Advances in APBD Research: High Content Screening, Antisense

Oligonucleotides and Computational Drug Design

Or Kakhlon

Department of Neurology Hadassah University Hospital

20 June 2012 APBD Research Foundation Annual Meeting, New York, NY

Funding: APBD Research Foundation

Glycogen biosynthesis involves chain elongation by Glycogen Synthase (GS) and chain branching by Glycogen Branching Enzyme (GBE). If chain elongation outbalances chain branching, glycogen could form starch-like precipitates made up of long, non-branched chains called polyglucosans.

Normal glycogen, branchedPolyglucosan, non-branched

GS/GBE activity ratio

Glycogen build up is normally suppressed in neurons by a well-regulated system, which inactivates (phosphorylates) and degrades Glycogen Synthase (GS)

What causes Adult Polyglucosan Body Disease (APBD)?

Vilchez et al (2007) Nat Neurosc

Glycogen build up is suppressed in neurons by a well-regulated system

Nevertheless, over time glycogen could precipitate as polyglucosan bodies if chain elongation is not adequately balanced by its branching.

Striano et al (2008) Nat Clin Pract NeurolWierzba-Bobrowicz et al (2008) Pholia Neuropathol

APBD

Two experimental approaches for curing APBD:

1. Ameliorating APBD, or slowing down the progress of the disease by reducing the GS/GBE activity ratio.

2. Clearance of polyglucosan bodies.

1. Ameliorating APBD, or slowing down the progress of the disease by reducing the GS/GBE activity ratio.2. Clearance of polyglucosan bodies.

There are pre-existing PG which cannot be removed by any GS/GBE modulating strategy.

Y329S

Keto 3d

Y329S

Gluc/Rap 3d

PG In

tens

ity/c

ell

Gluc/Rap

Gluc/Co

Y329S

Gluc/Co 3d

GFP LC3 Glysh

GBE

1sh

GBE

1/Ra

psh

GBE

1/Ra

p/3-

MA

Rapamycin can induce autophagy and also reverse polyglucosan accumulation. However, only in neurons transduced with shGBE1 lentiviruses.

Therefore, rapamycin could only suppress de novo PG synthesis, not degrade pre-existing PG

Nevertheless, there are three therapeutic strategies for reducing the GS/GBE ratio:

1A. Injection of Antisense Oligonucleotides against PTG & GS in collaboration with ISIS Pharmaceuticals. This approach is already in Phase I clinical trials for treating other disorders such as Spinal Muscular Atrophy (SMA)

1. Ameliorating APBD, or slowing down the progress of the disease by reducing the GS/GBE activity ratio.2. Clearance of polyglucosan bodies.

Plan: 1. In vitro screening for the identification of antisense oligonucleotides (ASO) to PTG is currently in progress (expected to be completed by July 2012).

2. Scaling up the drug and screening it in vivo both by inntracerebroventricular and systemically by subcutaneous injection (a few months).

3. Lead ASOs identified by the in vivo screens will be tested in the APBD mouse model (expected by fall of 2012). Hua et al (2010) Genes Dev

1B. Candidate Testing

1. Ameliorating APBD, or slowing down the progress of the disease by reducing the GS/GBE activity ratio.2. Clearance of polyglucosan bodies.

Testing three types of compounds known to reduce the GS/GBE ratio:

1. GS inhibitors (AMP Kinase (AMPK) and GSK3β activators). Examples: 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), PI3K inhibitors (e.g., wortmanin, Akt inhibitor IV), Berberine (herbal drug) etc.

2. GBE stabilizers.

3. Compounds predicted by solvent mapping to replace mutated Tyr329 in GBE1, or to destabilize GS and PTG.

Candidate testing

Compounds predicted by solvent mapping (in collaboration with Dima Kozakov, Boston University) to replace mutated Tyr329 in GBE1, or to destabilize GS and PTG.

Candidate testing

Designing drugs using protein solvent mapping GBE, GS and PTG were checked for "druggability" (activators for GBE and inhibitors for GS and PTG).

Kozakov et al (2012) PNAS

The Y329S site is druggable, as it is not conserved (less off target binding) and is concave and with a hydrophobic functional group.

Plan:

a. Testing candidate binding using calorimetry.

b. Testing the effect of bound drugs on GBE and GS activity using our established biochemical assays.

c. Testing library compounds for binding and activity modulation.

1C. Screening both for compounds which reduce GS/GBE ratio, or clear PG: High Content Screening using the IN CELL 2000 Analyzer

1. Ameliorating APBD, or slowing down the progress of the disease by reducing the GS/GBE activity ratio.2. Clearance of polyglucosan bodies.

Ctrl

Y329S

keto keto/Rap

Gluc Gluc/Rap Gluc/Co

Optimization by epifluorescence and confocal microscopes: Some representative photos

Integrated intensity of punctate PAS fluorescence (PAS/IP) is significantly higher in APBD patient (Y329Shomozygous) skin fibroblasts than in control fibroblasts.

PAS/IP in fibroblasts treated for 3 days with a ketogenic and diastase pre-digested is higher in patient-derived fibroblasts as compared to control fibroblasts. This means that patient-derived fibroblasts have pre-existing polyglucosans.

Patient-derived fibroblasts have pre-existing polyglucosans.

Rapamycin decreases PAS/PI. This reduction also only becomes statistically significant after diastase digestion.

Fibroblasts treated with glucose, rapamycin and cobalt have lower PAS/PI than fibroblasts treated with glucose and cobalt only, but equal PAS/PI to fibroblasts treated with glucose and rapamycin only.

The effect of rapamycin in reducing PAS/PI overrides the effect of cobalt in increasing it.

There is no significant difference in PAS/PI between fibroblasts treated with ketogenic medium and ketogenic medium supplemented with rapamycin.

Rapamycin couldn't degrade pre-existing polyglucosans (but only reduce de novo synthesized).

Cobalt increases PAS/PI staining in glucose fed fibroblasts with or without diastase digestion.

Diastase digestion amplifies the difference between fibroblasts treated with glucose and ketogenic medium.

Most PAS/PI staining is attributable to polyglucosans.

PAS/PI intensity in glucose fed fibroblasts is not significantly different between diastase digested and not digested conditions.

Polyglucosan analysis in control and Y329S fibroblasts by HCA using the IN CELL 2000

Analyser

Leonardo J. Solmesky, Ph.D.Cell Screening Facility for Personalized Medicine,

Lab for Neurodegenerative Diseases and Personalized Medicine,Department of Cell Research and Immunology,

Wise Faculty of Life Sciences,Tel Aviv University, Israel

Control Y329S/Gluc Y329S/Gluc/RapY329S/Gluc/Co

19Leonardo J. Solmesky, Ph.D. Cell Screening Facility for Personalized

Medicine

• High level of diffuse, non granular staining in control. Need more aggressive diastase treatment.

• Co increases PG intensity and rapamycin decreases it, reverting PAS staining to the diffuse pattern observed in control cells

ctrl Y329S

An example of an improved diastase treatment:

Polyglucosan granules count/cell. Populational distribution in different treatments

21

Glu

cG

luc/

Co

Glu

c/R

ap

• Rapamycin skews distribution to the left, i.e., reduces PG number.• Rapamycin increases area under the curve, i.e., increases cell count suggesting rescue against PG

toxicity.• Co appears to be toxic in this experiment.

• We need to analyze PG integrated intensity!

Polyglucosan granules mean area populational distribution in different treatments

22

Glu

cG

luc/

Co

Glu

c/R

ap

• Conclusion: Treatments do not affect mean PG size. Consistent with rap blocking de novo synthesis

Polyglucosan granules total area populational distribution in different treatments

23

Glu

cG

luc/

Co

Glu

c/R

ap

• As compared to mean PG size, total PG size correlates better with PG number

Nuclear area populational distribution in different treatments

24

Glu

cG

luc/

Co

Glu

c/R

ap

• Treatments did not affect nuclear size

Nuclear IxA populational distribution in different treatments

25

Glu

cG

luc/

Co

Glu

c/R

ap

• Treatments did not affect nuclear size

26

Population distribution among different phases of cell cycle under different treatments

Gluc Gluc/Co Gluc/Rap• Gluc/Co treatment led to growth arrest, while rapamycin slightly accelerated growth

High Throughput Screening

Readouts: PG integrated intensity (PAS/PI).

Positive hits analysis

1. If positive hits are suspected activators of AMPK, or GSK3β, testing activation of purified enzymes.

2. Testing for undesirable chemical liability (covalent protein binding, thioether adduct formation) consequent to metabolic processing.

3. Ranking compounds according to their effectiveness. Clusters of highest and lowest scorers will be aligned to predict a pharmacophore and toxicophore, respectively.

pharmacophore

in silico screening of pharmacophore and toxicophore (using Similarity Ensembl Approach (SEA))

Similar structures with known mechanism of action (future lead compounds)

Phar

mac

opho

re

Molecules with liability

Toxicophore

Possible targets

Candidates for therapeutic modulation.

Summary

• APBD can be ameliorated by reduction of the GS/GBE activity ratio.

• This approach is tested by a. Antisense Oligonucleotide injection; b. Solvent mapping of GBE, GS and PTG in order to find binding drugs; c. High Throughput Screening.

• High Throughput Screening is also used to find potential compounds which would clear PG and thus could not only ameliorate, but alleviate APBD pathogenesis.

Manifesting heterozygotes conundrum

Wt ctrl

Manifesting heterozygote lymphoblastsHomozygote

Manifesting heterozygote whole blood

Genomic DNA indicates a heterozygous p.Y329S mutation. However, cDNA reveals only one mutated allele, indicating that the second allele is missing.

cDNA sequencing:

Test MH carrier parents for mRNA homozigosity of cDNA v heterozygosity of gDNA

Yes

Need to explain the manifesting heterozygotes phenomenon by other ways – alternative to differential allelic expression

No

Manifesting hetreozygotes (MH) phenomenon – is it due to differential allelic expression?

First and foremost need control:

Is there reduced/no expression of WT allele?

Quantitate GBE expression by RT-PCR

GBE expression in MH is roughly 50% of normal

Suggests WT allele is suppressed

Polymorphism: Check: Methylation (Bisulfite sequencing), compare haplotypes between MH and WT, Linkage analysis to MH trait, whole gene analysis, exome analysis, CGH to check copy number differences, exome analysis, whole genome analysis, transcription factor binding, (exclude small deletions?)

GBE expression in MH is roughly the same as normal

Suggests compensation by overexpression

Look for post transcriptional modifications of GBE in MH

Collaboration: Corroborate results by PAS-based genome-wide siRNA screen in wt MEFs

Thanks

Hadassah Medical Center

Alexander Lossos

Tel Aviv University

Leonardo SolmeskyMiguel Weil

Boston University

Dima Kozakov

Columbia University

Orhan AkmanSalvatore DiMauro

ISIS Pharmaceuticals

Tamar Grossman

Apoptosis is also reported in glycogen synthase-activated neurons

Stauro

sporin

e

Prote

in ta

rget

ing

to G

lyco

gen

Suggestion: Polyglucosan accumulation induces apoptosis

Vilchez et al (2007) Nat Neurosc

Sarkar et al (2009) Cell Death Differ

Inclusion bodies (PBs) formed. Can induction of autophagy facilitate their clearance?

Can autophagy be cytoprotective against apoptosis?

Maiuri et al (2007) Nat Rev Mol Cell Biol

Test autophagy enhancers as a therapeutic strategy against APBD

Using the model to test therapeutic approaches: Induction of autophagy

Jaeger & Wyss-Coray (2009) Mol Neurodegen

Autophagy can be stimulated and inhibited in neurons by rapamycin and 3-methyl adenine, respectively

Rapamycin

mTOR

(through Ulk1/2 inhibition)IM

Autoph. MVB

Amphisome

Autolysosome

=LC3

Classical mode of action – autophagic engulfment followed by autolysosomal degradation (by acid maltase?)

How can PB be cleared by autophagy?

Vin

Rapamycin

mTOR

(through Ulk1/2 inhibition)IM

Autoph. MVB

Amphisome

Autolysosome

Can the effects of rapamycin be reproduced if autophagosome maturation to autolysosomes is inhibited?

DMSO RapRapPI

RapVin

RapVinPI Vin St

arve

dFi

brob

last

s-LC3 I-LC3 II

LC3 LC3 LC3

DMSO Rap Rap+Vin

Untr Rap Rap/Vin

0

50

100

150

200

1.1±0.2 1.5±0.1 2.0±0.2 2.2±0.1 1.9±0.3 1.7±0.2 6.2±0.5

* *

Saponized neurons: Autophagic flux is fast.

Rapamycin induces autopahgy and thus slows down autophagic flux.Vinblastine blocks autophagic maturation, further slowing down autophagic flux

Confirmation of vinblastine’s effect:

1. Reproduction of the block in autophagic flux induced by lysosomal protease inhibitors (PI).

2. Blunting the sensitivity of rapamycin induced neurons to PI.

GFP Gly mergeC

nt/Rap shGBE1/Rap shGBE1/Rap/3-MA shGBE1/Rap/Vin

FL-2

PI

FL-1 Annexin V

D

E

Fig. 5

Blocking autophagic maturation by vinblastine did not reverse down-modulation of polyglucosan accumulation and apoptosis by rapamycin.

Rapamycin protection of GBE1-knocked-down neurons did not depend on autophagic maturation and polyglucosan degradation in autolysosomes.

Rap+Vin

Q: Why does 3-MA antagonize Rap?A: Probably not via activation of autophagy or GSK3. Perhaps via PFK inhibition

As opposed to neurons, in APBD patient derived fibroblasts Rap probably does clears PB by autophagy

Control Gbe1Y329S/Y329S patient Gbe1Y329S/Y329S/Rap

Uranyl-Et-OH

Vin

Rapamycin

mTOR

(through Ulk1/2 inhibition)

?DGKα

IM

Autoph. MVB

Amphisome

Autolysosome

Suggestion: Rap-stimulated PB clearance is mediated by exosome release, bypassing autophagosomal maturation

A B

C a b

PBs were not found in either MVB (A), amphisomes (B &C), apparent exosomes (C), or cytosol (A-C) in GBE1-knocked down neurons treated with rapamycin.

shGBE/Rap shGBE/Rap

shGBE/Rap

Relatively small PBs were observed in untreated GBE1-knocked down neurons (D). Suggestion: Neurons not living with other cell types might succumb to cell death once glycogen deposits appear

D

shGBE

mTOR

Rapamycin

Vin

IM Autolysosome

GSK3β

PP active GS

Autoph.

Amphisome

MVB

X

XX

=LC3

=Polyglucosan Body

?

nt shGBE1 shGBE1 Rap

nt Rap shGBE1 Rap 3-MA

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1-G6P

+G6P

GS a

ctivi

ty (

nmol

/min

/mg

Prot

ein)

-75

-50

GS

Tubulin

Main conclusion: Rap positive effect was probably mediated by GS inhibition.

None of the treatments affected G-6-P-stimulated GS activity, suggesting it overrode GS phosphorylation state.

Testing three types of compounds known to reduce the GS/GBE ratio:

1. GS inhibitors (AMP Kinase (AMPK) and GSK3β activators). Examples: 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), PI3K inhibitors (e.g., wortmanin, Akt inhibitor IV), Berberine (herbal drug) etc.

2. GBE stabilizers.

3. Compounds predicted by solvent mapping to replace mutated Tyr329 in GBE1, or to destabilize GS.

Candidate testing

LO

D

Sco

re

Another direction: Microtubule-mediated transport :

A nerve biopsy from Hereditary Spastic Paraplegia reveals PB.

Linkage analysis along chromosome 2 shows the locus where markers for the diseased state co-segregated only in patients and not in healthy family members. This locus encodes the MT motor KIF1A

Do microtubule motors mediate PB clearance?

Summary

• We have shown that GBE1 knockdown causes polyglucosan accumulation and apoptosis in a pure neuronal model.

• These phenotypes can be rescued by rapamycin via inhibition of Glycogen Synthase and not via induction of autophagy

• We conclude that polyglucosan accumulation is causal for APBD. Therapeutic search should therefore focus on restricting polyglucosan accumulation.

Main objectives

• Establishing a neuronal model of APBD in which GBE1 is repressed and PB are observed.

• Using the model to test pharmacological and biochemical methods for correcting adverse phenotypes associated with GBE1 deficiency.

APBD Neuronal Model Produced by transduction with lentiviral particles encoding for shRNA against GBE1

Demonstration of GBE1 knockdown:

nt shGBE10

50

100

150

200

250

0

0.2

0.4

0.6

0.8

1

1.2

GBE1 activity

GBE1 mRNA

GB

E1

Ac

tiv

ity

(n

mo

l/min

/mg

pro

tein

)

GB

E1

mR

NA

(re

lati

ve

va

lue

s)

A RT-PCR & activity

Tubulin

GBE1

Con

trol

Y32

9S

hom

ozyg

ous

nt

shG

BE

1

-75 kD -50 kD

B

Western: Reduction comparable to that found in patients

GFP GBE1 mergeC

nt

shGBE1

Indirect immunofluorescence

GFP Gly merge

nt

shGBE1

shGBE1

GFP DIC Gly merge

A

B

C

Polyglucosan accumulates in GBE1 knocked down neurons

Lower expressers of shGBE1 lentiviruses are less affected:

Polyglucosan is the culprit

Vilchez et al (2007) Nat Neurosc

Glycogen detected as polyglucosan punctae similar to GSK3 inhibition or

PTG over expression.

nt

shGBE1

A

B

C

D

FL-1 Annexin V

SNP

FL-2

P

I

GBE1 knockdown increases apoptosis