identify compounds that rescue disease relevant mutant membrane proteins
TRANSCRIPT
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Identify Compounds that Rescue Disease Relevant Mutant Membrane Proteins
Dana Haley-Vicente, PhD
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• Introduction• Overview of membrane protein
trafficking & protein misfolding diseases
• Assay Technology • Monitoring of protein trafficking in cells
• Screening for Pharmacological Chaperones
• Examples using mutant GPCRs & ion channels
• Summary
Agenda
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Transmembrane Protein Trafficking
Protein defects lead to failure of proper folding and trafficking
Cell Membrane
Membrane Protein
(e.g. GPCR)
Golgi
Nucleus / ERMisfolded
Protein
Proteasome
Misfolded
Protein
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Protein Misfolding Diseases
Protein misfolding leads to loss of protein function and disease phenotypes
Disease/Abnormality Target Class
Hypogonadotropic hypogonadism GnRHR GPCR
Nephrogenic diabetes insipidus V2R GPCR
Retinitis pigmentosa Rhodopsin GPCR
Obesity MC4R, MC3R GPCR
Familial hypocalciuric hypercalcemia CaSR GPCR
Premature ovarian failure FSHR GPCR
Congenital hypothyroidism TSHR GPCR
Cystic Fibrosis CFTR Ion channel
Long QT Syndrome hERG Ion channel
Brugada Syndrome Nav1.5 Ion channel
Pain insensitivity Nav1.7 Ion channel
Hyperekplexia (startle disease) GlyT2 Glycine transporter
Dicarboxylic aminoaciduria SLC1A1 Glutamate transporter
Childhood (recessive) parkinsonism-dystonia DAT Dopamine transporter
Review Article: Stoy & Gurevich 2015 “How genetic errors in GPCRs affect their function: Possible therapeutic strategies”
Representative GPCR showing disease causing mutations reported from 5 select receptors (TSHR, LHCGR, FSHR, MC4R, and V2R).
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Rescuing Protein Misfolding
Pharmacochaperones (pharmacological chaperones) facilitate trafficking
Cell Membrane Membrane protein with
bound Pharmacochaperone
Golgi
Nucleus / ER
Pharmacochaperone binds
to Misfolded Protein
Pharmacochaperone
PharmacochaperoneSmall molecule compounds that
promote protein folding to
facilitate forward trafficking of
mutant/misfolded proteins
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“This is a new approach to therapeutics… Now we are controlling the access to the target and recognizing that many mutants are functional, but misrouted and can be returned to function, reversing the course of the disease.”
-- P Michael Conn, at Texas Tech University Health Sciences Center, US (2013)
Therapeutic Value of Pharmacochaperones
Successfully treating diseases with pharmacochaperones
Sources: http://www.news-medical.net/whitepaper/20160202/New-approach-to-treating-cataracts-uses-pharmacological-chaperone.aspx, https://www.michaeljfox.org/foundation/grant-detail.php?grant_id=738, & http://www.rsc.org/chemistryworld/2013/12/drug-fix-misfolded-proteins-promises-hope-incurable-diseases-alzheimers
Cataracts
Parkinson’s Disease
Alzheimer’s, Cancer,
Cystic Fibrosis, …
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• Introduction• Overview of membrane protein
trafficking & protein misfolding diseases
• Assay Technology • Monitoring of protein trafficking in cells
• Screening for Pharmacological Chaperones
• Examples using mutant GPCRs & ion channels
• Summary
Agenda
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Current Approaches
Need for easy-to-use HTS tools for discovery of pharmacochaperones
High Content Imaging (IHC, ICC)
FACS Analysis Functional Cell-based Assays
Pros Visualize target localization Detects trafficking to membrane Detect functionality of rescued membrane protein
HTS compatible
Cons Low throughput Difficulty detecting target re-
distribution Need for antibodies, epitope or
fluorescent protein tags Need for specialized, expensive
equipment
Low throughput Need for good antibodies or
addition of epitope tags on extracellular domains
Specialized, expensive equipment
Lack of Assays Difficulty developing & performing assays Indirect measure of pharmacotrafficking Difficulty distinguishing basal levels of
trafficking from induced trafficking Potential for non-specific activity Not all are HTS friendly
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• Robust assay with large signal-to-background and high precision
• Easy-to-follow, simple (mix & read) assay format with no wash protocols
• Highly scalable – Miniaturize to 3456-well
• Luminescence readout that is easily quantified and no specific instrument required
• Industry validated – 1000s of peer-reviewed publications
PathHunter® Assay Technology
A simple solution to study protein-protein interactions and more
Hydrolysis
Split β-Galactosidase Enzyme based Technology
Enzyme Donor (ED)[ProLink™ (PK)]
Enzyme Acceptor (EA)
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Application to Forward Trafficking
PathHunter® Pharmacotrafficking Assays for detecting membrane protein trafficking and screening of pharmacochaperone
Enzyme Donor (ED)
= PK (ProLink™)
1st assay format
Enzyme Acceptor (EA)
Two convenient
EA assay
formats
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Application to Forward Trafficking
PathHunter® Pharmacotrafficking Assays for detecting membrane protein trafficking and screening of pharmacochaperone
Two convenient
EA assay
formats
Enzyme Donor (ED)
= PK (ProLink™)
2nd assay format
Enzyme Acceptor (EA)
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• Introduction• Overview of membrane protein
trafficking & protein misfolding diseases
• Assay Technology • Monitoring of protein trafficking in cells
• Screening for Pharmacological Chaperones
• Examples using mutant GPCRs & ion channels
• Summary
Agenda
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Pharmacotrafficking assay successfully showed stabilization and proper trafficking of the mRHO(P23H) mutant receptor in the presence of 9-cis-retinal
Retinitis Pigmentosa – Protein Folding Disease
Screen for pharmacochaperones that stabilize mRHO(P23H) GPCR mutant
Rhodopsin P23H ref. Chen et al 2015: http://www.ncbi.nlm.nih.gov/pubmed/25783607
Mouse RHO(P23H)
Rhodopsin
1 0 -1 1 1 0 -1 0 1 0 -9 1 0 -8 1 0 -7 1 0 -6 1 0 -5 1 0 -4
0
1 0 0 0 0 0
2 0 0 0 0 0
3 0 0 0 0 0
4 0 0 0 0 0
5 0 0 0 0 0
9 -c is -re t in a l [M ]
RL
U
ED/PK
EAPharmaco-
chaperone
Immunostaining assay
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Other Mutant GPCR Protein Folding Diseases
Screen for pharmacochaperones using the PathHunter® Pharmacotrafficking Assay
1 0 -1 1 1 0 -1 0 1 0 -9 1 0 -8 1 0 -7 1 0 -6 1 0 -5 1 0 -4
0
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
1 0 0 0 0
S R 4 9 0 5 9 [M ]
RL
U
AVPR2(S167T)Vasopressin V2
Nephrogenic diabetes insipidus
aka Relcovaptan
Severe early-onset morbid obesity
MC4R(T162I)Melanocortin MC4
T H IQ [M ]
RL
U
1 0 -1 1 1 0 -1 0 1 0 -9 1 0 -8 1 0 -7 1 0 -6 1 0 -5 1 0 -4
0
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
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CFTR-F508 mutant is the most common cause of Cystic Fibrosis affecting over 70,000 people worldwide
Ion Channel and Transporter ApplicationsOther mutated membrane proteins that lead to disease phenotypes
Disease/Abnormality Target Target Class
Cystic Fibrosis CFTR Ion channel
Long QT Syndrome hERG Ion channel
Brugada Syndrome Nav1.5 Ion channel
Pain insensitivity Nav1.7 Ion channel
Hyperekplexia (startle disease) GlyT2 Glycine transporter
Dicarboxylic aminoaciduria SLC1A1 Glutamate transporter
Childhood (recessive) parkinsonism-dystonia DAT Dopamine transporter
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Ion Channel CFTR-F508 Application
Vertex Pharmaceuticals screening study reveals several pharmacochaperones and successful rescuing of CFTR-F508
1 0 -1 1 1 0 -1 0 1 0 -9 1 0 -8 1 0 -7 1 0 -6 1 0 -5 1 0 -4
0
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 0
2 0 0 0 0 0
2 5 0 0 0 0
C o m p o u n d [g /m L ]
RL
U
C 2 C o rre c to r
C 3 C o rre c to r
C 4 C o rre c to r
C 7 C o rre c to r
C 1 8 C o rre c to r
ImmunostainingMutant CFTR-F508
Pharmacochaperone = CorrectorCompounds courtesy of CF Foundation Therapeutics compound collection
1 0 -1 1 1 0 -1 0 1 0 -9 1 0 -8 1 0 -7 1 0 -6 1 0 -5 1 0 -4
0
2 0 0 0 0 0
4 0 0 0 0 0
6 0 0 0 0 0
8 0 0 0 0 0
1 0 0 0 0 0 0
C o m p o u n d
RL
U
V X -8 0 9 + 1 0 g /m L C 4
V X -8 0 9 [M ]
C 4 [g /m L ]
• Compound rank order was the same as indicated in the literature
• Additive effect was revealed for VX-809 and C4 corrector
PathHunter® Pharmacotrafficking CFTR-F508 Assay
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Ion Channel KCNH2(G601S) Application
• 100+ Mutations in KCNH2 (human ERG)• Defects in intracellular transport/trafficking
• Reduced functional K+ current
• Long QT syndrome
• Cardiac arrhythmias/cardiotoxicity
• Pharmacological rescue of KCNH2(G601S) by clofilium
Toxicology application related to cardiac arrhythmias
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Calcium Channel, Cav2.2
653960 907
6293
0
1000
2000
3000
4000
5000
6000
7000
A1B-PK A1B-PK/2D1 A1B-PK/B3 A1B-PK/2D1/B3
Subunit combination
U2OS EA-MEM + Cav2.2 subunitsBasal activity
Bas
al R
LU
ER
PK
ER
PK
PK
ER
PK
Multi-subunit Ion Channel Assembly Application
Unique application of the PathHunter® Pharmacotrafficking Assay
Subunit Combination
Ba
sa
l R
LU
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Disease Relevant Mutant GPCRs & Ion Channels
Growing menu of PathHunter® Pharmacotrafficking Assays
Mutant Membrane Protein
Description Target Disease Relevance Readout
ADRB2(W158A) ** Adrenergic receptor β2 GPCR Endosome - EA
AVPR2(S167T) Vasopressin receptor 2 GPCR Nephrogenic diabetes insipidus
Endosome - EA
CFTR-ΔF508 Cystic fibrosis transmembrane conductance regulator
Ion Channel Cystic fibrosis Membrane - EA
KCNH2(G601S) Potassium voltage-gated channel, subfamily H (eag-related), member 2
Ion Channel Long QT syndrome (Cardiac arrhythmias)
Membrane - EA
MC4R(T162I) Melanocortin 4 receptor GPCR Obesity Endosome - EA
mRHO(P23H) Rhodopsin GPCR Retinitis pigmentosa Endosome & Membrane - EA
SMO(W535L) Smoothened frizzled family receptor GPCR Basal skin cell carcinomas Membrane – EA
** Adrenergic receptor β2 ADRB2(W158A) can be used for validation experiments
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• Identify pharmacochaperones that ..
• Rescue disease relevant mutant membrane proteins
• Correct/stabilize membrane protein misfolding
• Lead to proper trafficking to the cell membrane and functional rescue
• Applications for disease relevant mutant GPCRs, ion channels, and transporter
• Disease relevant mutants - functional rescue
• Ion channel - assembly-related pathologies and assembly disruption
Summary
Detect forward trafficking using PathHunter® Pharmacotrafficking Assays