identification of new inhibitors of plasmodium falciparum enoyl- acp reductase symposium on:...
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Symposium on: Advances in Parasitology “Education and Research in Parasitology in the service of Mankind “
Malaria, what is it?Malaria, what is it?
A parasitic infection caused by the Plasmodium parasite and transmitted by female Anopheles mosquitoes.
Why do we need to focus on malaria?Why do we need to focus on malaria?
• Infects 200-300 million people every year!
• Kills one million peoples annually!!!
• Kills one child every 30 seconds!!!!
Gambian child with severe malaria Gambian child with severe malaria anemiaanemia
Drug Introduction In vivo resistance confirmed
Origin
Chloroquine 1945 Early 1960s
Southeast Asia,
South America
Sulfadoxine and pyrimethamine(Fansidar)
1967 Late 1960s Southeast Asia,
South America
Mefloquine (Lariam) 1985 Early 1990s Southeast Asia
Atovaquone and proguanil (Malarone)
2000 2002 Africa
Artemether and lumefantrine (Coartem)
2001 2008 Southeast Asia
The Challenge of Resistance!
How could the problem of resistance be tackled
• Three approaches are under investigation:
1/ The novel use of older drugs ( use in combination)
2/ The re-design of existing drugs ( develop analogues, discovery of natural products)
3/ Validation of novel parasite- specific drug targets ( benefit from the better understanding of the parasite biology and genomics)
e.g. targeting a parasitic enzyme, which is not present in mammals, or which has significant structural differences from the corresponding enzyme in mammals
Example: The chosen target, may over time, lose its sensitivity to the drug
Example: The penicillin-binding-protein (PBP) known to be the primary target of penicillin in the bacterial species Staphylococcus aureus has evolved a mutant form that no longer recognizes penicillin.
PfENR Inhibition assay was established, standardized and optimized for screening
Classification of PfENR inhibitors
PfENR inhibitors
Compound code Concentration (mM) % inhibition IC50 (µM) ± SEM
1 0.007 86.9 2.0 ± 0.023 0.05 89.1 7.6± 0.803 0.05 81.5 33.4± 0.34 0.10 88.2 35.2 ± 0.25 0.05 57.0 42.7 ± 0.36 0.07 66.9 58.9 ± 0.57 0.12 81.0 59.9 ± 6.211 0.25 64.6 62.8 ± 3.213 0.25 80.1 91.13± 3.816 0.25 80.9 195.0 ± 1.021 0.25 51.1 248.3 ± 0.7242529
0.350.501.00
50.560.454.5
329.5 ± 8.4 345.3 ± 1.4 920.2 ± 3.2
% of inhibition and IC50 values of PfENR inhibitors from natural
sources
HO O
OOH
OH
OHO
OH O
OH
2
3
456
7
89
10
1'
2' 4'
6'
2a
3a
4a 5a
6a
7a
8a9a
10a
1'a
2'a4'a
6'a
11
O
O
OH
OH
OMe
OMe
O
O
OH
OH
MeO
MeO
CH2
MeO
OMe
AB
C
D E
F
O
OOH
HO
OH
OH
3 13 21
O
OOH
HO
OH
OH
O
O
OH
OH
HO
O
O
O
OCH3
HO
OH
H3CO OCH3
O
OOH
HO
OH
OH
O
9 25 16 7
OH
OH
HO
Gossypol (compound 1) 27 28
Kinetic studies on gossypol (compound 1):
IC50= 2.0 µM
Ki= 4.0
Mechanism of Inhibition: competitive with regard to the substrate, crotonyl CoA
Lineweaver-Burk
1/[Crotonyl CoA] (µM)
-0.015 -0.010 -0.005 0.000 0.005 0.010 0.015
1/R
ate
(µm
ol/m
in/m
g)
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
I = 0.5I = 3.5I = 7
Vmax = 27.9 Km = 66.5 Ki = 4.
Dixon
[IGossypol] (µM)
-15 -10 -5 0 5 10
1/R
ate
(µm
ol/m
in/m
g)
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
S = 80S = 120S = 200S = 500
Vmax = 27.9 Km = 66.5 Ki = 4.
Kinetic Behavior of Gossypol
O
OH
O
O
O
O
O
O
H
H
H
H
H
Gly104
Ile105
Gly106
Asp107
Gly110
Tyr111
Gly112
Trp113
Ser215
Leu216
Ala217
Leu265
Thr266
Tyr267 Lys
285
Ala312
Gly313
Pro314
Leu315
Ser317Arg
318
Ala319
Ala320
Schematic representation of the binding pocket of Gossypol on the surface of PfENR.
Michaelis-Menten
[Crotonyl CoA] (µM)
0 100 200 300 400 500 600
Ra
te (µ
mo
l/min
/mg
)
0
2
4
6
8
10
12
14
16
I = 0I = 50I = 75I = 85
Vmax = 17.1 Km = 80.6 Ki = 177.4
O
OOH
HO
OH
Lineweaver-Burk
1/[Crotonyl CoA] (µM)
-0.020 -0.015 -0.010 -0.005 0.000 0.005 0.010 0.015
1/R
ate
(µ
mo
l/min
/mg
)
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
I = 0I = 50I = 75I = 85
Vmax = 17.1 Km = 80.6 Ki = 177.4
H
O
O
O O
O O
O
O
O
H
H
H
H
H
H
O
O
O
H H
-
-
-Gly104
Ile105
Gly106
Gly110Tyr
111
Ile130
Trp131
Val134
Ser215
Leu216
Ala217
Asn218
Ala219
Tyr267
Tyr277
Gly313
Pro314
Leu315
Ser317
Ala319 Ala
320
Ala322
Binding models of PfENR in complex with compound 4
Score value = 10.34
O
O
O
O
OO
H
HH
H
Gly104
Gly106
Asp107
Gly110
Trp131
Val134
Asp168
Ala169
Ser170
Leu216
Asn218
Ser317
Arg318
Ala319
Representation of compound 21 docked to the active site of PfENR. Score value =7.20/ IC50 = 100 µM
CONCLUSION AND FUTURE DIRECTIONS The current study has resulted in the identification of new PfENR inhibitors with chemical diversity and broad range of potency
We have identified here for the first time the well know natural compound ‘Gossypol’ as a potent PfENR inhibitor with IC50 and Ki values of 2.0 µM and 4 respectively
Gossypol was know for its antimalarial activity but through the inhibition of Plasmodium falciparum Lactate Dehydrogenase (PfLDH). If we consider its newly identified PfENR inhibitory activity, gossypol could be described as dual-target inhibitor, a critical feature for a hit to be developed into potential antimalarial lead
The kinetic behaviour and the analysis of the inhibitors-PfENR complexes would pave the way for the development of these inhibitors into antiplasmodial agents
• Sudan's diverse and virgin flora
• Rich traditional medicine knowledge
• Well-equiped drug discovery facility
• Enzymology and enzyme inhibition Rsearch
• Antioxidant discovery research
• Structural computational chemistry research.
Medical Biochemistry Research Lab. (http://medicalbiochem.googlepages.com)
ACKNOWLEDGEMENTS
Many people have contributed to this work
Prof Asaad Khalid Members of ICCBS
Prof Mohammed Galal
Prof Hassan Alsubki Members of lab # 404, HEJ
Prof Mohamed Iqbal Members of MAPRI
Dr. Mohamed A. Mesaik Financial fund of the IDB
Dr. Omer M. Abdellah Financial fund of the Ministry
Dr. Talal A.Awad of Higher Education
Mr. Elteab Fadul Omdurman Islamic University
Miss Fatima Elfatih
Miss Ghada
THANK THANK YOUYOU
THANK THANK YOUYOU