Los días 11 y 12 de diciembre de 2014, la Fundación Ramón Areces celebró el Simposio Internacional 'Neuropatías periféricas hereditarias. Desde la biología a la terapéutica' en colaboración con CIBERER-ISCIII y el Centro de Investigación Príncipe Felipe. El tipo más común de estas patologías es la enfermedad de Charcot-Marie-Tooth, un trastorno neuromuscular hereditario con una prevalencia estimada de 17-40 afectados por 100.000 habitantes. Durante estos dos días, investigadores mostraron sus avances en la mejora del diagnóstico y el tratamiento y, por ende, de la aproximación clínica y la calidad de vida de las personas afectadas por estas patologías.

1. Drug Screening and Discovery for Charcot-
Marie-Tooth Disease INTERNATIONAL SYMPOSIUM
Nerve biology and inherited peripheral neuropathy. From biology to
therapy 2014 Madrid

2. Drug Screening and Discovery for
Charcot-Marie-Tooth Disease
INTERNATIONAL SYMPOSIUM
Nerve biology and inherited peripheral neuropathy. From
biology to therapy 2014 Madrid
Waisman Center
University of Wisconsin-Madison

3. • Identification of Genetic Causes
• Mechanistic Insight
– Biochemistry and Interacting Proteins/Pathways
– Animal Modeling
– Informatics
• Drug Discovery
– Identification of Drug Target
– Drug Screens
– Medicinal Chemistry and Pharmacology
Pathway to a CMT Treatment

4. Inoue K et al. Genome Res. 2001;11:1018-1033
Saporta et al., 2011
©2001 by Cold Spring Harbor Laboratory Press
–A 1.4 Mb duplication containing the Peripheral Myelin Protein 22 (Pmp22) gene is the
most common cause of Charcot-Marie-Tooth Disease (CMT1A)
–>35% of all CMT cases, >55% of all genetically diagnosed CMT
–Several rodent models using Pmp22 overexpression exhibit CMT1A-like symptoms
–Lowering Pmp22 expression leads to improved nerve function in rodent models of
CMT1A
Pmp22 and CMT1A

5. Cell-based assays for
Pmp22 modulation
Cell Type
Building a Reporter
Performing Screens
Validation Assays
Creating Cell Based Assays

6. • Criteria for a cell line
– Schwann cell
– Expresses high levels of
Pmp22
– Easily grown
– Regulation of Pmp22
dependent on
physiological regulators
– Conformation of the
Pmp22 genes is correct.
• S16 cell line: Richard
Quarles/NIH
Creating Cell Based Assays: Cell Type

7. • Genes are not just
“lines” or “sequences”
• They are
Protein/nucleic acid
complexes that have 3D
structures, enzymatic
activity and cell type-
specific ligands (known
as transcription factors)
Ramon y Cajal drawing, used in
Court et al., Nature 2004
Form Follows Function

8. HHistone H3 K27 acetylation marker of active enhancers
Egr2/Krox20 transcription factor
Sox10 transcription factor
ChIP-Seq Annotation
of the Pmp22 gene

9. Loss of H3K27ac-marked
enhancers after nerve injury
Pmp22

10. Cell-based assays for
Pmp22 modulation
Cell Type
Building a
Reporter
Performing Screens
Validation Assays
Creating Cell Based Assays

11. Mechanistic Targets of Pmp22 Regulation
PMP22 gene
3
Nucleosomes
1. Transcriptional and Chromatin Regulation
Protein
3. Translation
AAAAAAAAAA
4. Protein Stability
2. RNA processing and Stability

12. Published in: James Inglese; Patricia Dranchak; John J. Moran; Sung-Wook Jang; Rajini Srinivasan; Yolanda Santiago; Lei Zhang; Rajarshi Guha;
Natalia Martinez; Ryan MacArthur; Gregory J. Cost; John Svaren; ACS Chem. Biol. 2014, 9, 2594-2602.
DOI: 10.1021/cb5005492
Copyright © 2014 American Chemical Society
Genome Editing and Reporter Insertion
Rationale: Gene in its natural habitat will reflect not only
transcriptional, but also epigenetic (miRNAs) regulation
FMDV 2a “ribosome-stuttering” peptide allows production of native Pmp22 and
unfused reporter from a common transcript.

13. FLuc
NanoLuc
The Coincidence Reporter: Using two
reporters in one cell line.
Problem: >80% of all active compounds from reporter gene assays
are false positives; they modulate reporter itself
Problem: >80% of all active compounds from reporter gene assays
are false positives; they modulate reporter itself
Solution: Deploy a flexible coincidence reporter that utilizes 2 or
more dissimilar reporter genes in tandem
Solution: Deploy a flexible coincidence reporter that utilizes 2 or
more dissimilar reporter genes in tandem
Reporter 1 2A Reporter 2
R1 R2
Target pathwayTarget pathway
Genomic DNA
Protein
Promoter TALEN-mediated genomic integration
Example: Firefly
Luc–2A–NanoLuc
integrated down-
stream of gene
promoter relevant
to Parkinson’s
disease
Cheng & Inglese (2012) Nat. Meth.
Hasson, S. (unpublished)
FLuc
NanoLuc

14. response vs. concentration
14
Cytotoxicity firefly luciferase nanoluciferase
Coincidence Reporter Data

15. • S16 assay: secreted nanoluciferase/GFP
• RT4 assay: firefly luciferase
• S16: secreted nanoluc/Fluc
• S16: nanoluc/Firefly luciferase
• RT4: secreted nanoluc/Firefly luciferase
RT4 is an independent Schwann cell line that
expresses Pmp22
Developing New Assays

16. Cell-based assays for
Pmp22 modulation
Cell Type
Building a Reporter
Performing
Screens
Validation Assays
Implementing Cell Based Assays

17. NCATS: National Center for Advancing
Translational Sciences
• Founded 2004 as part of National Institutes of Health Roadmap
• State-of-the-art drug screening facility dedicated to finding new
drugs for disorders that currently have no treatment
• Directed by James Inglese, leader in new technology development
for drug screening,
• Screening has been initiated (40,000 cmpds/week)!

18. • 1536-well plates, inter-plate dilution series
• Assay volumes 2-5 μL
• Assay concentration ranges over 4 logs (high:~ 100 μM) • Automated curve fitting and classification
• Establish nascent SAR, pharmacological
dependence
• Reconstruct
concentration-
response data
A
B
C
Inglese et al. (2006) PNAS 103, 11473-11478
D
• Combined with cross-validating orthogonal
assays should allow rapid identification of
biologically relevant modulators
FLuc BlaScreening chemical libraries at a single
concentration will lose some potent compounds
that are toxic at higher doses.
Quantitative High Throughput Screening

19. What have we found so far?
• Performed screen of >3000 approved drugs,
using two genome edited assays
• Pilot screen for eventual screen of entire NIH
collection (~400,000 cmpds., ongoing)
Results of Drug Screen

20. Progesterone/Glucocorticoids have been
shown to activate Pmp22 expression.
Progesterone Antagonist showed therapeutic
promise in CMT1A rat model (Sereda/Nave).
Screen identified steroid activators: GR/PR
antagonist: mifepristone.
Steroid Receptor Agonist and Antagonists

21. Bryostatin
• Developed for treatment of myeloid cancers
• Proteasome Inhibitors reduce Pmp22 in vitro and
in vivo
• Prototype of proteasome inhibitors,
Bortezomib/Velcade, is associated with
peripheral neuropathy
• Newer generation of PIs appear to lack
association with peripheral neuropathy
• Need to determine if chronic dosing is nontoxic
and effective in CMT1A models
Proteasome Inhibitors

22. Bryostatin
• Bryostatin 1: Isolated from marine invertebrate
(bryozoan)
• Bryostatin 1 modulates protein kinase C (PKC).
• Bryostatins compete for the PKC DAG-binding
site with very high affinity (≈1.35 nM) producing
a brief activation period followed by a prolonged
downregulation.
• All PKC activators trigger downregulation due to
degradation of membrane-bound PKC
• May work to inhibit long term activation of
Mek/Erk signaling.
PKC modulators

23. Cell-based assays for
Pmp22 modulation
Cell Type
Building a Reporter
Performing Screens
Validation
Assays
Validation Assays

24. Reporter Assays
Gene Expression
Profiling
Tertiary Screens
Primary SC’s
In vitro myelination
Stem Cell Models
Rodent Models
Drug Screening Overview
Do compounds that reduce Pmp22 change the whole myelination program?

25. Selectivity of Effects on Pmp22

26. DAPI MBP NF MERGE
NoTreatment10nM
In Vitro Myelination
(L. Feltri, Univ. of Buffalo)

27. 0
0.2
0.4
0.6
0.8
1
1.2
A B A B A B A B A B A B A B
Bort
1uM
Bort
100nM
MLN
1uM
MLN
100nM
Oproz
1uM
Oproz
100nM
IVC
PMP22 total
P1
P2
1a 1b 2 3 4 5Pmp22
P1 P2
Human iPSC-derived Schwann cells

28. Is Gene Expression a Druggable Target?
Ligand/Receptor Interactions
Signaling Pathways (e.g. kinases)
Gene Expression
Mechanism X (microRNAs,
translation efficiency transcript
stability)
Mechanism Y
(epigenetic mechanisms?)

29. Reporter Assays of Gene Expression
Potential Uses
• Designed for use in Gene Dosage Disorders, such as
CMT1A
• Could be used to induce a related family member than
compensates for mutant protein
• Ongoing Efforts for many disorders will identify modifier
loci in genetic models or human genetics studies
• Genome edited reporter assays can be designed to
modulate genes that can compensate for CMT-
associated mutation

30. Svaren lab
Erin Jones
Rajini Srinivasan
Courtney Krueger
Camila Lopez-Anido
John Moran
James Inglese lab
Sung-Wook Jang
Ryan McArthur
Patricia Dranchak
Waisman Center,
University of Wisconsin
NCATS/NIH Megan Brewer
Anthony Antonellis
Anita Bhattacharrya
Laura Feltri

31. • Fact?
• Internet Search
• Secondary Screen to
pick relevant results
• Tertiary Screen: Click
and read
• Compound to treat
CMT1A? (Effective,
Safe)
• Chemical Library screen
– Approved drugs, natural
products, synthetic
compounds
• Secondary Validation
Assays
• Clinical Trials
Searching for a relevant……

32. • For secondary validation of assays, using an
independent Schwann cell lines
• To test if primary assay is typical, or whether it
is an outlier
• To determine which combination of secondary
assays are most predictive of a response at
the gene level, ideally selective for Pmp22 and
not other genes.
Why more assays?

33. Activity
relative to
bortezomib
at 0.6uM
Log M
Gluc-biological
Gluc-biochemical
inhibition
Cytotoxicity
Mifepristone

34. RT-qPCR
Criteria
1a
intermediate
gene array
fail
Criteria
2
fail
Primary cell
model
Criteria
3
fail
Pmp22-Fluc
qHTS
Pmp22-βLac
qHTS
Cell TiterGlo
2,779 cpds
Criteria 1a (dual activity &
>70% Eff): 37 cpds
Criteria 1b (non-
toxic):
11 cpds pass
26 cpds
Criteria
1b
fail
Criteria 2 (> -60% in
Pmp22 mRNA
expression):
4 cpds pass6 cpds
pass
pass
pass
pass
pass
Western
analysis
cytotoxicity
Fluc
BLA
RT-qPCR in S16
Schwann cells
Primary data from orthologous reporter screen
Jang et al., ACS
Chemical
Biology, 2012

35. Assay Validation
• Reporter Activity
• Response to microRNAs
• Regulation by Transcription Factors
Nanoluc Activity shown for
siSox10 cells relative to control
siRNA transfection
Transfection at O hours
Medium changed at ~24 hours
Supernatant assayed at 48 hours

36. 0
secNanoLuc Response to Bortezomib Treatment
-12 -11 -10 -9 -8 -7 -6 -5 -4
-125
-100
-75
-50
-25
25
50
0
Clone E1
Clone E7
Log [Bortezomib], M
%ActivityNormalizedto10mMBortezomib
Treatment
secNanoLuc Total Luminescence
C
lone
E1
N
o
Treatm
entClone
E1
D
M
SO
C
lone
E1
B
ortezom
ib
C
lone
E7
N
o
Treatm
entC
lone
E7
D
M
SO
Clone
E7
Bortezom
ib
0
10000
20000
30000
40000
secNanoLucLuminescence(RLU)
Response of Locus-embedded reporters to
modulators of Pmp22 expression

37. P2P1
1a 1b 3 4 52Exons:
49,300,000 49,310,000 49,320,000 49,330,000 49,340,000
Egr2 ChIP Seq
Sox10 ChIP Seq
+11kb-7kb
Intron elementSox10 element
Identification of Pmp22 Enhancers
using Egr2 and Sox10 ChIP-Seq in vivo
Rationale for Assay Development
• ChIP analysis identified major regulatory element of Egr2/Sox10 binding in
large intron of Pmp22 locus in vivo.
• Human element drives peripheral nerve-specific expression in mice,
(Jones et al., J. Neuroscience 2011

38. Refining Screen Strategy
S16-secNanoLuc qHTS to
discover modulators of Pmp22
Medicinal
Chemistry
Optimization
in vitro ADMET
in vivo PK
S16-FLuc reporter
assay
Purified NanoLuc
assay
Confirmatory
assays
Cytotoxicity S16 cells (cell
viability by Cell Titer-Glo)
Counterscreen
Optimization/
characterization
Primary
screen
S16-qRT-PCR for
endogenous
Pmp22 level
S16-qRT-PCR
intermediate gene
array
Secondary
assays
Primary rat
Schwnn cells-qRT-
PCR
Tertiary assays
Chemical Probe
Hit rates >1%
Low rate of validation
on endogenous genes
(~12%)
Primary Screen
Eliminating Toxic compounds

40. Published in: James Inglese; Patricia Dranchak; John J. Moran; Sung-Wook Jang; Rajini Srinivasan; Yolanda Santiago; Lei Zhang; Rajarshi Guha;
Natalia Martinez; Ryan MacArthur; Gregory J. Cost; John Svaren; ACS Chem. Biol. 2014, 9, 2594-2602.
DOI: 10.1021/cb5005492
Copyright © 2014 American Chemical Society

41. Pharmacologic correlation between S16 GLuc and S16 secNLuc reporter lines. (A) EC50 correlation for 121 library compounds
and related chemotypes or bioactive classes obtained from reporter lines. For bell curves EC50 was derived from ascending
portion of the concentration–response curve (CRC). NC indicates error threshold exceeded in one of the assays (see
Methods). Data are the mean of n = 4, error is the SD. (B, C) Examples CRCs used to derive EC50 values for compounds
falling on the correlation plot diagonal and showing opposite pharmacological responses, bortezomib and fluorometholone.
Published in: James Inglese; Patricia Dranchak; John J. Moran; Sung-Wook Jang; Rajini Srinivasan; Yolanda Santiago; Lei Zhang; Rajarshi Guha;
Natalia Martinez; Ryan MacArthur; Gregory J. Cost; John Svaren; ACS Chem. Biol. 2014, 9, 2594-2602.
DOI: 10.1021/cb5005492
Copyright © 2014 American Chemical Society

42. Molecular and pharmacological characterization of Pmp22 reporter assay using genome editing. (A) Assay of TALEN pairs
using a Cel-1 endonuclease assay demonstrates cleavage at the Pmp22 stop codon with 9.6% efficiency. (B) The S16
Schwann cell line with insertion of Gaussia luciferase (GLuc) was exposed to bortezomib at the indicated concentrations, and
relative luciferase activity was measured at 24 h after exposure. (C) The S16 reporter line was transfected with either Sox10
siRNA or a scrambled control siRNA, and luciferase activity was measured at 48 h after transfection using two different cell
plating concentrations. The medium was replaced at 24 or 2 h prior to measurement as indicated. (D) Lysates from the S16
reporter line were analyzed by immunoblot for PMP22 expression. The doublet of PMP22 is observed in the control siRNA
lanes but is absent after transfection with siRNA for Sox10. Background bands at higher molecular weights are indicated.
Molecular weight markers (kDa) are shown on the left side. (E) The S16 reporter line with secNLuc was exposed to the
indicated concentrations of bortezomib, and NLuc activity was measured at 24 h. (F) Three independent clones of the NLuc
assay were transfected with siRNA for Sox10, and activity was measured at 48 h after transfection. The resulting activity is
shown relative to the same clones transfected with a control siRNA, which was set as 1.
Published in: James Inglese; Patricia Dranchak; John J. Moran; Sung-Wook Jang; Rajini Srinivasan; Yolanda Santiago; Lei Zhang; Rajarshi Guha;
Natalia Martinez; Ryan MacArthur; Gregory J. Cost; John Svaren; ACS Chem. Biol. 2014, 9, 2594-2602.
DOI: 10.1021/cb5005492
Copyright © 2014 American Chemical Society

43. Characterization of PKC modulators. (A) Activity of modulators of Pmp22 transcription by bryostatin and bortezomib, the NLuc
DHP ligand, cilnidipine, and the cytotoxic agent digitonin in the S16 NLuc assay. (B) NLuc enzyme obtained from S16 secNLuc
cell culture media and incubated with the compounds in panel A for 24 h prior to determination of NLuc enzyme activity. (C)
Phorbol ester, PMA activity on reporter and Pmp22 transcript levels. The secNLuc S16 cell line was plated and exposed to the
indicated concentrations of the PMA. At 24 h, NLuc activity was measured from the medium, and RNA was purified from the
cells to perform quantitative RT-PCR analysis of Pmp22. Activities and mRNA levels are shown relative to vehicle-treated cells,
which were set at 1. (D) Comparative analysis of the compounds in panel A and PMA in the S16 intronic enhancer FLuc assay.
Error bars indicate standard deviation of two replicates. (E) Expression analysis of Pmp22 and co-regulated mRNA transcripts
with byrostatin treatment. S16 cells were treated with byrostatin at 1 nM (gray bar) or 10 nM (black bar) or DMSO (0.6%) only
(white bar) for 24 h and then applied to qRT-PCR targeting major myelin genes and their regulators. Data were first normalized
to ActB and then plotted relative to the untreated sample set to 1 for each target gene. Error bars indicate the SD of three
replicates.
Published in: James Inglese; Patricia Dranchak; John J. Moran; Sung-Wook Jang; Rajini Srinivasan; Yolanda Santiago; Lei Zhang; Rajarshi Guha;
Natalia Martinez; Ryan MacArthur; Gregory J. Cost; John Svaren; ACS Chem. Biol. 2014, 9, 2594-2602.
DOI: 10.1021/cb5005492
Copyright © 2014 American Chemical Society