Drug discovery for neurological disorders: A focus on bioassay development / Anél Petzer

Drug discovery for

neurological disorders:

A focus on bioassay

development

Anél Petzer LogoType

Content

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What is research and the drug discovery process

Biological evaluation Research focus:

Neurological diseases

Research highlights Concluding remarks

Medicinal Chemistry ….?

Medicinal/Pharmaceutical chemistry deals with the discovery, design, development and both

pharmacological and analytical

characterisation of drug substances.

Medicinal chemists are indispensable in the preclinical stages of drug

development, and again as

pharmaceutical chemists in drug quality control.

Idea Lead compound Biological evaluation Computer modelling Research topic

Drug design plan/strategy (mostly an idea)

Synthesis of novel compounds/ screening of libraries

Drug-like properties (solubility, permeability etc.)

Biological evaluation in vitro (laboratory) and in vivo (animals) Computer aided drug design and modelling

Properties

A scientist in his laboratory is not a mere

technician: he is also a child confronting

natural phenomena that impress him as

though they were fairy tales….

Alzheimers’s Disease

• Reduced

acetylcholinesterase production

• Dementia

• AChE, BuChE and NOS

Neurological Disorders

A neurological disorder is any disorder of the nervous system caused by structural, biochemical or electrical abnormalities (Wikipedia)

Parkinson’s Disease

• Reduced dopamine

production

• Motor symptoms, dementia, depression

• MAO-B, COMT, NOS

Depression

• Reduced serotonin and noradrenalin production • Common but serious mood

disorder (different types) • MAO-A and NOS

Biological assays

Why is biological assay development important?

The big question…. Why is it necessary to focus on bioassay

development?



Scarce skill



Backbone of drug discovery process



Good assay leads to reliable results



Assay should be robust, quick and cheap to perform



Alternatives available - troubleshoot

Biological evaluation

Structure and functions of enzymes

-4 -2 0 2 4 6 20 40 60 80 100 log [I] R at e %

1. IC

₅₀

determination

(Test the activity of the chemical compound for the specific enzyme)

Biological evaluation

Assay development procedure

M

mM (10-3 _M)

µM (10-6 _M)

nM (10-9 _M) 50 %

2. Determine the mode of inhibition

(competitive, uncompetitive, non-competitive etc.)

Lineweaver-Burk plots and Michaelis Menten enzyme kinetics

Biological evaluation

Assay development procedure

Drug vs. Substrate Drug + Substrate/Enzyme Allosteric binding site

3. Determine the reversibility of the inhibition

(Dilution, time-dependent, dialysis)

Reversible inhibition Irreversible inhibition

Biological evaluation

Assay development procedure

0 25 50 75 100 dialysed undialysed NB NB NI depr MAO-B R a te ( % )

Research Highlights

1 6 5 4 Methylene Blue

analogues and similar dye compounds

Methylene Blue and Azure B projects (Dual MAO and

AChE)

Benzoquinone as

irreversible MAO-B and MAO-A inhibitors

Dual MAO and COMT inhibitors Sulfonamides (nM) Isatins 2 3 7 Propargylamine inhibitor (2-PAT)

Methylene Blue and Azure B projects

Methylene Blue IC₅₀ MAO-A: 0.07 µM MAO-B: 4.37 µM AChE: 0.214 µM BuChE: 0.389 µM Azure B IC₅₀ MAO-A: 0.011 µM MAO-B: 0.968 µM AChE: 0.486 µM BuChE: 1.99 µM

Azure B = reversible, competitive MAO inhibitor

Methylene Blue analogues and similar dye

compounds

ETC

Nile Blue

Neutral Red New MB

Dimethyl MB

Methylene Blue (MB)

Enzymology

Methylene Blue Analogues

MAO-B

IC

₅₀

Reversibility

_inhibition

Mode of

NB: 0.012 µM MB: 4.37 µM Reversible Competitive -4 -2 0 2 0 25 50 75 100 125 NR NB NMB CV DMMB MAO-B Log [I] R at e (% ) 0 25 50 75 100 dialysed undialysed NB NB NI depr MAO-B R at e ( % )           1/[S] 1/ V ( % )        [I],M S lo p e MAO-B Nile Blue

Enzymology

Methylene Blue Analogues

MAO-A

IC

₅₀

Reversibility

Mode of

inhibition

NB: 0.0077 µM CV: 0.0037 µM MB: 0.07 µM Competitive Reversible -4 -2 0 2 0 25 50 75 100 125 NR NB NMB CV DMMB MAO-A Log [I] R at e (% ) 0 25 50 75 100 dialysed undialysed NB NB NI parg MAO-A R at e ( % )           1/[S] 1/ V ( % )        [I],M S lo p e MAO-A Nile Blue

MAO-A

MAO-B

Molecular docking

Methylene Blue Analogues

Methylene Blue

Benzoquinones as irreversible MAO-A and MAO-B

inhibitors

O O O O O TMN Benzoquinone

Enzymology

Benzoquinone as irreversible MAO-A and MAO-B inhibitors

IC

₅₀

Reversibility

Mode of

inhibition

MAO-A: 5.84 µm Irreversible Not determined

-3 -2 -1 0 1 2 3 0 25 50 75 100 MAO-B - 4 MAO-A - 5d Log[I] R a te ( % ) 0 25 50 75 100 dialysed undialysed 5d 5d NI parg R a te ( % ) Not applicable to irreversible inhibitors

Enzymology

IC

₅₀

Reversibility

Mode of

inhibition

Competitive Reversible ? 0 25 50 75 100 dialysed undialysed 4 4 NI depr R at e ( % ) -3 -2 -1 0 1 2 3 0 25 50 75 100 MAO-B - 4 MAO-A - 5d Log[I] R a te ( % ) Concluded from previous studies MAO-B: 10.2 µM

MAO-A (irreversibility)

Proposed mechanism

Isatins

N O O H N O O H O N O O H O N O O H 5 6

Enzymology

Isatins

IC

₅₀

Reversibility

Mode of

inhibition

MAO-B: 0.00066 µM 0.66 nM Competitive Reversible -3 -2 -1 0 1 2 3 1 2 3 4 Log[I] R at e 0 15 30 60 0 1 2 3

Incubation time (min)

R at e -0.02 0.00 0.02 0.04 0.06 0.0 0.4 0.8 1.2 1.6 1/[S] 1/ V

Enzymology

Isatins

IC

₅₀

Reversibility

Mode of

inhibition

MAO-A: 0.562 µM Reversible Competitive

-3 -2 -1 0 1 2 3 5 10 15 20 25 30 Log[I] R at e 0 15 30 60 0 2 4 6 8

Incubation time (min)

R at e -0.02 0.00 0.02 0.04 0.06 0.00 0.15 0.30 0.45 0.60 1/[S] 1/ V N O O H

Dual MAO and COMT inhibitors

O HO HO NO2 Tolcapone Entacapone NO2 HO HO CN N O

Enzymology

Dual MAO and COMT inhibitors

IC

₅₀

Reversibility

Mode of

inhibition

MAO-B: 13.9 µM Reversible (MAO-B) Competitive

-3 -2 -1 0 1 2 3 0 25 50 75 100 Log[I] R a te ( % ) 0 25 50 75 100 dialysed undialysed 1d 1d NI sel R a te ( % )           1/[S] 1/ V ( % )           [I],M S lo p e O HO HO NO2 Br

Enzymology

Dual MAO and COMT inhibitors

IC

₅₀

COMT IC₅₀: 0.07 to 0.29 µM

A chromatogram routinely obtained for the detection and quantitation of normetanephrine

generated through the COMT-catalysed

methylation of (-)-norepinephrine. The retention

times of (-)-norepinephrine and

normetanephrine are 2.8 min and 4.1 min, respectively -3 -2 -1 0 1 2 3 0 25 50 75 100 Log[I] R at e (% ) -3 -2 -1 0 1 2 3 0 25 50 75 100 Log[I] R at e (% ) -3 -2 -1 0 1 2 3 0 25 50 75 100 Log[I] R at e (% ) -3 -2 -1 0 1 2 3 0 25 50 75 100 Log[I] R at e (% ) -3 -2 -1 0 1 2 3 0 25 50 75 100 Log[I] R at e (% ) -3 -2 -1 0 1 2 3 0 25 50 75 100 Log[I] R at e (% ) -3 -2 -1 0 1 2 3 0 25 50 75 100 Log[I] R at e (% ) -3 -2 -1 0 1 2 3 0 25 50 75 100 Log[I] R at e (% ) -3 -2 -1 0 1 2 3 0 25 50 75 100 Log[I] R at e (% ) -3 -2 -1 0 1 2 3 0 25 50 75 100 Log[I] R at e (% ) -3 -2 -1 0 1 2 3 0 25 50 75 100 Log[I] R at e (% ) 1a 1b 1c 1d 1e 1f 1g 1h 1i 1j 1k

Sulfonamides (nM)

IC

₅₀

MAO-B

= 0.00051 µM

Propargylamine inhibitor (2-PAT)

Selegiline/deprenyl Rasagiline Pargyline

2-PAT

MAO-A: 0.721 µM MAO-B: 14.6 µM

Propargylamine inhibitor (2-PAT)

2-PAT 0 25 50 75 100 dialysed undialysed 2-PAT 2-PAT NI parg MAO-A R a te ( % ) 0 25 50 75 100 dialysed undialysed 2-PAT 2-PAT NI seleg MAO-B R a te ( % ) MAO-A MAO-B

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Basic research – contribution to specialised research field/topic

•

Training of postgraduate students (MSc and PhD) – researchers of the

future

•

_{International collaboration (establish international collaboration that}

leads to recognition)

•

Builds on the image not only for the NWU but also for South Africa

We like what we do! That is our passion.

Purpose of research

• Aan God al die eer

• My man Jacques, my seuntjies Adriaan en Phillip, my ouers en broers

• My familie en vriende

• Studente

• Proff. Jeanetta du Plessis en Sandra van Dyk

• Kollegas by die Skool vir Farmasie, Pharmacen en die NWU

• Medewerkers

• Dekaan, Skooldirekteur en Mev. Yolande Avenant

Special thanks /

Dankie sê…….