Quantifying the antimalarial activity of compounds through

High-Content Imaging Screening

Sowmya Gowda; Leonardo Lucantoni; Vicky M. Avery

Discovery Biology, Eskitis Institute for Drug discovery
Griffith University, Nathan 4111, Queensland, Australia
Sowmya.basavarajgowda@griffithuni.edu.au.

Abstract

Results

Malaria is caused by protozoan parasite called Plasmodium falciparum is the most

widely recognized parasitic disease in the world and leads to 438,000 deaths every
year. Development and spread of resistance, combined with decreasing efficacy of
the partner drugs used in Artemisinin Combination Therapy ,are the major concerns
in malaria therapy. New antimalarial compounds need to be discovered to identify
new drugs that are active against emerging resistant strains of the parasite with novel
mechanisms of action to replace the existing drugs. We have tested 560 compounds
from large scaffold library using high-content imaging assay and found 10 hits were
active against plasmodium 3D7 strains (drug sensitive) and Dd2 strains (multidrug
resistant) strains and showed no cytotoxicity against HEK293 mammalian cells and
therefore are good for starting point for further drug discovery.

Aims
The goal of this project was to evaluate the antimalarial activity of a collection of
representative compounds from a large scaffold chemical library, using high content
imaging-based assay, with the purpose of identifying new antimalarial candidates.

Introduction
Malaria is the most widely recognized parasitic disease in the world transmitted by
the female A nopheles mosquito and responsible for killing of millions of people .
Diseases caused by Plasmodium falciparum rapidly leads to clinical symptoms related with 48-hour asexual parasite
life cycle (Figure 1). Emergence
of parasite resistance to available
antimalarial drugs is the major
problem for malaria eradication.
High-throughput
screening
(HTS) methods for antimalarial
drugs have become available in
recent years. Nevertheless new
antimalarial compounds need to
be discovered to identify new
drugs that are active against
emerging resistant strains of the
parasite with novel mechanisms
of action to replace the existing
drugs (Wells et al., 2009) .

Screening of a scaffold library set GDB21.

Screening hits were defined as wells showing greater than 50% inhibition. 10 hits were found of which6 inhibited the parasites
(SN00797498, SN00775298, SN00786699, SN00784248, SN00790702, SN00791542 ) in atleast one biological replicate were identified as confirmed hits by analysis (Figure 4).Having found compounds with significant inhibition against the drug-sensitive parasite
strain 3D7 and multidrug resistant strain Dd2 at 5 M, to confirm the activity and potency
these compounds, they were tested against HEK293 mammalian cells (Figure 6).
Table 1: Average of reference antimalarals IC50 (n=2).
Antimalarial
Compounds

IC50

SEM

Chloroquine

15.55

2.85

Artemisinin

16.65

3.4

Pyrimethamine

16.1

1.75

Puromycin

143.65

22.95

Primaquine

4637.6

989.35

Table 2:IC50 of Screening hits and associated analogues against 3D7 and DD2

Methylene blue

7.75

1.1

strains and HEK cells (n=2).

Figure 5: Fluorescence images of positive (left)

and negative (right) control.

Screening Hits

Characterisation for screening hits and assessment of antimalarial activity of hit scaffolds.
All the 10 hits had more than 50% inhibition
against 3D7 and Dd2, except
SN00802052 . None of the active scaffolds
and analogues were found to be cytotoxic. All
the tested hits were found to be similarly active
against the P.falciparum 3D7 and Dd2 strains .
All the 10 hits confirmed activity on retest
against 3D7, Dd2 and HEK 293 mammalian
cells. IC50 ratio of Dd2/3D7 <1M is the best
hit. Best potency of scaffold hit belongs to included
analogues
like
SN00775286,
SN00775300 . Dose response curve of these
two compounds are plotted (Figure 7).

SN00797498

METHODS

This is a selection subset of

larger scaffold
library.GDB21
screening library,560
compounds
screened at
5M

Data analysis
IC50.Potency determination
against drug
sensitive 3D7
and multidrug
resistant Dd2
and for cytotoxicity against
HEK293 cells.

Hit determination >

50% inhibition.

Testing 2-3
representatives from the
big library
that share the
scaffold as
hits.

CL9375

SN00781285

0.5

0.64

SEM

1.01

1.4

0.15

99%

0.48

30.26%
2.50%
6.00%
82.50%

SN00783211
SN00781292
SN00781301
SN00769485

SC000718

1.8

1.8

0.53

SC001236

1.1

1.28
1.68

0.19

SN00769491
SN00769505
SN00769487
SN00775298
SN00775286
SN00775295
SN00775300
SN00786699

CL9254

0.63

1.34
1

CL1623

1.94

2.9

2.39

0.97

% In h ib it io n

SN00783385
SN00783346
SN00794343

3D 7
DD2
H E K C e lls

SN00802052

CMI409

Stock Plate

Data
Analysis

1/25

Water

Plate imaging on opera

1/10

Imaging empty
assay plate

45l of cultures

Incubation for 72
hrs
Apply DAPI

Figure 3:Plate handling and Screening method.

Sorbitol synchronized parasites were seeded in 45l of culture at 0.3% heamtocrit
and 2% parasitemia was seeded in the assay plates (already containing 5l of diluted
compounds) to achieve an overall dilution of 250 with final top concentration of
40M at 0.4% DMSO. After 72-hour incubation at standard conditions, the fluorescent stain DAPI in a PBS based permeabilization buffer was added to the plates after a wash with PBS. The plates were left overnight at room temperature and were
imaged on an Opera High Content Screening Platform (PerkinElmer) with 20x
objective, 420-450 nm emission and 405 nm excitation.
Cytotoxicity
HEK293 cells were seeded at 2000 cells/well and incubated for 24hrs before addition of compounds. Resazurin-based assay was utilized to determine the cell viability. Plates were incubated for 6 hrs before reading on Envision plate reader.
Data Analysis
Automated primary image analysis was performed simultaneously with the imaging
process using an Acapella software. Screening data was normalized in Microsoft
excel. The Z parameter was used to determine the quality of the assay (Zhang et al.,
1999) . Dose response data were normalized and used to calculate the IC50 values
by 4 parameter nonlinear regression analysis using Graphpad prism. All the assays
were run in two biological replicates.

Dd2-IC50
(M)

10%
47.50%
-0.50%
89.95%
1%
-6.50%
10.50%
82.44%
75.50%
1%
76%
99.05%
16%
24.50%
100%
16%
4.50%
3.50%
4.93%

SN00802056
SN00802062
SN00790702

CL7232

1.54

7.82

1.40

CL6662A

1.75

2.63

1.41

SN00790698
SN00790687
-4

-3

-2

-1

SN00791542

SN00791552
SN00791562

L o g C o n c e n t r a t io n ( M )

Figure 6: Dose response plot of

SSN00797498 against 3D7, Dd2 and HEK293 cells.

SN00783960

CL3097

3.03

SN00802965
SN00783671

2.66

2.50

-4.50%
1.50%
53.14%
1.50%
24.50%
55.77%
46.50%
98.50%
81.72%

SEM

Dd2 Inhibition
at 5M

HEK293
IC50
(M)

0.77

98.31%

>5

19%
7%
0%
79.35%

>5
>5
>5
>5

26%
16.50%
47%
87.58%
17.50%
-1.50%
16%
71.50%
69.50%
6%
84.50%
99.32%
43%
24.50%
93.53%
22.55%
1%
-10.5
10.87%

>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5

3%
0.50%
68.72%
0%
6.50%
66.49%
64%
97.50%
87.67%

>5
>5
>5
>5
>5
>5
>5
>5
>5

6.50%
0.50%

>5
>5

0.76 0.75

28.4 37.4
4

0.60 1.31

1.80

2.06 1.42
2.4

0.48

2.1
0.56 0.63

0.96

5.3

59.57

0.00
5

50

2.7

1.15

0.68

9.45 1.04

0.53

1.8
3219
.3

1%
15%

0.64

*SEM=Standard

mean error. Screening hits were tested alone for conformation of screening results
(A) and together with analogues in (B).

Figure 2:Screening hits and analogues.

Dd2

3D7
SN 00775298

100

100

SN 00775300

50

( A n a lo g )
SN 00775286

( A n a lo g )

-5 0

% In h ib itio n

( S c r e e n in g h it)
% In h ib it io n

The GDB21 screening library was available as two dilution ready plates provided by
Compounds Australia (35,000 diverse compound library), containing 1 l of 1.25
mM stocks in 100% DMSO screened at 5M against the P.falciparum 3D7 strain.
For screening, a validated high throughput imaging assay was used (Duffy and
Avery, 2012) . Screening hits and analogues were also provided by Compounds Australia as dilution ready plates containing 1 l of serially diluted stocks in 100%
DMSO (final concentration range 0.25 nM - 5M) to be tested against 3D7 and
DD2 strains and also against HEK293 cells to test cytotoxicity of the compounds. In
each plate, two full columns of 14 wells each containing a final concentration of 5
M puromycin and 0.4% DMSO were used as positive and negative controls, respectively (Figure 5). All compound containing plates were stored at -20C until
use.6 reference antimalarials was run in parallel as an additional control with every
assay at 40M in top dose (Table 1).

3D7
Inhibition at
5M

1.28

SC013994

SN00784248

SN00797498
100

Dose Response

3D7-IC50
(M)

SN00786679
SN00786702

(http://www.cddep.org/sites/default/files/malaria-life-cycle_3.jpg)

Screening

Scaffold
ID

SN00797519
SN00797517
SN00797493

Figure 1: Malaria life cycle.

Screening Cascade

Analogues

Figure 4 : Compounds screened and hits

found.

SN 00775298
( S c r e e n in g h it )
SN 00775300

50

(A n a lo g )
SN 00775286

(A n a lo g )
-5 0

-4

-3

-2

-1

-4

L o g C o n c e n t r a t io n ( M )

-3

-2

-1

L o g C o n c e n tr a t io n ( M )

Figure 7: Dose response plot of Screening hit SN00775298 and its analogues against Plasmodium 3D7 and Dd2
strains.
Due to practical limitations, we could only test 2-3 analogues for each screening hit compound. The test, however, could still provide
valuable preliminary information to hypothesize whether or not the anti-malarial activity is a common feature of the scaffold, and test
even more analogues in the future to find better actives.

Conclusion
The objective of malaria eradication has determined goals for the treatment and prevention of malaria, which currently cannot be
reached by the available antimalarials. New antimalarial compounds need to be discovered to identify new drugs that are active
against emerging resistant strains of the parasite with novel mechanisms of action to replace the existing drugs . By screening a collection of novel scaffolds we found 6 promising scaffold. The remaining hits and analogues did not show any activity. However given
the number of analogues tested , we cannot exclude the other analogues as they might have antimalarial activity. The next steps of this
research should include testing the scaffolds against gametocytes and liver stages, and further studies on the mechanisms of action and
identification of the targets.

Acknowledgements
We thank Australian Red Cross for providing provision of human blood for the experiments.

References
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