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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