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Drug discovery for neurological disorders: A focus on bioassay development
Anél Petzer Content
What is research and the 01 drug discovery process
Research focus: 02 Neurological diseases
03 Biological evaluation
04 Research highlights
05 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. Drug Discovery Process: Research topic Idea Drug design plan/strategy (mostly an idea) Lead Synthesis of novel compounds/ compound screening of libraries
Drug-like properties (solubility, Properties permeability etc.)
Biological Biological evaluation in vitro evaluation (laboratory) and in vivo (animals)
Computer Computer aided drug design and modelling modelling 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….
…..Marie Curie Neurological Disorders A neurological disorder is any disorder of the nervous system caused by structural, biochemical or electrical abnormalities (Wikipedia) Parkinson’s Disease Alzheimers’s Disease Depression • Reduced dopamine • Reduced • Reduced serotonin and production acetylcholinesterase noradrenalin production • Motor symptoms, dementia, production • Common but serious mood depression • Dementia disorder (different types) • MAO-B, COMT, • AChE, BuChE and NOS • MAO-A and NOS 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 • Proteins that act as the body’s catalysts (speed up a chemical reaction) Computer aided drug design Biological evaluation Assay development procedure
1. IC50 determination (Test the activity of the chemical compound for the specific enzyme)
100 M 80 60 mM (10-3 M) 50 %
Rate % Rate 40 20 µM (10-6 M)
-4 -2 0 2 4 6 log [I] nM (10-9 M) Biological evaluation Assay development procedure
2. Determine the mode of inhibition (competitive, uncompetitive, non-competitive etc.) Lineweaver-Burk plots and Michaelis Menten enzyme kinetics
Drug vs. Substrate Drug + Substrate/Enzyme Allosteric binding site (same active site) complex (different binding site) Biological evaluation Assay development procedure
MAO-B 3. Determine the reversibility of the inhibition 100 75
(Dilution, time-dependent, dialysis) 50 Rate (%) Rate
25
0 NINB depr NB dialysed undialysed
Reversible inhibition Irreversible inhibition Research Highlights
Propargylamine inhibitor (2-PAT) 7 Methylene Blue and Azure B projects (Dual MAO and 1 AChE) Sulfonamides (nM) 6
Methylene Blue 2 analogues and similar dye compounds Dual MAO and 5 COMT inhibitors Benzoquinone as 3 irreversible MAO-B and Isatins 4 MAO-A inhibitors Methylene Blue and Azure B projects
Methylene Blue IC50 Azure B IC50
MAO-A: 0.07 µM MAO-A: 0.011 µM MAO-B: 4.37 µM MAO-B: 0.968 µM
AChE: 0.214 µM AChE: 0.486 µM BuChE: 0.389 µM BuChE: 1.99 µM Azure B = reversible, competitive MAO inhibitor Methylene Blue analogues and similar dye compounds
ETC Dimethyl MB
Nile Blue Methylene Blue Cresyl Violet (MB)
Neutral Red New MB Enzymology Methylene Blue Analogues Nile Blue MAO-B MAO-B MAO-B 100 125 Slope NR 75 100
75 NB 50 [I], M DMMB Rate (%) Rate 50 Rate (%)
NMB 25 (%) 1/V 25 CV
0 0 -4 -2 0 2 NINB depr NB Log [I] dialysed undialysed 1/[S]
MAO-B Mode of Reversibility IC50 inhibition NB: 0.012 µM Reversible Competitive MB: 4.37 µM Enzymology Methylene Blue Analogues Nile Blue
MAO-A MAO-A MAO-A 100 125 Slope 100 75
75 DMMB 50 [I], M
50 (%) Rate Rate (%)
NMB NR (%) 1/V NB 25 25 CV
0 0 NINB parg NB -4 -2 0 2 Log [I] dialysed undialysed 1/[S]
MAO-A Mode of Reversibility IC50 inhibition NB: 0.0077 µM Reversible Competitive CV: 0.0037 µM MB: 0.07 µM Molecular docking Methylene Blue Analogues
MAO-A MAO-B
Methylene Blue
Nile Blue Benzoquinones as irreversible MAO-A and MAO-B inhibitors
Benzoquinone O
O O
TMN
O
O Enzymology Benzoquinone as irreversible MAO-A and MAO-B inhibitors
100
100 MAO-A - 5d 75 MAO-B - 4 75 Not applicable to 50
50 (%) Rate
Rate (%) Rate 25 irreversible 25 0 inhibitors 0 NI5d parg 5d -3 -2 -1 0 1 2 3 Log[I] dialysed undialysed
Mode of IC Reversibility 50 inhibition
MAO-A: 5.84 µmIrreversible Not determined Enzymology Benzoquinone as irreversible MAO-A and MAO-B inhibitors
100 100 MAO-A - 5d MAO-B - 4 75 75
50 50
Rate (%) Rate Concluded from Rate (%) Rate 25 25 previous studies 0 0 NI4 depr 4 -3 -2 -1 0 1 2 3 Log[I] dialysed undialysed
Mode of IC Reversibility 50 inhibition
MAO-B: 10.2 µM Reversible ? Competitive Proposed mechanism Nucleophiles react with 1,4-benzoquinones to form covalent adducts
MAO-A (irreversibility) Isatins
O
O N H
O 5 O 6 N O
H O O N H
O O O N H Enzymology Isatins
4 3 1.6 3 1.2 2 2 0.8 Rate 1/V Rate 1 0.4 1 0.0
0 -3 -2 -1 0 1 2 3 0 15 30 60 -0.02 0.00 0.02 0.04 0.06 Log[I] Incubation time (min) 1/[S]
Mode of IC Reversibility 50 inhibition
MAO-B: 0.00066 µM Reversible Competitive 0.66 nM O Enzymology O N Isatins H
30 0.60 25 8
20 0.45 6 15 0.30 Rate
4 1/V Rate 10 0.15 5 2
0 0.00 -3 -2 -1 0 1 2 3 0 15 30 60 -0.02 0.00 0.02 0.04 0.06 Log[I] Incubation time (min) 1/[S]
Mode of IC Reversibility 50 inhibition
MAO-A: 0.562 µMReversible Competitive Dual MAO and COMT inhibitors
O Tolcapone HO HO
NO2 O HO N CN HO
NO2 Entacapone O HO Enzymology
HO Dual MAO and COMT inhibitors NO2 Br
100
100 75
75 Slope
50
50 (%) Rate [I], M Rate (%) Rate
25 (%) 1/V 25
0 0 -3 -2 -1 0 1 2 3 NI1d sel 1d 1/[S] Log[I] dialysed undialysed
Mode of IC Reversibility 50 inhibition
MAO-B: 13.9 µMReversible (MAO-B) Competitive Enzymology Dual MAO and COMT inhibitors
1a 1b 1c 1d 100 100 100 100
75 75 75 75
50 50 50 50 Rate(%) Rate(%) Rate(%) Rate(%) 25 25 25 25
0 0 0 0 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 Log[I] Log[I] Log[I] Log[I]
1e 1f 1g 1h 100 100 100 100
75 75 75 75
50 50 50 50 Rate(%) Rate(%) Rate(%) Rate(%) 25 25 25 25
0 0 0 0 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 Log[I] Log[I] Log[I] Log[I] A chromatogram routinely obtained for the detection and quantitation of normetanephrine 1i 1j 1k 100 100 100 generated through the COMT-catalysed 75 75 75
50 50 50
Rate(%) Rate(%) Rate(%) methylation of (-)-norepinephrine. The retention 25 25 25 0 0 0 times of (-)-norepinephrine and -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 Log[I] Log[I] Log[I] normetanephrine are 2.8 min and 4.1 min, respectively
IC50
COMT IC50: 0.07 to 0.29 µM Sulfonamides (nM)
IC50 MAO-B = 0.00051 µM = 0.51 nM Propargylamine inhibitor (2-PAT)
Selegiline/deprenyl Rasagiline Pargyline
2-PAT
MAO-A: 0.721 µM MAO-B: 14.6 µM Propargylamine inhibitor (2-PAT)
MAO-A MAO-BMAO-B 100 100
75 75
50 50 Rate (%) Rate (%) Rate 2-PAT 25 25
0 0 NI2-PAT parg 2-PAT NI2-PAT seleg 2-PAT dialysed undialysed dialysed undialysed Purpose of research (why do we still do research?)
• 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. Thank you Special thanks / Dankie sê……. • 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