Post-Translational Modification Enzymes:
How to Interrogate Key Drug Targets
Hicham Zegzouti, Ph.D.
Group Leader, Assay Design Group
Promega Corporation
Promega Corporation
©2016 Promega Corporation.
[email protected]
Post Translational Modifications (PTMs) Play Key
Roles in Biological Regulation
• Cellular networks are orchestrated by reversible post translational
modifications (PTMs).
• PTMs act as signaling pathway switches and regulate important cellular
processes:
• Cell-cell communications
• Enzyme activity regulation
• Transcription and translation
• Protein-protein interactions
• Protein folding and degradation
• Protein localization
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PTMs are diverse and so are the enzymes
involved
PTM Enzymes
•
Large Families
Kinases
Palmitoyltransferase
Ubiquitin Ligases
• > 500 Kinases
• > 100 Methyltransferases
• > 800 Ubiquitin ligases
Glycosyltransferase
Methyltransferases
• ~ 60 Fe(II)/α-KG hydroxylases
•
Regulated expression
and activity
•
Acetyltransferases
Sumotransferases
Variety of substrates
Demethylases
Hydroxylases
PTMs amplify the diverse functions of the proteome by covalently adding
functional groups to proteins.
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The number of experimentally verified PTMs has
expanded over the years.
The PTMcode Data Set
• 316,546 experimentally verified PTMs
• 69 different types (P, Ub, Me, Ac, GlcNac…)
• Over 45,361 proteins modified
• 19 eukaryotes
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Pablo Minguez et al. Nucl. Acids Res. 2015;43:D494-D502
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PTM: Nature’s escape from genetic imprisonment*
•
PTMs change the properties of amino acids in response to developmental or
physiological requirements of the cell.
•
Multisite PTM leads to a combinatorial explosion in the number of potential
molecular states of a protein.
•
Distinct states triggered by PTM can elicit distinct downstream responses.
• One Protein
• Different PTMs
• Different Forms
• Different cell responses
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Prabakaran et al. WIREs Syst Biol Med 2012. doi: 10.1002/wsbm.1185
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Reversible PTMs and Metabolic Processes
Governing Donor Supply
•
PTMs require a constant supply of donor molecules to be able to process
upstream information in timely manner for proper regulation of biological
processes.
•
Two different kinds of donor supply:
•
Small molecular groups, e.g., phosphoryl, acetyl, glycosyl supplied by metabolic
donors such as ATP, acetyl-CoA, UDP-GlcNAc.
•
Polypeptide-based groups such as ubiquitin, and ubiquitin-like modifications
(SUMO, NEDD) are made by gene transcription and used in PTM through a chain
of enzymatic reactions.
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AKT-Lipid Kinase Signaling Example
Crosstalk between Signal Transduction pathways
Small molecular
group modifications
Transferase
(ADP)
(ATP)
(Pi)
Hydrolase
Regulation of:
• Cell survival • Proliferation • Insulin‐dependent metabolic responses
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Manning and Cantley (2007) Cell 129, 1261-1274.
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Glycosylation in different areas of biology
Glycosylation is central to many biological processes, including:
• Protein and enzyme functions
• Plant and bacterial cell wall structures
• Pathogen mechanism of infection
• Biological function of therapeutics.
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UDP-GlcNAc
Naked Protein
UDP
OGT
G
GlcNac.ted
Protein
OGA
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Ubiquitination and Protein Degradation Example
Canonical NF-kB pathway
Polypeptide-based
modifications
Crosstalk between phosphorylation and ubiquitination
Stimuli
(TNFα,IL‐1β)
Receptor
(Ub)
Ligase
Cytoplasm
Pol II
VVVVVVVVVV
VVVVV
Hydrolase
Regulation of:
• Cell Survival
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• Inflammation
Nucleus
• Immunity
• Cell Proliferation
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PTM Enzymes and Human Diseases
Dysfunction
• Implication of PTM enzymes in many diseases:
o Cancer, inflammation, diabetes…
• Many PTM enzymes have become validated drug targets
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JAK/STAT Signaling Pathway: Driver for
Inflammation
• Rheumatoid arthritis is an autoimmune disease that typically affects the small joints in the hands and feet • It can cause pain, swelling and deformity • Tissue lining joints become inflamed and thickened, fluid builds up and joints erode and degrade
bcr
JAK/STAT signaling
Very High Activity
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Tofacitinib: JAK Inhibitor for Rheumatoid
Arthritis
FDA approved in 2012
Aberrant activation of JAK/STAT signaling can be reduced with JAK inhibitor Tofacitinib
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Tofacitinib reduces RA by inhibiting JAK activity
Mori et al. International Immunology, Vol. 23, No. 11, pp. 701–712
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Overexpression of SCF E3 Ligase Complex
Components: Driver for Liver Cancer
Liver cancer
X
• Nedd8 modification of the SKP1‐
Cullin‐F‐box (SCF) E3 ligase is required for its activity (Ub transfer)
• The Neddylation complex (Nedd 8 PTM similar to Ubiquitination):
• Nedd8‐activating enzyme E1 (NAE)
• Nedd8‐conjugating enzyme E2 (Ubc12)
• NEDD8‐E3 ligase
Normal Cell
• NAE is a drug target to decrease the bcr
activity of SCF complex to treat liver Very High Activity
cancer
Ubiquitination
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Nedd8-Activating Enzyme Inhibitor MLN4924
Suppresses Liver Cancer Cell Growth
Tumor growth measured by fluorescence imaging
• Inhibiting Nedd8 modification through NAE inhibition decreases the activity of E3 ligase overexpressed in liver cancer
X
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• Nedd8‐activating enzyme inhibitor MLN4924 induces autophagy and apoptosis to suppress liver cancer cell growth
Luo et al 2012, Cancer research. DOI: 10.1158/0008‐5472.CAN‐12‐0388
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PTM Enzymes in Basic Research and Drug
Discovery
PTM enzymes Studies and Screening Approaches
Biochemical assay
• In Vitro Biochemical
• Immunological
• Cell based assays
• In Vitro Biochemical
Biochemical assay
Biochemical assay
•• In Vitro Biochemical
Cell based assays
•• Cell based assays
Other
•• Other
Stages of Drug Discovery Need for a Universal Enzyme Assays that can be applied to all
members of a PTM enzyme family
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Detection of PTM Enzyme Activity
Donor substrate
PTM
+
e.g. ATP
Luminescence
e.g. ATP detection
Acceptor
Substrate
Enzyme
e.g. Protein
e.g. Kinase
PTM
Acceptor
Substrate
Modified‐Substrate
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2nd Product
e.g. ADP
Radioactive 32P or 33P‐ATP
e.g. electrophoresis, SPA,
Filter binding.
Fluorescence
Antibody based:
e.g. Transcreener, Adapta
Fluorescence
Antibody based:
e.g. HTRF/LANCE, Alphascreen
Antibody free:
e.g. ADP‐Quest, HPLC
Antibody free:
e.g. Caliper, Z’‐Lyte, Mass spectrometry
Substrate consumption
+
PTM Detection
Luminescence
ADP‐Glo (ADP), MTase‐Glo (SAH)
Product Formation
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PTM enzymes utilize nucleotide substrates or
generate them as products
ATP ADP
An assay that detects
the universal product
of a PTM enzyme
family can be applied
to all members of the
family.
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α‐KG Succinate
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Nucleotide enzyme targets
Ubiquitin Ligases
Aminoacyl tRNA synthetases
cAMP‐PDEs
DNA Ligases
Direct detection
Kinases
ABC transporters
Heat Shock proteins
ATPases, Helicases
ATP AMP
ADP
ATP
GTP
GTPases
GEFs
GAPs
Bioluminescence
Galactosyltransferase
O‐GlcNAc transferase (OGT)
GalNAc transferase
UDP
Fucosyltransferase
Mannosyltransferase
UMP Phosphoglycosyltransferases
GDP CMP Sialyltransferases
Bioluminescent Nucleotide
detection technologies
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Glo
Powerful research toolbox for
various enzyme characterizations
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Nucleotides enzyme targets
Epigenetics and more
Indirect detection*
JumonjiC Demethylases
DNA Hydroxylases
Dioxygenases
*Reaction products converted to an intermediate then to ATP:
S‐Adenosylhomocysteine – Succinate ‐ Coenzyme A
Succinate
Histone Acetyltransferases
Acyltransferases
Palmitoyltransferase
Bioluminescent nucleotide
detection technologies
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Glo
SAH
Methyltransferases
e.g. Histone, protein, DNA…
CoA
Powerful research toolbox for
various enzyme characterizations
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What we aim for during assay development?
“Any”-Glo Assay target performance:
 Sensitivity: Detect low levels of product with high Signal-to-background ratios
 Linearity: Good range of product concentrations to fit low and high enzyme activity
 Signal Stability: Luminescent signal should be stable (2-3hours)
 Ease-of-Use: Very few steps. Add-and-read assay type desirable
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Bioluminescent Glycosyltransferase assay
Platform
Direct detection
 One Step Detection: After the GT reaction, the detection reagent is added in 1:1 ratio.
Luminescence signal is recorded after 60 min incubation.
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Bioluminescent GT assays are very sensitive with a
broad range of detection
Linearity
Sensitivity
•
•
•
Nucleotide Assay UDP GDP Signal to Background ratios (fold) at each nucleotide concentration (µM) 25 12.5 6.25 3.13 1.56 0.78 0.39 0.20 0.10 0.05 0.02 0 UDP‐Glo 12368
6803
3588
1828
917
459
227
119
60
30
16
1
GDP‐Glo 41700 24917 13317
7028
3533
1788
898
436
208
110
54
1
Bioluminescent glycosyltransferase assays are linear up to 25-50µM with high
dynamic range.
The assays can detect nucleotide concentration as low as 10nM with > 2-fold S:B.
The bioluminescent signal generated from the assays is stable over time (HTS).
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Universal Activity Assays to measure most
Glycosyltransferase Enzymes
GDP‐Glo
UMP/CMP‐Glo
Luminescence (RLU)
Luminescence (RLU)
Luminescence (RLU)
UDP‐Glo
Luminescence (RLU)
Luminescence (RLU)
Luminescence (RLU)
Luminescence (RLU)
Acceptor: Phospholipid
Das et al 2016, Scientific Reports 6, 33412
Phosphoglycosyltransferase (PglC)
 Detect the activity of any nucleotide-sugar utilizing GT regardless of substrate chemical structure.
 Detects the activity of low amounts of GT enzymes with high S:B
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Profiling glycosyltransferase donor and acceptor
substrate specificity
Testing GT specificities for different sugar donor and acceptor substrates
UDP-Glo assay can be used to:
 Study specificity of transfer of different sugars by diverse GTs.
 Find specific sugar acceptor substrates for GTs of interest.
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Substrate Km determinations using nucleotide
detection
UDP
GDP
UMP
CMP
XcbA
80
25ng/µl NmX, 40ng XcbA
60
40
20
Km ~ 212 ± 15µM
0
0.0
0.5
1.0
1.5
2.0
2.5
UDP-GlcNAc, mM
XcbA
30
CMP Produced
(pmol/min/ug)
150µM UDP-GlcNAc, 40ng XcbA
25
20
15
10
5
0
Km ~ 0.69 ± 0.18ng/µl
0
10
20
30
NmX, ng/µl
Bioluminescent assays accurately determine biochemical values for sugar donors and acceptors of different GTs
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“Add-and-Read” Homogeneous Formats
Direct detection
White Plates
Format 1:1:2 (µl)
1,536‐well
2.5/2.5/5
384‐well
5/5/10
10/10/20
96‐well
25/25/50
50/50/100
 Two step detection: After the kinase reaction, the ATP is depleted with the first reagent
then ADP is detected with a second reagent addition.
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One universal assay platform - many applications
ADP‐Glo™ is a Universal in vitro
Biochemical Assay for all types of Kinase Studies
High‐Throughput Screening
Mode of action studies
ADP‐Glo™ Assay Platform
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Kinase inhibitor
profiling
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Screening for Kinase inhibitors using luminescent
ADP detection
Identification of true Kinase inhibitors vs false positives using
Luminescence Kinase assay
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Determination of Inhibitor’s Mechanism of Action
PKA ATP Competitive inhibitor H‐89
PKA ATP non Competitive inhibitor PKI
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Cellular Levels mM
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Profiling inhibitors against a subset kinase
panel with ADP-Glo kinase assay
EGFR
EGFR
HER2/4
p38α
PI3K α/β/δ
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Complex biochemical reactions can be
assessed with universal assays
Ubiquitination: Multiple
enzymes involved
E3 ligase activity drives the AMP production by E1 forward and can be measured
using AMP-Glo assay
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Importance of Methylation/Demethylation in
Epigenetics
• Stable epigenetic inheritance is essential for maintenance of specific tissue and cell type functions
• Epigenetic changes are reversible, and they are mediated by groups of enzymes known as writers, erasers and readers
• Methylation is a common posttranslational modification in histones, and provides marks for protein complexes involved in gene expression or silencing
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Universal Methyltransferase and Demethylase/
Dioxygenase Assays
Indirect detection
Methylation
SAH detection
Demethylation
Succinate
detection
 Add-and-Read Format: After the enzymatic reaction, the product formed is converted to
ATP, which then drives a luminescent reaction.
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Variety of substrate types can be used with
universal assays
Evaluation of different potential high structure substrates for DOT1L methyltransferase using
MTase-Glo™ Methyltransferase Assay.
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Variety of substrate types can be used with
universal assays
Small molecule
Small molecule methylation detection using MTase-Glo™
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Variety of substrate types can be used with
universal assays
Succinate-Glo™ detects the activity of any JMJC demethylase or dioxygenase
regardless of substrate methylation state and position or chemical nature
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Bioluminescent nucleotide assays for PTM
enzymes activity detection
ADP
AMP
SAH
Succinate
CoA
UDP
GDP
UMP, CMP
The activity of each PTM enzymes class can be assessed with a universal
bioluminescent nucleotide detection assay
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Bioluminescent Nucleotide Assays Summary
Bioluminescent nucleotide assays have the following important features
required for a robust PTM enzyme activity detection:
 Universal assays: can be used with all the members of a PTM enzyme family.
Adequate for profiling inhibitors
 Highly sensitive assays that will allow the detection of low activity enzymes or
use low amounts of purified enzymes
 Easy to use. 1-2 step additions and read
 Bioluminescent detection is resistant to chemical interference. Ideal for
compound screening
 HTS friendly: sensitive in low volume format and the signal is stable for batch
processing
 Bioluminescent PTM enzyme detection is adequate for studying acceptor and
donor substrates biochemical properties (e.g. Km)
 No substrate limits: Assays allow study of inhibitor mode of action
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Thank you
Questions?
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