Identification of Novel Anticonvulsant Drugs using a Larval … · 2016. 12. 15. · F3394-0676 •...

Simon Hand Vincent Cunliffe (Department of Biomedical Science) Joseph Harrity (Department of Chemistry) Val Gillet (Information School)

Identification of Novel Anticonvulsant Drugs using a Larval Zebrafish Model

Epilepsy Our assay Screening of kinase inhibitors Future work

Epilepsy

• Estimated 50 million cases worldwide

• A neurological syndrome with many clinically distinct subtypes, all of which are characterised by seizures

• Seizures are periods of acute, dysregulated synaptic activity in the brain

Epilepsy

• The currently available anti-epileptic drugs are not effective enough

• 30% of people with epilepsy fail to respond satisfactorily to first line treatments

• Those drugs that are affective have unwanted side effects

Epilepsy

Seizures are attributed to imbalances of excitatory and inhibitory neurotransmitter activities in the brain; glutamate and γ-amino butyric acid (GABA) respectively

Glutamate (+) GABA (-)

Epilepsy

GABA (inhibitory)

Glutamate (excitatory)

Normal neurotransmission

Epilepsy

GABA (inhibitory)


In a seizure...

Epilepsy

GABA (inhibitory)


In a seizure...

Seizure

Zebrafish model

– Mapped genome

– Rapid development

– High through-put vertebrate model

– Compounds can be absorbed through aqueous media

– Transparency allows whole mount visualisation of gene expression via in situ hybridisation

• Pentylenetetrazole (PTZ) is a convulsant. Exposure to PTZ triggers a seizure by strong antagonistic action at the GABA A receptor

Santoriello et al. J Clin Invest. 2012;122(7):2337-2343.

http://www.jci.org/122/7

The Assay

The Assay

GABA (inhibitory)


transmission of the inhibitory GABA signal is blocked by Pentylenetetrazole (PTZ) inducing a seizure

Seizure

In a PTZ-induced seizure...

The Assay

In a PTZ-induced seizure...

GABA (inhibitory)


Seizure Motor

transmission of the inhibitory GABA signal is blocked by Pentylenetetrazole (PTZ) inducing a seizure

Screening for Anticonvulsants

• A seizure “switches on” particular pattern of gene expression in the brain e.g. cfos gene (right)

• cfos expression after PTZ exposure is not observed if an effective anticonvulsant is administered

• Positive control is the known anticonvulsant Valproic Acid (VPA)

Valproic acid (VPA)

Baxendale et al. Disease Models and Mechanisms, 5, 000-000 (2012)

Screening of Kinase Inhibitors

• The specific pathways involved in seizures are still largely unknown, but are likely to include kinase enzymes

• Therefore, we tested the hypothesis that kinase inhibition is a valid anticonvulsant strategy

• Target focussed library screening of over 1000 kinase inhibitors

Presence of phosphate group

can be detected by other proteins/cells

Protein Protein

ATP ADP

Kinase activity

+


Compounds screened in a 96 well plate, initially at 25μM

In each well…

In Situ

Miss

or

Hit

PTZ (convulsant)

Potential Anticonvulsant


A hit in the in situ

screen tells us…

Compound has prevented the

seizure

OR

The compound interferes

with gene expression

OR

The larvae are dead

A hit in the locomotor

screen tells us…

Larvae has not has a seizure

OR

Compound is sedative

OR

Compound paralysed larvae

OR

The larvae are dead

Miss (cfos expression)

or

Hit (no cfos expression)



screen tells us…


seizure

OR



OR

The larvae are dead


screen tells us…

Larvae has not has a seizure

OR


OR


OR

The larvae are dead

Miss (cfos expression)

Hit (no cfos expression)

or


42 hits confirmed by in situ repeat screens

As cfos expression can be repressed independently of stopping a seizure (false positive by in situ) anticonvulsant activity must be confirmed independently

10 of the most potent were bought in to test in in locomotor assays


Miss

or

Hit

PTZ (convulsant)



Miss

or

Hit

PTZ (convulsant)


PTZ-induced seizures trigger a large observable increase in motor activity, which can be tracked.

no convulsant PTZ PTZ + effective anticonvulsant


Effective anticonvulsant compounds reduce the distance travelled after PTZ exposure



Effective anticonvulsant compounds reduce the distance travelled after PTZ exposure


Non anticonvulsant Anticonvulsant


6 of 10 hit compounds show significant dose dependant reduction in locomotor activity after PTZ exposure no convulsant PTZ PTZ + effective

anticonvulsant

Non anticonvulsant Anticonvulsant


A hit in the In situ

screen tells us…

Brain has not/had a seizure

OR



OR

The larvae are dead


screen tells us…

Compound has prevented the seizure

OR


OR


OR

The larvae are dead


What does each screen tell us?


screen tells us…


seizure

OR



OR

The larvae are dead


screen tells us…


OR


OR


OR

The larvae are dead



screen tells us…


seizure

OR



OR

The larvae are dead


screen tells us…


OR


OR


OR

The larvae are dead

If a compound stops both the transcriptional and behavioural seizure responses then it is likely that that compound is a true hit



screen tells us…


seizure

OR



OR

The larvae are dead


screen tells us…


OR


OR


OR

The larvae are dead

OR…



screen tells us…


seizure

OR



OR

The larvae are dead


screen tells us…


OR


OR


OR

The larvae are dead


In Situ

Miss

or

Hit

PTZ (convulsant)



Miss

or

Hit


• 48hpf embryos have a functional, visible heart

• Effect of compounds on both heart rate and morphology can be observed under microscope


Normal heart rate

tells us…

Compound is not

toxic


screen tells us…


OR


OR



screen tells us…

Compound has

prevented the seizure

OR

The compound

interferes


- thus locomotor and in situ results are valid


Normal heart rate

tells us…

Compound is not

toxic

AND

Compound has



screen tells us…


OR


OR



screen tells us…

Compound has


OR

The compound

interferes



Normal heart rate

tells us…

Compound is not

toxic

AND

Compound has



screen tells us…


OR


OR



screen tells us…

Compound has


OR

The compound

interferes


A validated hit compound is one that is not toxic and prevents the behavioural and transcriptional responses to a seizure

Locomotor response to PTZ

In Situ’s

Media alone

PTZ 20uM 10uM 5uM 2.5uM

Hit 9

Heart rates

Toxicity

E3

DM

SO

20

uM

1

10

uM

1

2.5

uM

1

5u

M 1

20

uM

2

10

uM

2

5u

M 2

2.5

uM

2

20

uM

3

10

uM

3

5u

M 3

2.5

uM

3

20

uM

4

10

uM

4

5u

M 4

2.5

uM

4

20

uM

5

10

uM

5

5u

M 5

2.5

uM

5

20

uM

6

10

uM

6

5u

M 6

2.5

uM

6

20

uM

7

10

uM

7

5u

M 7

2.5

uM

7

20

uM

8

10

uM

8

5u

M 8

2.5

uM

8

20

uM

9

10

uM

9

5u

M 9

2.5

uM

9

20

uM

10

10

uM

10

5u

M 1

0

2.5

uM

10

0

2 0

4 0

6 0

8 0

1 0 0

A ll C k 1 H its T o x ic ity - 2 4 h p o s t e x p o s u re (3 d p f)

T re a tm e n t

% o

f la

rva

e p

er

co

nd

itio

n

N o rm a l

O dem a

C u rv e d S p in e

O d e m a + C u rv e d s p in e

N o H e a rtb e a t

Best so far – F3394-0676

Media alone


cfos

F3394-0676

• 10 analogues of F3394-0676 selected

• Tested in in situ, locomotor and toxicology assays

Summary

1. Hit identification =

2. Hit confirmation = 3. Hit validation =

4. Hit investigation =

5. Lead identification =

Tim

esc

ale

Can kinase inhibition prevent PTZ-induced seizures?

Are you sure?

Are you really sure?

How do these compounds work?

Can we develop novel compounds that prevent seizures better than those we start with?

Yes

Yes

Something like this…

Yes

Summary

1. Hit identification =

2. Hit confirmation = 3. Hit validation =

4. Hit investigation =

5. Lead identification =

Tim

esc

ale

Can kinase inhibition prevent PTZ-induced seizures? (in situ’s)

Are you sure? (locomotor assays)

Are you really sure? (heart rates)

How do these compounds work?

Can we develop novel compounds that prevent seizures better than those we started with?

Yes

Yes

This way…

Sure enough

Investigation of F3394-0676

• 10 analogues of F3394-0676

• Currently being tested via in situ, locomotor and toxicology assays

Moe 2014.09

Future Work

qPCR- to quantify the transcriptional responses

F3394-0676 kinase inhibition profiling

Searching for compounds that match our pharmacophore of F3394-0676 analogues

Must develop selection criteria to select ligands from, for example, ChEMBL to promote structural diversity

In vivo (zebrafish)

In Silico (modelling)

In situ (Chemistry)

Acknowledgements

Particular thanks to

Vincent Cunliffe

Sarah Baxendale

Joe Harrity

Val Gillet

Acknowledgements

Particular thanks to

Vincent Cunliffe

Sarah Baxendale

Joe Harrity

Val Gillet

…Any Questions?

In Situ’s

Media alone


Hit 9

Hit 4

Identification of Novel Anticonvulsant Drugs using a Larval … · 2016. 12. 15. · F3394-0676 •...

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Transcript of Identification of Novel Anticonvulsant Drugs using a Larval … · 2016. 12. 15. · F3394-0676 •...