Screening for small molecule modulators of Hsp70 chaperone activity using protein aggregation suppression assays: Inhibition of the plasmodial chaperone PfHsp70-1

Article abstract of DOI:10.1515/BC.2011.040

Plasmodium falciparum heat shock protein 70 (PfHsp70-1) is thought to play an essential role in parasite survival and virulence in the human host, making it a potential antimalarial drug target. A malate dehydrogenase based aggregation suppression assay was adapted for the screening of small molecule modulators of Hsp70. A number of small molecules of natural (marine prenylated alkaloids and terrestrial plant naphthoquinones) and related synthetic origin were screened for their effects on the protein aggregation suppression activity of purified recombinant PfHsp70-1. Five compounds (malonganenone A-C, lapachol and bromo-β-lapachona) were found to inhibit the chaperone activity of PfHsp70-1 in a concentration dependent manner, with lapachol preferentially inhibiting PfHsp70-1 compared to another control Hsp70. Using growth inhibition assays on P. falciparum infected erythrocytes, all of the compounds, except for malonganenone B, were found to inhibit parasite growth with IC₅₀ values in the low micromolar range. Overall, this study has identified two novel classes of small molecule inhibitors of PfHsp70-1, one representing a new class of antiplasmodial compounds (malonganenones). In addition to demonstrating the validity of PfHsp70-1 as a possible drug target, the compounds reported in this study will be potentially useful as molecular probes for fundamental studies on Hsp70 chaperone function.

Full text of DOI:10.1515/BC.2011.040

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Biol. Chem., Vol. 392, pp. 431–438, May 2011 • Copyright ꢀ by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2011.040
Screening for small molecule modulators of Hsp70 chaperone
activity using protein aggregation suppression assays:
inhibition of the plasmodial chaperone PfHsp70-1
Ingrid L. Cockburn1, Eva-Rachele Pesce1, Jude M.
Pryzborski2, Michael T. Davies-Coleman3, Peter G.K.
Clark4, Robert A. Keyzers3,4, Linda L. Stephens1 and
Gregory L. Blatch1,*
Introduction
Of the five Plasmodium species known to infect humans,
Plasmodium falciparum, which can cause cerebral malaria,
results in the highest number of deaths (Haldar and Mohan-
das, 2007) and thus is the focus of most malaria research.
Although uncomplicated malaria is curable if treated early,
current antimalarials are fast becoming ineffective due to
increased drug resistance (Tuteja, 2007). This problem makes
the search for effective new therapies and, in particular, nov-
el drugs with new mechanisms of action and hence new tar-
gets, an extremely important and worthwhile research area.
Molecular chaperones, and more specifically heat shock
proteins, are known to play an important role in the survival
and virulence of many protozoan parasites, including P. fal-
ciparum (Neckers and Tatu, 2008). Heat shock proteins
70 kDa in size (Hsp70s) facilitate the correct folding and
assembly of newly synthesized proteins, the refolding of
incorrectly folded or aggregated proteins and the transloca-
tion of proteins across membranes (Mayer and Bukau, 2005).
Structural features of Hsp70s include an approximately
45 kDa ATPase domain, an approximately 15 kDa peptide-
binding domain and an approximately 10 kDa C-terminal
domain ending in a conserved EEVD motif (Mayer and
Bukau, 2005), which interacts with proteins containing a
tetratricopeptide repeat domain (Blatch and La¨ssle, 1999;
Scheufler et al., 2000). Heat shock proteins 40 kDa in size
(Hsp40s), characterized by the presence of a conserved
approximately 70 amino acid sequence termed the J-domain
(Cheetham and Caplan, 1998), act as co-chaperones by inter-
acting, via their J-domains, with the Hsp70 ATPase domain.
Hsp40 co-chaperones can deliver peptide substrates to Hsp70
and stimulate the ATPase activity of Hsp70 (Cheetham et al.,
1994).
The complex life cycle of P. falciparum is associated with
many potential stresses to the parasite, including significant
temperature increases due to febrile episodes in the human
host (Acharya et al., 2007) and attack by host defense mech-
anisms (Sharma, 1992). The ability of the parasite to survive
these stresses has been attributed to heat shock proteins
(Kumar et al., 2003). In P. falciparum, as much as 2% of all
genes encode molecular chaperones (Acharya et al., 2007),
with six encoding Hsp70s (Shonhai et al., 2007) and at least
43 encoding Hsp40s (Botha et al., 2007). The major cyto-
solic plasmodial Hsp70, PfHsp70-1 (PF08_0054), has been
shown to have chaperone activity using both ATPase and
aggregation suppression assays (Matambo et al., 2004;
Ramya et al., 2006; Shonhai et al., 2008; Misra and Rama-
chandran, 2009). Furthermore, PfHsp70-1 has been shown
1 Biomedical Biotechnology Research Unit, Department of
Biochemistry, Microbiology and Biotechnology, Rhodes
University, Grahamstown 6139, South Africa
2 Department of Parasitology, Faculty of Biology, Philipps
University Marburg, D-35043 Marburg, Germany
3 Department of Chemistry, Rhodes University,
Grahamstown 6139, South Africa
4 Centre for Biodiscovery, School of Chemical and Physical
Sciences, Victoria University of Wellington, Wellington,
New Zealand
*Corresponding author
e-mail: g.blatch@ru.ac.za
Abstract
Plasmodium falciparum heat shock protein 70 (PfHsp70-1)
is thought to play an essential role in parasite survival and
virulence in the human host, making it a potential antima-
larial drug target. A malate dehydrogenase based aggregation
suppression assay was adapted for the screening of small
molecule modulators of Hsp70. A number of small mole-
cules of natural (marine prenylated alkaloids and terrestrial
plant naphthoquinones) and related synthetic origin were
screened for their effects on the protein aggregation sup-
pression activity of purified recombinant PfHsp70-1. Five
compounds (malonganenone A-C, lapachol and bromo-b-
lapachona) were found to inhibit the chaperone activity of
PfHsp70-1 in a concentration dependent manner, with lapa-
chol preferentially inhibiting PfHsp70-1 compared to another
control Hsp70. Using growth inhibition assays on P. falci-
parum infected erythrocytes, all of the compounds, except
for malonganenone B, were found to inhibit parasite growth
with IC50 values in the low micromolar range. Overall, this
study has identified two novel classes of small molecule
inhibitors of PfHsp70-1, one representing a new class of anti-
plasmodial compounds (malonganenones). In addition to
demonstrating the validity of PfHsp70-1 as a possible drug
target, the compounds reported in this study will be poten-
tially useful as molecular probes for fundamental studies on
Hsp70 chaperone function.
Keywords: heat shock proteins; malaria; malonganenones;
molecular chaperones; naphthoquinones; Plasmodium
falciparum.
2010/297
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432 I.L. Cockburn et al.
to suppress the thermosensitivity of a mutant strain of Esche-
richia coli lacking DnaK (Shonhai et al., 2005). PfHsp70-1
is localized to the cytosol and the nucleus of the parasite
(Kumar et al., 1991; Pesce et al., 2008) and is expressed at
high levels in all the blood stages of the P. falciparum life
cycle, with expression levels increasing after heat shock
(Kumar et al., 1991). Taken together, these data suggest that
PfHsp70-1 plays an important role in parasite viability in the
human host, making it an attractive antimalarial drug target.
Hsp70s have been implicated in a number of diseases and
illnesses including cancer, viral infections and protein con-
formational diseases such as Parkinson’s disease (Brodsky
and Chiosis, 2006). For this reason, there are increasing num-
bers of studies aimed at identifying small molecule modu-
lators of Hsp70, both inhibitors and activators, for use in the
development of chemotherapeutic agents (Jinwal et al.,
2009). Compared with Hsp90, inhibition or modulation of
Hsp70s (specifically the ATPase activity) by small molecules
is thought to be more challenging. This, as reviewed by Mas-
sey (2010), has been attributed to various structural and bio-
chemical properties of the chaperones, including the more
hydrophilic ATP binding site of Hsp70 compared to that of
Hsp90 in addition to a very high affinity of Hsp70 for ATP
and ADP, making inhibition by competitive binding more
difficult than for Hsp90, which has a lower affinity for
nucleotides.
A number of Hsp70 inhibitors have been identified,
including adenosine analogs (ATP mimics), spergualins,
pyrimidinones, fatty acids and peptides (Evans et al., 2010).
ATP mimics of Hsp70 have been found to be relatively selec-
tive and have been shown to have cytotoxic effects on
HCT116 (human colon cancer) cells, which in some cases
was attributed to Hsp70 modulation by these compounds
(Williamson et al., 2009). 15-Deoxyspergualin (DSG), an
immunosuppressive drug belonging to the spergualins, has
been found to modulate Hsp70 chaperone activity, including
both ATPase and aggregation suppression activity (Brodsky,
1999; Ramya et al., 2006). Structural analogs of DSG, the
pyrimidinone-peptoids, have also been tested as modulators
of Hsp70 chaperone activity, resulting in the identification of
inhibitors of either the Hsp40-stimulated ATPase activity of
Hsp70 or the basal Hsp70 ATPase activity with certain inhib-
itors exhibiting Hsp70 inhibition that correlated with inhi-
bition of breast cancer growth (Fewell et al., 2004; Wright
et al., 2008). These pyrimidinones have proven useful in
rational design and testing of novel Hsp70 inhibitors, in
which, it was revealed that aromatic or hydrophobic groups
were important features for inhibition of Hsp70 ATPase
activity (Wise´n et al., 2008). Recently, a pyrimidinone com-
pound was found to act as an artificial co-chaperone to
Hsp70, stimulating its chaperone activity and binding adja-
cent to the site on Hsp70 involved in interaction with the J-
domain of Hsp40. When steric bulk was added to this
compound, inhibition of Hsp70 chaperone activity was
observed (Wise´n et al., 2010). These findings provide useful
information on the possible mechanism of modulation of
Hsp70 by pyrimidinones and related compounds. Finally, a
number of pyrimidinones were tested as potential inhibitors
of the ATPase activity of PfHsp70-1 and for inhibitory
effects on P. falciparum growth. Of the compounds screened,
nine were found to have significant inhibitory effects on par-
asite growth, in addition to effects on the ATPase activity of
PfHsp70-1 (Chiang et al., 2009).
In this study, two classes of compounds were screened for
potential effects on PfHsp70-1 chaperone activity. The first
class, the marine tetraprenylated alkaloids malonganenones
A, B and C, are natural products extracted from Leptogorgia
gilchristi, a Mozambique sea fan. These novel marine com-
pounds were found to have moderate cytotoxicity towards a
number of oesophageal cancer lines (Keyzers et al., 2006).
The second class of compounds is the 1,4 naphthoquinones,
specifically lapachol and its derivatives, of both natural and
synthetic origin. Lapachol is a common naturally occurring
naphthoquinone found in the heart-wood of various tree spe-
cies belonging to the bigonaceous family, found in Brazil
and other tropical regions of the western hemisphere (da Sil-
va Ju´nior et al., 2009). Lapachol and its derivatives have
been shown to have both anticancer (Bonifazi et al., 2010)
and antiplasmodial activity (Pe´rez-Sacau et al., 2005). Both
compound classes were selected for this study because of
their established biological activity and the structural simi-
larities that they share with ATP (purine analogs) and with
known inhibitors of Hsp70 and Hsp90 chaperones, like gel-
danamycin (quinone analogs). The tetraprenylated alkaloid
class of compounds screened in this study also contain pep-
toid components, which in addition to pyrimidinone groups
have been found to be important for Hsp70 interaction and
modulation (Wright et al., 2008). In this study we report
the identification of two novel classes of small molecule
inhibitors of PfHsp70-1, one representing a new class of
antiplasmodial compounds.
Results
PfHsp70-1 protein aggregation suppression activity
is inhibited by selected marine prenylated alkaloids
and naphthoquinone compounds
PfHsp70-1, as previously reported (Shonhai et al., 2008), was
found to be effective in suppressing the thermally induced
aggregation of malate dehydrogenase (MDH), reducing the
aggregation of MDH by more than half at substoichiometric
levels (Figure 1). A non-chaperone protein (BSA) and the
solvent (DMSO) used to dissolve the test compounds had no
effect on the aggregation of MDH (Figure 1). Following the
testing of our pure compound repository (Table 1), five com-
pounds (malonganenones A-C, lapachol and bromo-b-lapa-
chona) showed notable inhibition of the aggregation
suppression activity of PfHsp70-1 (Figure 2). With the
exception of bromo-b-lapachona, PfHsp70-1 aggregation
suppression activity was almost completely inhibited ()90%
MDH aggregation) by each of the five compounds at
300 mM. Furthermore, in most cases the inhibition of Pf-
Hsp70-1 occurred in a concentration dependent manner (Fig-
ure 2), indicative of a specific interaction. Control assays in
which no MDH was present and no detectable aggregates
were observed suggested that the reversal of the aggregation
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Inhibition of the chaperone activity of PfHsp70-1 433
P. falciparum growth in culture. All five compounds, except
for malonganenone B, were able to inhibit parasite growth
with IC50 values of -20 m
M (Table 2). These IC50 values
were found to be comparable to that of MAL3-39 (Table 2),
an Hsp70 small molecule modulator previously shown to
have inhibitory effects on P. falciparum growth (Chiang et
al. 2009). Malonganenone A and malonganenone C, in par-
ticular, were found to have the highest inhibitory effects on
parasite growth.
Discussion
Figure 1 PfHsp70-1 suppresses the thermal aggregation of MDH.
Progress curve showing MDH aggregation over time (0.72 m , closed
squares) and aggregation suppression by PfHsp70-1 (0.36 m
closed circles). Controls performed show that the observed aggre-
gation suppression is due to chaperone activity and not the presence
M
To our knowledge, this is the first report of a screening for
Hsp70 inhibitors using the MDH aggregation suppression
assay. This study has resulted in the identification of two
novel classes of small molecule inhibitors of PfHsp70-1, one
representing a new class of antiplasmodial compounds
(malonganenones). In addition to demonstrating the validity
of PfHsp70-1 as a possible drug target, these compounds will
be potentially useful as molecular probes for fundamental
studies on Hsp70 chaperone function. The latter will be
enhanced by the elucidation of the binding sites and mech-
anism of action for these classes of compounds. These com-
pounds could be triggering the release of MDH from
PfHsp70-1 simply by competitive binding to the substrate
binding site. However, it is also possible that some of the
compounds are associating with the ATP-binding site and
causing an allosteric effect that lowers the affinity of
PfHsp70-1 for MDH, as occurs on ATP binding (Shonhai
et al., 2008).
M
,
of a second protein (BSA, 0.36 mM; open circles) and showing that
DMSO, at the maximum concentration used (1%) in any subsequent
inhibitor screening reactions, has no effect on MDH aggregation
(open squares). These curves represent a typical set of control reac-
tions using one batch of purified PfHsp70-1 and which was repeated
at least twice on separate batches of purified protein.
suppression activity was not due to aggregates formed by
either the compounds themselves or the compounds in com-
bination with PfHsp70-1 or by PfHsp70-1 itself (data not
shown). Additional control assays were conducted with
MDH in the presence of each of the compounds, without
chaperones. All of the compounds had no affect on MDH
aggregation, except at the highest concentration (300 mM),
where malonganenone A caused a slight enhancement of
MDH aggregation (data not shown) and all the other com-
pounds had minor effects on MDH aggregation (for example,
lapachol, Figure 3). Overall these data suggest that marine
prenylated alkaloids and naphthoquinones represent two nov-
el classes of small molecule modulators of PfHsp70-1.
In this study, relatively high compound concentrations
were required for high levels of inhibition of PfHsp70-1
(100–300 mM). In a study by Chiang et al. (2009), similar
concentrations of pyrimidinone compounds (300 m
M) were
required for inhibition of the ATPase activity of PfHsp70-1.
However, these same compounds were shown to inhibit at
Lapachol differentially affects Hsp70s from different
organisms
relatively low concentrations (IC50-2 mM) the growth of P.
falciparum using parasite-infected erythrocyte cultures
(Chiang et al., 2009).
Recombinant human Hsp70 was found to be ineffective in
MDH assays, as the chaperone itself aggregated at 488C and
therefore was unable to suppress the thermal aggregation of
MDH at this temperature (data not shown). Thus, the com-
pounds were tested for effects on Medicago sativa (alfalfa)
Hsp70 as an alternative control Hsp70. Lapachol was shown
to reproducibly inhibit PfHsp70-1 protein aggregation sup-
pression activity in a concentration dependent manner but
had little to no effect on the alfalfa Hsp70 activity (Figure 3).
These data suggested that Hsp70 chaperones from different
organisms, despite relatively high sequence similarities, can
be differentially inhibited by selected small molecules.
The biological activities of lapachol and its derivatives
have been well studied. Naphthoquinones have been found
to be cytotoxic towards various human cancers including
ovarian, breast, lung and cervical cancers lines (Bonifazi et
al., 2010; da Silva Ju´nior et al., 2010), with many compounds
displaying IC50 values of -5 mM. This group of compounds
has also been shown to exhibit trypanocidal activity (Ferreira
et al., 2006; Silva et al., 2006; da Silva Ju´nior et al., 2008)
and antiplasmodial activity. A group of aminonaphthoqui-
nones was tested in P. falciparum (clones W2 and D6)
growth inhibition assays, with one of the aminoquinones dis-
playing higher potency than chloroquine towards the W2
clone (Kapadia et al., 2001). A number of phenazine deriv-
atives of lapachol were tested for inhibitory effects in both
P. falciparum growth inhibition assays and P. berghei infec-
tivity assays in mice, with certain compounds shown to have
inhibitory effects on both systems (de Andrade-Neto et al.,
Inhibitors of PfHsp70-1 also inhibit P. falciparum
growth
The five compounds found to inhibit PfHsp70-1 in vitro
chaperone activity were tested for their potential effects on
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434 I.L. Cockburn et al.
2004). In another study, lapachol and bromo-b-lapachona
were found to inhibit the growth of P. falciparum F32 with
IC50 values of 24.4 and 2.7 mM, respectively (Pe´rez-Sacau
et al., 2005). These values were comparable to the IC50 val-
ues for parasite growth inhibition determined in this study
using P. falciparum 3D7. When comparing malonganenones
A-C, in terms of their performance in PfHsp70-1 in vitro
chaperone assays vs. P. falciparum growth inhibition assays,
malonganenones A and C exhibited greater inhibitory activ-
ity than malonganenone B in both assays. Similarly, two sim-
plified synthetic analogs of malonganenone B (PGKC4_7C
and PGKC4_4I) were inactive in our assays. Integrating all
these data, it is tempting to speculate that Hsp70 is a poten-
tial cellular target of both the naphthoquinones and malon-
ganenones. Interestingly, celastrol (a quinine), was found to
activate the human heat shock response. This activation,
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Inhibition of the chaperone activity of PfHsp70-1 435
Figure 2 The protein aggregation suppression activity of PfHsp70-1 is inhibited in a concentration dependent manner by naphthoquinone
and marine prenylated alkaloid compounds.
MDH (0.72 m
300 m ). Compounds found to inhibit PfHsp70-1 were lapachol (A), bromo-b-lapachona (B), malonganenone A (C), malonganenone B
(D) and malonganenone C (E). For each set of compounds, progress curves were produced for: MDH only (closed squares); MDH and
PfHsp70-1 (open squares); MDH, PfHsp70-1 and 1 m compound (closed circles); MDH, PfHsp70-1 and 10 m compound (closed
triangles); MDH, PfHsp70-1 and 100 m compound (open triangles); MDH, PfHsp70-1 and 300 m compound (open circles). This set of
M) aggregation at 488C was monitored in the presence of PfHsp70-1 (0.36 mM) and various compounds (0, 1, 10, 100,
M
M
M
M
M
figures represents a typical series of experiments on one batch of purified PfHsp70-1 in each case and each compound was tested on at
least two separate batches of purified protein giving similar results.
however, was caused by specific activation of heat shock
transcription factor 1 and not a direct effect on chaperones
(Westerheide et al., 2004).
Materials and methods
Protein purification
The potential that these compounds might differentially
inhibit different Hsp70s needs to be investigated further,
particularly for the comparison of PfHsp70-1 and human
Hsp70s, such as Hsc70, Hsp70, BiP and Grp75. Because
human Hsp70 is not amenable to thermally based protein
aggregation suppression assays, we intend to test the differ-
ential effects of these compounds using other chaperone
assays such as the luciferase refolding assay, which has been
successfully used by others (Yamamoto et al., 2010). All of
these chaperone assays can be adapted to examine Hsp70
together with its co-chaperones and, therefore, it will also be
possible to analyze the effect of these compounds on Hsp40-
stimulated Hsp70 chaperone activity.
The expression and purification of (His)6-tagged PfHsp70-1 from
E. coli XL1 Blue wpQE30-PfHsp70-1x cells was carried out as
previously described (Matambo et al., 2004; Shonhai et al., 2008).
Purified protein was dialyzed into the MDH assay buffer.
Source of compound library
Lapachol (compound 1, Table 1) and several synthetic derivatives
of lapachol (compounds 2–5, Table 1) were kindly provided by
Professor Antonio Ventura Pinto (Nu´cleo de Pesquisas em Produtos
Naturais, UFRJ, 21944-971 Rio de Janeiro, RJ, Brazil).
The malonganenones were isolated from Leptogorgia gilchristi,
collected at a depth of 20 m from a reef near Ponto Malongane,
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436 I.L. Cockburn et al.
Figure 3 Lapachol differentially affects the aggregation suppression activities of Medicago sativa Hsp70 and PfHsp70-1.
Lapachol (0, 1, 100 and 300 m ) was tested for effects on the MDH (0.72 m ) aggregation suppression activities of PfHsp70-1 (0.36 m
compared with M. sativa Hsp70 (0.36 m ). White bars, M. sativa Hsp70; grey bars, PfHsp70-1. The X-axis labels refer to MDH only
(MDH) and lapachol concentrations in m (0, 1, 100 and 300), in the presence of MDH and Hsp70. A control assay (shaded bar; labeled
C) was conducted to assess the aggregation of MDH in the presence of lapachol (300 m ) without chaperone. The percentage MDH
M
M
M)
M
M
M
aggregation levels were calculated using the absorbance values after 1800 s, with the reaction of MDH alone taken as 100% MDH
aggregation. For the compound testing, the standard deviation is shown and the numbers associated with the bars indicate the average
percentage MDH aggregation.
Mozambique in 1995 with the aid of SCUBA. Freeze dried gor-
gonian (148 g) was exhaustively extracted with methanol that was
removed under reduced pressure. The dried extract was partitioned
between water (4.3 g) and ethyl acetate (3.1 g). A portion of the
organic partition (0.9 g) was re-dissolved in methanol that was then
defatted with hexanes, after which repeated bench-top chromatog-
raphy using both normal- (DIOL) and reversed- (HP20) phase mate-
rials, followed by normal-phase HPLC (DIOL) resulted in the
isolation of malonganenones A (29 mg; compound 7), B (14 mg;
compound 8) and C (4 mg; compound 9). The purification and
establishment of chemical structures of these compounds using mass
spectrometry and nuclear magnetic resonance has been described
(Keyzers et al., 2006, Table 1). Two synthetic analogs of malon-
ganenone B were also prepared (compounds 10 and 11, Table 1).
Protein aggregation suppression assays
MDH-based protein aggregation suppression assays using PfHsp70-
1 were performed according to previously described methods (Bos-
hoff et al., 2008; Shonhai et al., 2008). Briefly, the aggregation of
MDH in assay buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.4) at
488C was monitored using a Helios Alpha DB spectrophotometer
with a Peltier-controlled cell, measuring light scatter due to protein
aggregates at 360 nm over a 30 min period. As shown previously
(Shonhai et al., 2008), the aggregation suppression activity of
PfHsp70-1 was found to be ATP dependent, with ATP lowering the
affinity of PfHsp70-1 for MDH (data not shown). The MDH assay
was adapted for use as a screening tool for potential modulators of
PfHsp70-1 chaperone activity, specifically the ability to suppress the
thermal aggregation of a substrate protein. MDH (pig heart,
Roche, Mannheim, Germany) was used at a concentration of
Briefly, caffeine (1.00 g) was refluxed in a mixture of 1
M NaOH(aq)
(17 ml) and EtOH (8 ml) for one hour. Repeated recrystalization
from EtOAc resulted in the purification of PGKC4_7C (0.11 g; 40%
yield; compound 10). PGKC4_7C (0.28 g) was then refluxed in neat
formic acid (14 ml) to provide the formate PGKC4_4I (compound
11) that was purified by recrystalization from toluene (0.20 g; 60%
yield). The identity of both products was established from mass
spectrometry and nuclear magnetic resonance data (data not shown).
0.72 m
chased from Alfa Biogene International, Germany) were used at a
concentration of 0.36 m to give a level of aggregation suppression
M and recombinant PfHsp70-1 and M. sativa Hsp70 (pur-
M
which would allow detection of both stimulation and inhibition by
the various test compounds. Before screening, a spectral scan of
each compound was performed at a range of 200–900 nm, to ensure
that the compounds had no effect on absorbance at or around
360 nm. None of the compounds showed any significant absorbance
above 300 nm (data not shown). The compounds (marine prenylated
alkaloids and naphthoquinones) were dissolved in DMSO and
Table 2 IC50 values of test compounds on
P. falciparum 3D7 parasite growth.
screened at a final concentration of 300 m
pounds were briefly incubated with the chaperone prior to addition
of MDH. Only those compounds that showed an effect on the aggre-
M in the assay. The com-
Compound
IC50 (mM)
"
18.67 2.27
Lapachol
"
Bromo-b-lapachona
Malonganenone A
Malonganenone B
Malonganenone C
MAL3-39
17.29 4.44
gation suppression activity of PfHsp70-1 activity at 300 m
further assayed at a range of concentrations (0.36, 1, 10, 100, 200
and 300 m ) on at least two independently purified batches of
M were
"
0.81 0.24
)50
M
"
5.20 2.49
PfHsp70-1. DMSO was shown to have no effect on PfHsp70-1
activity in the assay in a reaction in which DMSO was included at
1% (the maximum final concentration in inhibition experiments).
Control reactions without MDH were carried out with DMSO alone,
the test compounds alone, the chaperones alone and the chaperones
with each compound in addition to DMSO. These were conducted
"
29.58 16.03
DMSO
NDa
aND (not determined): the DMSO IC50 value was
not determined due to the extremely low cytotox-
icity of the solvent.
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Inhibition of the chaperone activity of PfHsp70-1 437
to ensure that any reversal of aggregation suppression activity in a
test reaction was not due to aggregates formed by either the com-
pounds themselves or the compounds in combination with the chap-
erone or by the chaperone itself (data not shown). In addition, each
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M with MDH alone (no chaperone)
to assess the effect of the compounds on MDH aggregation.
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Chiang, A.N., Valderramos, J.C., Balachandran, R., Chovatiya, R.J.,
Mead, B.P., Scneider, C., Bell, S.L., Klein, M.G., Huryn, D.M.,
Chen, X.S., et al. (2009). Select pyrimidinones inhibit the prop-
agation of the malarial parasite, Plasmodium falciparum. Bioorg.
Med. Chem. 17, 1527–1533.
da Silva Ju´nior, E.N., de Souza, M.C.B.V., Fernandes, M.C., Menna-
Barreto, R.F.S., Pinto, M.C.F.R., Lopes, F.A., de Simone, C.A.,
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Growth inhibition assays
Plasmodium falciparum 3D7 parasites were cultured according to
Trager and Jensen (1976). Growth inhibition assays were conducted
on P. falciparum 3D7-infected erythrocytes using the LDH method
and a starting parasitaemia of 0.1% (Makler et al., 1993). A ‘no
compound’ control was included on each plate and was taken as
the ‘100% growth’ value for that plate. Each assay plate also includ-
ed cyclohexamide treated cultures to account for any initial or back-
ground LDH activity. These values were subtracted from all other
LDH activities. The reported IC50 values and the accompanying
variation represent the 95% confidence interval of IC50 values as
calculated using GraphPad Prism 4 (San Diego, CA, USA) software.
da Silva Ju´nior, E.N., Pinto, M.C.F.R., de Moura, K.C.G., de
Simone, C.A., Nascimento, C.J., Andrade, C.K.Z., and Pinto,
A.V. (2009). Hooker’s ‘lapachol peroxide’ revisited. Tetrahedron
Lett. 50, 1575–1577.
Acknowledgements
da Silva Ju´nior, E.N., de Deus, C.F., Cavalcanti, B.F., Pessoa, C.,
Costa-Lotufo, L.V., Montenegro, R.C., de Moraes, M.O., Pinto,
M.C.F.R., de Simone, C.A., Ferreira, V.F., et al. (2010). 3-Ary-
lamino and 3-alkoxy-nor-b-lapachone derivatives: synthesis and
cytotoxicity against cancer cell lines. J. Med. Chem. 53, 504–
508.
de Andrade-Neto, V.F., Goulart, M.O.F., da Silva Filho J.F., da Silva,
M.J., Pinto, M.C.F.R., Pinto, A.V., Zalis, M.G., Carvalho, L.H.,
and Krettli, A.U. (2004). Antimalarial activity of phenazines
from lapachol, b-lapachone and its derivatives against Plasmo-
dium falciparum in vitro and Plasmodium berghei in vivo.
Bioorg. Med. Chem. Lett. 14, 1145–1149.
This research was funded by a Deutsche Forschungsgemeinschaft
(DFG) German-African Cooperation Project in Infectology grant
(DFG wRef: LI 402/12-0x). In addition, funding from the National
Research Foundation (South Africa) and the Department of Envi-
ronmental Affairs through the SEACHANGE program is gratefully
acknowledged. ILC was awarded Rhodes University and Deutscher
Akademischer Austausch Dienst (DAAD) Masters bursaries, ERP
is a Claude Leon Foundation Postdoctoral Fellow, LLS was awarded
a South African Malaria Initiative (SAMI) Postdoctoral Fellowship
and PGKC holds VUW Masters and Woolf-Fisher Scholarships. The
authors would like to thank Dr. Stefan Baumeister (Philipps Uni-
versity Marburg, Germany) for his help with the GraphPad software.
Evans, C.G., Chang, L., and Gestwicki, J.E. (2010). Heat shock
protein 70 (Hsp70) as an emerging drug target. J. Med. Chem.
53, 4585–4602.
Ferreira, V.F., Jorqueira, A., Souza, A.M.T., da Silva, M.N., de Sou-
za, M.C.B.V., Gouveˆa, R.M., Rodrigues, C.R., Pinto, A.V., Cas-
tro, H.C., Santos, D.O., et al. (2006). Trypanocidal agents with
low cytotoxicity to mammalian cell line: a comparison of the
theoretical and biological features of lapachone derivatives.
Bioorg. Med. Chem. 14, 5459–5466.
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Received November 8, 2010; accepted December 17, 2010
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