Author affiliations

Development of Novel ALS Treatment on the Basis of Mechanisms of Cellular Chronological Life Span Control Michael Youdell1, Elizabeth Milway1, Jane Mellor1, 3, Huw Jones1, Phil Jordan2, Ewan Hunter2, Aroul Ramadass2, Merit Cudkowicz4, Alexandre Akoulitchev1,2 Background: Chronological life span (CLS) control is a highly conserved cellular mechanism responsible for network coordination of repair mechanisms capable of improved viability and amelioration of cellular pathologies in ageing cells. By deploying ChronoscreenTM, a phenotypic screen for extension of chronological life span control, together with RNAi we have identified a biologically active compound RDC5, with a mode of action dependent on autophagy, protein turnover, protein synthesis. Treatment with RDC5 showed amelioration of toxic effects of SOD1 mutants in C.elegans and in a neuroblastoma cell line. Further analysis of RDC5 revealed high efficacy in the turnover of several neurotoxic aggregating proteins. We are currently testing RDC5 treatment regimes in SOD1 and TDP43 models of ALS in mice. Epigenetic and metabolic links to CLS offer unique advantages for development of diagnostic, prognostic and predictive blood based ALS biomarkers, based on chromosome conformation signatures. On the basis of the previous successful biomarker development for AD patients by Oxford BioDynamics Ltd, we are conducting a screen and cross validation for chromosome conformation signatures biomarker panels associated with ALS, as well as their suitability for disease monitoring under RDC5 treatment.

Adapted from Fraser et al. Genome Biology 2009

Chronos Therapeutics Limited (UK)1, Oxford BioDynamics Limited (UK)2, Biochemistry Department, University of Oxford, UK3, Massachusetts General Hospital, ALS Clinic4

3C: Chromatin undergoes several layers of

compaction, but some of the conformations formed by the DNA also have regulatory functions. Cis and trans chromosomal contacts may help to regulate gene expression and epigenetic control. Physical DNA contacts can be identified using the chromatin confirmation capture (3C) technique, shown above for a cis interaction.

EpiSwitch Assay: EpiSwitch technology is based on the

chromosome conformation capture technique developed by Dekker et al. (2002). It is a non-invasive Industry Standard technology suitable for detecting specific chromosome conformation signatures in different disease states. 3C analysis was performed in accordance with manufacturer’s instructions for EpiSwitch 3C technology (Oxford BioDynamics Ltd). Briefly, cells were cross-linked in 32% paraformaldehyde, quenched, resuspended in hypotonic buffer and subjected to restriction digest (FastDigest, Fermentas) kit for 20 min, followed by ligation for 10 min at 16C. Following proteinase K treatment, 13 µl of the mix was analysed by nested PCR (25 cycles of 94°C for 1 min, 56°C for 45 sec, 72°C for 30 sec). PCR products were analyzed on a Caliper GX90 electrophoresis system (Perkin Elmer) in accordance with the manufacturers’ instructions.

Abstract Number: 6

Stage&I&Yeast&Oxanol&

Screen&

Stage&II&&Yeast&

Bioscreen&

Stage&III&C.#elegans#Screening&

ChronoscreenTM&

Automated&Screen&for&Measurement&of&CLS&&  High&throughput&pla6orm.&&  Measures&outgrowth&of&yeast&cultures&treated&with&compound.&  Used&to&confirm&hits&from&the&Oxanol&Screen.&&  QuanBtaBve&data&on&extension&of&Healthspan,&Median&Lifespan&&&Maximum&Lifespan&

Automated Screen for Measurement of CLS   bus-5 or bus-8 mutant (drug sensitive) background.   Timed egg laying generates synchronous population.   Worms assessed on a daily basis for viability by

microscopy.   Assays: Healthspan, Median Lifespan & Maximum

Lifespan   Conducted under GLP conditions.

High&Throughput&Screen&for&Viability&  High&throughput&system.&&  Fluorescent&assay&to&measure&viability&of&a&yeast&cell.&  Assesses&effect&of&a&compound&on&the&viability&of&postKmitoBc&yeast&Conducted&under&GLP&condiBons.&

CHRONOSCREENTM&

Phenotypic&ReadKOut&of&Chronological&Life&Span&(CLS)&Extension&

ModulaBon&of&The&Cellular&Processes&of&Ageing&&&Disease&

0 10 20 300

10

20

30

40

50

60

70

80

90

100

Days

Popu

la4o

n6Viability66(%) Control

Compound6A

Time6of6Last6Mito4c6Cell6Division

Time6of6Last6Mito4c6Cell6Division

HealthspanExtension

MaximumLifespanExtension

Median6LifespanExtension

 &Healthspan&&Time&at&which&populaBon&reaches&95%&Viability&&

 &Median&Lifespan&&Time&at&which&populaBon&reaches&50%&Viability&

 &Maximum&Lifespan&&Time&at&which&populaBon&reaches&5%&Viability&

Chronological&Lifespan&is&the&length&of&Ame&an&individual&or&populaAon&survives&aCer&the&last&mitoAc&cell&division.&&

SOD1%Worm%Lifespan

0 5 10 15 20 250

10

20

30

40

50

60

70

80

90

100

Days

Popu

la?o

n%Viability%%(%)

SOD1%(127X),%ControlSOD1%(127X),%RDC5SOD1%(WT),%Control

Chroneuro&is&able&to&significantly&improve&the&Median&&Lifespan&of&worms&expressing&a&mutant&form&of&SOD1&known&to&cause&ALS.&&

24%&&Median&Lifespan&

Extension&

SOD1&(G39A)&Survival&Data&in&NSC34&Neuroblastoma&Cells&

Amelioration of Toxic SOD1 aggregates by RDC5

Tryp

an B

lue

Stai

ning

(%)

Wild Typ

e

Wild Typ

e

SOD1 G93

A

SOD1 G93

A0

10

20

30

40

50ControlRDC5

Chroneuro&reduces&the&toxicity&of&ALS&SOD1&mutants&(G93A)&expressed&in&neuroblastoma&cell&line&NSC34&

Ageing&is&a&Highly&Regulated&Biological&Process&AGEING&

CHRONOSCREENTM&&&BEYOND&Work&Flow&

1°&Screen&Yeast&&

3°&Screen&Worm&HTP&

MOA&&Screen&&

Disease&Models&

2°&Screen&Yeast&&

Dose&Response&&

ChronoscreenTM&

M.O.A&Screen& GeneBc&Analysis& Worm&RNAi& Target&ID& Transcriptomics&

Disease&Models& Parkinson’s&Disease& HunBngton’s&Disease& Obesity& ALS&& Alzheimer’s&Disease& Neuronal&ProtecBon&& Lysosomal&Disorders& Mitochondrial&Disorders& Muscular&Dystrophy& Fibrosis&& Diabetes&&

YEAST&RESULTS& WORM&RESULTS&

Compound& Healthspan&&Median&Lifespan&

Healthspan&Median&Lifespan&

Standards&

Resveratrol& +5%& +20%& +16%& +6%&

Caffeine& +5%& +10%& +14%& +6%&

Chronos&Compounds&

RDC5& +50%& +80%& +80%& +33%&

CHR1& +25%& +70%& +25%& +27%&

Defined&pathways&influence&the&development&of&ageZrelated&phenotypes&and&are&highly&conserved&among&species,&from&yeast&to&humans.&&

  Resegng&ageing&controls&in&yeast&and&worms&&

  High&efficacy&in&mouse&disease&models&  TherapeuBc&value&in&humans&&

10 proprietary leads, 67 repositioned leads Focused&Library&of&30,000&&

Small&Molecules&

Library&of&1200&&FDA/EU&approved&drugs&

SOD1&&Survival&Data&

ALS Diagnosis 2-8/100,000 Population

Riluzole: Glutamate antagonist

9% chance of 1 year

improved survival**

Average gain 2-3 months.

No symptom improvement

Tracheotomy Artificial

Ventilation, Death

FVC reduction Supportive care,

Nasogastric feeding,

Supportive care drugs:

Tizanidine, Baclofen

Botulinum toxin

Anticholinergics

Antidepressants

Phase 2 Goal: Lead compound delays reduction in FVC (breathing difficulties), +ve effect on

ALSFRS-R.

Phase 3 Goal: Lead compound delays reduction in FVC (breathing difficulties) .

Reduces ventilation need and extends ventilator free and absolute lifespan, together with ALS Disease Activity Index improvement via ALSFRS-R.

Problem/SoluBon&in&ALS&

Average time to death: 39 Months, 10 Year Survival <10%

Chronological&Controls

ReplicaBve&Controls

p&values&generated&from&tKtest,&6&week&treatment&regime&&

A53T&Survival&Data&

Trial&ongoing&–&further&data&accumulaBon&from&52&weeks&onwards&expected&

Effect&of&RDC5&on&PolyQ&Expressing&C".elegans

0 5 10 15 20 250

10

20

30

40

50

60

70

80

90

100

Days

Popu

laCo

n&Viability&&(%)

WT&+&ControlQ35&+&ControlQ35&+&RDC5

17%&&Median&Lifespan&

Extension&

PolyQ&&Survival&Data&

ReducBon&of&AggregaBon&in&Murine&Striatum&

ALS&TRIALS&

InZvivo&Brain&Stem&Analysis&  MRI:&NeurodegeneraBon&&  MRS:&Metabolic&changes&

OpBon&for&early&cull&group&to&observe&disease&progression&3&doses,&oral&gavage,&6days/week,&main&cohorts&n=20,&early&cull&

groups&n=5&&&

ALS&SOD1&Mice&&(TgNZSOD1ZG93AZ1Gur)&&

60&days&

175&days&

Chroneuro&or&Vehicle&Dosing&

Survival,&Body&Weight,&Disease&Onset&&&Stage&Scoring&

120&days&

Early&Cull&Cohort&

G93A&SOD1&OverKExpression&

Rotarod,&Grip&Strength&&&&&Open&Field&Tests&

90&days&

ExZvivo&Analysis&  Histology:&Motor&neuron&

counts&  RNA&processing&efficiency&

Background:&C57BL/6.&3&dose&groups,&aged&and&young&cohorts,&oral&gavage,&6days/week&

WT&Mice&&(Aged&3m&&&18m)&

Month&0& Month&6&

Chroneuro&or&Vehicle&Dosing&

Survival,&Body&Weight,&Disease&Onset&&&Stage&Scoring&

Young&&&Aged&Mice&

Biochemical&Readouts&  TDPK43&

localizaBon&  UbiquiBn&

localizaBon&&  RNA&processing&

efficiency&  RNA&ediBng&&

Histology&  TDPK43&localizaBon&  UbiquiBn&deposits&  Motor&neuron&counts&Other Ex-vivo Analysis   RNA processing efficiency

InZvivo&Brain&Stem&Analysis&  MRI:&

NeurodegeneraBon&&  MRS:&Metabolic&

changes&

3&doses,&oral&gavage,&6days/week,&cohorts&n=20,&4&week&duraBon&

Week&0&

Week&4&

Chroneuro&or&Vehicle&Dosing&

Survival,&Body&Weight,&Onset&&&Stage&Scoring&

TDPK43&Model&

Rotarod,&Grip&Strength&&&Open&Field&Tests&

ALS&TDPZ43&Mice&(virally&

transfected)&

Control Chroneuro

TDP-43 3/27

ALS Non-ALS

Tissue sample Band present at correct size?

(±10%) 151 ALS YES (231 bp)

152 ALS YES (232 bp)

153 Non-ALS NO

154 Non-ALS NO

Primer& ALS& NonZALS&151& 152& 153& 154&

TARDBP1&5/9& 1& 0& 1& 1&TARDBP1&5/13& 1& 1& 0& 0&TARDBP1&5/17& 1& 1& 1& 1&TARDBP1&5/21& 1& 1& 1& 0&TARDBP1&5/25& 1& 0& 1& 1&TARDBP1&9/13& 1& 1& 0& 1&TARDBP1&9/17& 1& 1& 1& 1&TARDBP1&9/21& 1& 0& 1& 1&TARDBP1&9/25& 1& 0& 0& 1&TARDBP1&13/21& 0& 0& 0& 0&TARDBP1&13/25& 1& 0& 0& 1&TARDBP1&17/21& 1& 1& 1& 1&TARDBP1&17/25& 1& 1& 1& 1&TARDBP1&21/25& 1& 1& 1& 1&

Primer& ALS& NonZALS&151& 152& 153& 154&

TARDBP1&3/11& 1& 1& 1& 1&TARDBP1&3/15& 1& 0& 1& 0&TARDBP1&3/19& 0& 0& 0& 0&TARDBP1&3/23& 1& 1& 0& 1&TARDBP1&3/27& 1& 1& 0& 0&TARDBP1&11/15& 1& 0& 1& 0&TARDBP1&11/19& 0& 0& 1& 1&TARDBP1&11/23& 1& 1& 1& 1&TARDBP1&11/27& 1& 1& 1& 1&TARDBP1&15/23& 1& 1& 1& 1&TARDBP1&15/27& 1& 1& 1& 1&TARDBP1&19/23& 1& 1& 1& 0&TARDBP1&19/27& 0& 0& 1& 1&TARDBP1&23/27& 1& 1& 1& 1&

TDP-43 19/27

ALS Non-ALS

Tissue sample Band present at correct size?

(±10%) 151 ALS NO

152 ALS NO

153 Non-ALS YES (366 bp)

154 Non-ALS YES (365 bp)

11022000 11105000 3 5 9 11 13 15 19 21 23 25 27

17

ALS

Non-ALS

11022000 11105000 3 5 9 11 13 15 19 21 23 25 27

17

Key Present in 2 of 2 ALS samples and not in any non-ALS samples

Present in 2 of 2 non-ALS samples and not in any ALS samples