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