: many enzymes: compe ve 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 Cataly cally dead Accurate IC50 less protein than enzymes give no determina on fluopol-ABPP signal Assembly of a diverse 97 human serine hydrolase panel DPP4 Acetylcholinesterase
Purified, ac ve enzymes
Thrombin Uses of a high-throughput, mul plexed enzyme ac vity assay
1. Selec vity profiling of drugs and chemical probes
2. Inhibitor discovery based on both potency and selec vity
Representa ve 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 selec vity 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, func on is poorly understood
-PRCP globally expressed, regulates several bioac ve pep des
Roujeau et al. JAMA Dermatol, 2013 Telaprevir-PRCP interac on underscores the value of family-wide profiling PRCP: α/β hydrolase fold, cleaves a er proline
NS3 protease and CELAs: chymotrypsin-like folds, cleave a er hydrophobic residues Bortezomib highlights comprehensiveness of EnPlex compared to exis ng methods
Bortezomib, first-in-class proteasome inhibitor approved for mul ple 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 serine protease targets • EnPlex found those 6, plus 7 addi onal targets Uses of a high-throughput, mul plexed enzyme ac vity assay
1. Selec vity profiling of drugs and chemical probes
2. Inhibitor discovery based on both potency and selec vity
Inhibitor discovery from a boronic acid library
O Y OH Boronic acids drive potency, X N B H but achieving selec vity 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 Inhibi on (%) Inhibi on (%)
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 selec ve 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 selec ve inhibitors ARI-2408 vs. ARI-2243: Similar in efficacy, but drama cally 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 applica ons EnPlex: a mul plexed, high-throughput enzyme ac vity assay that can profile 100s of enzymes against 1000s of compounds Applica ons: • Aid lead selec on and op miza on in drug discovery (DPP4, APEH, RBBP9)
• Profile exis ng drugs for selec vity (sitaglip n, bortezomib)
• Discover off-target interac ons media ng toxicity (telaprevir)
• Repurpose drugs and probes
• Mechanis c insights into enigma c 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 Tu s Medical School: John Davis Jack Lai Wengen Wu NIH/NIAID: Sarah Poplawski Robert Munford William Bachovchin David Sanford
Current Ac vity-Based Probe Repertoire
• Directed approaches - Cysteine proteases (Bogyo et al) - Metalloproteases (Crava et al, Yao et al) - Kinases, Phosphatases (Ac vx, Taunton, Zhang) BHAcATP probe - Histone deacetylases (Crava et al) (Kinases) - Cytochrome P450s (Crava et al) - Protein Arginine Deiminases (Thompson et al) - Protein deubiqui nases (Kessler et al)
• Non-directed approaches (Crava et al) - Alcohol/aldehyde dehydrogenases - Enoyl hydratases - Epoxide hydrolases - Glutathione S-transferases Phenyl sulfonate probe - Oxidoreductases (ac vated cysteines; GSTO1) - Transglutaminases