Cytostatic versus cytocidal activities of chloroquine analogues and inhibition of hemozoin crystal growth

Article abstract of DOI:10.1128/AAC.01709-12

We report an improved, nonhazardous, high-throughput assay for in vitro quantification of antimalarial drug inhibition of β-hematin (hemozoin) crystallization performed under conditions that are more physiological relative to previous assays. The assay uses the differential detergent solubility of crystalline and noncrystalline forms of heme and is optimized via the use of lipid catalyst. Using this assay, we quantify the effect of pH on the crystal growth-inhibitory activities of current quinoline antimalarials, evaluate the catalytic efficiencies of different lipids, and test for a possible correlation between hemozoin inhibition by drugs versus their antiplasmodial activity. Consistent with several previous reports, we found a good correlation between hemozoin inhibition potency versus cytostatic antiplasmodial potency (50% inhibitory concentration) for a series of chloroquine (CQ) analogues. However, we found no correlation between hemozoin inhibition potency and cytocidal antiplasmodial potency (50% lethal dose) for the same drugs, suggesting that cellular targets for these two layers of 4-aminoquinoline drug activity differ. This important concept is also explored further for QN and its stereoisomers in the accompanying paper (A. P. Gorka, K. S. Sherlach, A. C. de Dios, and P. D. Roepe, Antimicrob. Agents Chemother. 57:365-374, 2013). Copyright

Full text of DOI:10.1128/AAC.01709-12

Cytostatic versus Cytocidal Activities of Chloroquine Analogues and
Inhibition of Hemozoin Crystal Growth
a,b,c
a,b,c
a,b,c
c
a,b,c
a,b,c
a,b,c
Alexander P. Gorka,
Jonathan P. Brower,
John N. Alumasa,
* Angel C. de Dios, Paul D. Roepe
* Katy S. Sherlach,
Lauren M. Jacobs,
Katherine B. Nickley,
*
a,b,c
a
b
c
Department of Biochemistry and Molecular & Cellular Biology, Center for Infectious Diseases, and Department of Chemistry, Georgetown University, Washington, DC, USA
We report an improved, nonhazardous, high-throughput assay for in vitro quantification of antimalarial drug inhibition of
␤
-hematin (hemozoin) crystallization performed under conditions that are more physiological relative to previous assays. The
assay uses the differential detergent solubility of crystalline and noncrystalline forms of heme and is optimized via the use of
lipid catalyst. Using this assay, we quantify the effect of pH on the crystal growth-inhibitory activities of current quinoline anti-
malarials, evaluate the catalytic efficiencies of different lipids, and test for a possible correlation between hemozoin inhibition by
drugs versus their antiplasmodial activity. Consistent with several previous reports, we found a good correlation between hemo-
zoin inhibition potency versus cytostatic antiplasmodial potency (50% inhibitory concentration) for a series of chloroquine
(
(
CQ) analogues. However, we found no correlation between hemozoin inhibition potency and cytocidal antiplasmodial potency
50% lethal dose) for the same drugs, suggesting that cellular targets for these two layers of 4-aminoquinoline drug activity dif-
fer. This important concept is also explored further for QN and its stereoisomers in the accompanying paper (A. P. Gorka, K. S.
Sherlach, A. C. de Dios, and P. D. Roepe, Antimicrob. Agents Chemother. 57:365–374, 2013).
here are an estimated 350 million to 500 million Plasmodium since lipid nanospheres have been shown to be closely associated
T
falciparum malaria infections annually, with at least 1 million with nascent Hz crystals within the DV of the parasite (11, 28).
of these being fatal (1). The continued spread and evolution of Also, a variety of lipids isolated from the DV of malarial parasites
resistance to currently used antimalarials (2–5) have generated have been shown to efficiently catalyze Hz crystal growth in vitro
renewed effort to design drugs effective against chloroquine (11, 28, 30, 31). Although the concentration and exact identity of
(
CQ)-resistant (CQR) malaria. This effort benefits from further the lipids extracted from the DV is still not fully known (11, 28),
development of rapid, inexpensive screens that more reliably es- phosphatidylcholine (PC), monoacylglycerols (MAGs), diacylg-
timate antiplasmodial activity.
lycerols (DAGs), and triacylglycerols (TAGs) are among those
During the trophozoite stage of the intraerythrocytic cycle, P. identified to be present within this acidic compartment.
falciparum actively degrades host red blood cell hemoglobin, pro-
Several plate-based and non-plate-based assays have been de-
ducing millimolar quantities of toxic ferriprotoporphyrin IX veloped to quantify Hz crystal growth and inhibition of that
FPIX) heme within the digestive vacuole (DV) of the parasite (6, growth by drugs (12, 22, 23, 25, 32–37). Most previously reported
). Due to the lack of a heme oxygenase pathway (8), the malarial assays require centrifugation and/or filtration and are performed
(
7
parasite maintains a low concentration of toxic free heme by se- under nonphysiological conditions (an exception being low-
questration into inert, nontoxic crystalline hemozoin (Hz) (9– throughput assays performed previously [11]). Some of these
1
2). The Hz crystallization pathway is an important target for studies have hinted at a correlation between Hz inhibition potency
antimalarial chemotherapy. Quinoline antimalarials, such as CQ and antiplasmodial IC , but contrasting results have also been
50
and amodiaquine (AQ), are known to bind to multiple precrys- reported (19, 21, 38). We recently reported a significant correla-
talline forms of heme in different ways and thus presumably in- tion between Hz inhibition and IC50 across a series of structurally
hibit crystal growth by sequestration of monomeric and/or di- related quinine (QN) analogues (39) but found that it does not
meric heme (13–18). Quantifying drug inhibition of Hz
formation in vitro has often been viewed as a faster and more
economical way to predict and even quantify drug antiplasmodial
activity in vivo (15, 16, 19–26). However, the assays that have been
used in this manner are typically done under conditions that are
far from physiological. Also, it is becoming increasingly clear that
some antimalarial drugs possess both cytostatic (growth-inhibi-
tory; quantified by use of the 50% inhibitory concentration
Received 17 August 2012 Returned for modification 5 October 2012
Accepted 26 October 2012
Published ahead of print 31 October 2012
Address correspondence to Paul D. Roepe, roepep@georgetown.edu.
*
Present address: John N. Alumasa, Department of Biochemistry & Molecular
Biology, The Pennsylvania State University, University Park, Pennsylvania, USA;
Katherine B. Nickley, Georgetown University School of Medicine, Washington, DC,
USA; Jonathan P. Brower, Tufts University School of Medicine, Boston,
Massachusetts, USA.
[
IC ]) and cytocidal (parasite cell kill; quantified by use of the
50
5
0% lethal dose [LD ]) activity. To our knowledge, no studies
50
have tested how Hz inhibition potency is related to both IC50
and LD .
Although Hz crystallization chemistry is not completely eluci-
dated, it is known that preformed Hz, certain proteins, and some
lipids can facilitate heme crystallization (11, 27–29). Current evi-
dence strongly supports lipid catalysis of crystallization in vivo,
A.P.G. and J.N.A. contributed equally to this article.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
5
0
/
AAC.01709-12.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.01709-12
356 aac.asm.org
Antimicrobial Agents and Chemotherapy p. 356–364
January 2013 Volume 57 Number 1

IC50 versus LD50 for CQ Analogues
FIG 1 Structures of common quinoline antimalarials and 7-chloroquinoline drug analogues used in this study. MQ, mefloquine; PQ, primaquine.
hold when cytocidal activity (LD ) is considered the antiplasmo- Sterile polystyrene 96-well tissue culture plates and other laboratory plas-
5
0
dial measurement (see the accompanying paper [40]). In previ- tics were purchased from Fisher Scientific. 1-Monopalmitin (pMAG) and
1
-monostearin (sMAG) were purchased from Doosan Serdary Research
Laboratories (Toronto, ON, Canada). 1,2-Diolyl-rac-glycerol (18:1, cis-9)
DO), 1,2-dipalmitoyl-sn-glycerol-3-PC (DG), and 1,2-dioctanoyl-sn-
ously reported high-throughput assays, multiple steps involved in
the processing of Hz crystals are cumbersome and can add addi-
tional error. Some use hazardous reagents or require expensive
equipment, such as microfiltration plates.
Additional development of several classes of antimalarial drugs
would benefit from a more complete understanding of the rela-
tionship between drug potency against malarial parasites and their
ability to inhibit Hz formation. As part of this effort, high-
(
glycerol-3-PC (DT) were purchased from Avanti Polar Lipids Inc. (Ala-
baster, AL). Kieselgel 60 silica was purchased from Selecto Scientific (Su-
wanee, GA). Sodium dodecyl sulfate (SDS) was purchased from Bio-Rad
Laboratories (Hercules, CA). Hemin was from Fluka (Buchs, Switzer-
land). CDCl was purchased from Cambridge Isotope Laboratories Inc.
3
(Andover, MA). SYBR green I nucleic acid dye was purchased from Invit-
throughput assays that more closely mimic the physiological con- rogen (Eugene, OR).
ditions of Hz formation would be helpful. We have developed
Antimalarial drugs QN monohydrochloride dihydrate, QD hydro-
such an assay that uses physiologically relevant lipid catalysts, chloride monohydrate, CQ diphosphate, and AQ dihydrochloride dihy-
ionic strength, and temperature and that incorporates inexpen- drate were purchased from Sigma-Aldrich. Synthesis and chemical char-
acterization of CQ analogues 1 to 5 have been reported previously (24,
sive, readily attainable, nonhazardous reagents. Using this assay,
4
1–43). Compounds 6 to 9 were synthesized as described below.
General methods. Flash chromatography was performed on Kieselgel
0 (particle size, 0.032 to 0.063 mm). Thin-layer chromatography (TLC)
we quantified the effect of pH on Hz inhibition for common an-
timalarials, CQ, QN, quinidine (QD), and amodiaquine (AQ),
and evaluated the catalytic efficiencies of different lipids. We also
tested for a correlation between Hz inhibition and antiplasmodial
IC50 and LD50 for a series of CQ analogues with a wide range of
antiplasmodial activities (Fig. 1) (24, 41–43).
6
analyses were performed on Selecto Scientific flexible TLC plates (silica gel
0, F 254, 200 ␮m). Nuclear magnetic resonance (NMR) spectra were
6
1
13
obtained on a 400-MHz ( H) and 100-MHz ( C) Varian Fourier trans-
form (FT)-NMR spectrometer (Santa Clara, CA) using CDCl as the sol-
3
vent and tetramethylsilane (TMS) as the external standard.
MATERIALS AND METHODS
Synthesis of compounds 6 to 9 (Fig. 2). (i) N-(7-Chloro-4-quinolyl)-
Materials and chemicals. Routine chemicals, media, and solvents were 1,4-diaminobutane (compound 6). 4,7-Dichloroquinoline (4.0 g, 20.3
reagent grade or better, purchased from Sigma-Aldrich (St. Louis, MO) or mmol) was dissolved in 1,4-diaminobutane (10.7 g, 121.4 mmol) under
Fisher Scientific (Newark, DE), and used without further purification. N and heated to 80°C for 1 h, followed by 100°C for 5 h. A white precip-
2
FIG 2 Synthesis of compounds 6 to 9. NaBH(OAc) , sodium triacetoxyborohydride.
3
January 2013 Volume 57 Number 1
aac.asm.org 357

Gorka et al.
itate was observed after 1 h. After cooling to room temperature, 1 M with an Agilent 8453 UV-visible spectrophotometer (Santa Clara, CA).
NaOH (40 ml) was added and the reaction mixture was extracted with Also, a 5 mM FPIX stock was diluted to 1 mM in 0.1 M bicarbonate buffer
CH Cl . The organic layer was washed with brine and H O, dried over (pH 9.1) containing different concentrations of SDS (0, 0.5, 1.0, 2.5, and
2
2
2
anhydrous Na SO , and concentrated in vacuo to afford the product as a 5%, wt/vol). These solutions were then serially diluted in 1.5-␮l micro-
2
4
1
white solid (3.4 g, 13.5 mmol) in 67% yield. H NMR (400 MHz, CDCl ) centrifuge tubes such that the final FPIX concentration ranged from 0 to
3
␦
(
1.19 (bs, 2H), 1.64 (m, 2H), 1.87 (m, 2H), 2.83 (t, 2H), 3.30 (q, 2H), 6.00 20 ␮M. Equal volumes (200 ␮l) of these solutions were then transferred in
bs, 1H), 6.38 (d, 1H), 7.33 (dd, 1H), 7.73 (d, 1H), 7.94 (d, 1H), 8.52 (d, triplicate to microtiter plate wells. Absorbance readings from the wells
13
1
1
H). C NMR (100 MHz, CDCl ) ␦ 26.26, 30.99, 41.70, 43.41, 99.02, were obtained at 405 nm using a BioTek ELx800 absorbance microplate
3
17.55, 121.62, 125.18, 128.92, 134.82, 149.42, 150.16, 152.28.
ii) N-(7-Chloro-4-quinolyl)-N=-(N؆-t-Boc-aminoethyl)-1,4-
reader (Winooski, VT).
(
Assay optimization. The heme concentration was optimized by com-
diaminobutane (compound 7) (44). A solution of N-(7-chloro-4-quino- paring the Hz yield upon various starting heme concentrations. The op-
timal concentration of catalyst was determined in the presence of 100 ␮M
FPIX, propionate buffer (1 M) at 37°C in a 16-h assay format (see Results).
We compared the Hz yield in the presence of sonicated lipids, phospha-
tidylserine (PS), PC, pMAG, sMAG, DO, DG, and DT, as well as mixtures
of PC, pMAG, and sMAG, versus pH and at a range of lipid concentra-
tions. PC, pMAG, and sMAG produced the highest Hz yields when used at
Ն0.5 mg/ml (see Fig. S1 in the supplemental material). Additionally, cat-
alytic efficiency was assessed at pH 5.2 versus pH 5.6 (45) in kinetic assays
using PC, pMAG, or sMAG. The optimal buffer concentration was deter-
mined in the presence of 100 ␮M FPIX, propionate buffer (pH 5.2), and
lyl)-1,4-diaminobutane (compound 6; 0.9 g, 3.7 mmol), N-tert-butoxy-
carbonyl (N-t-Boc) glycinal (1.0 g, 6.3 mmol), and sodium
triacetoxyborohydride (1.4 g, 6.7 mmol) in anhydrous CH Cl was al-
2
2
lowed to stir at room temperature for 24 h. The reaction was quenched by
the addition of H O, and the organic layer was extracted with saturated
2
NaHCO , dried over anhydrous Na SO , and concentrated in vacuo to
3
2
4
give a yellow oil. This was further purified by column chromatography
over silica gel (5% methanol [MeOH], CH Cl ), with a gradual increase to
2
2
3
0% and, finally, 100% MeOH. The product eluted as a yellow oil (1.1 g,
1
2.7 mmol) in 75% yield. H NMR (400 MHz, CDCl ) ␦ 1.38 (s, 9H plus
3
0.5 mg/ml PC at 37°C using a 16-h assay as described below, with the
solvent peak overlap ϭ 13H), 1.52 (m, 2H), 1.70 (m, 2H), 2.47 (m, 4H),
.11 (m, 2H), 3.25 (m, 2H), 5.06 (bs, 1H), 5.63 (bs, 1H), 6.33 (d, 1H), 7.25
greatest Hz yield achieved at 1 M propionate (see Fig. S2 in the supple-
mental material).
Hz inhibition assay. As described in Results, the optimized assay is
based on the differential solubility of crystalline and noncrystalline forms
of FPIX in 2.5% (wt/vol) SDS (86.7 mM) and alkaline bicarbonate buffer
3
13
(
dd, 1H), 7.79 (d, 1H), 7.78 (d, 1H), 8.42 (d, 1H). C NMR (100 MHz,
CDCl ) ␦ 24.62, 25.98, 28.24, 38.33, 42.92, 49.90, 53.52, 79.06, 98.50,
3
117.01, 121.87, 124.90, 127.37, 134.78, 148.16, 150.32, 151.02, 156.19.
(
iii) N-(7-Chloro-4-quinolyl)-N=-(N؆-t-Boc-aminoethyl)-N=-(3-hy-
(0.1 M, pH 9.1). Hemin (2 mM) in 0.1 M NaOH was titrated to the desired
droxypropyl)-1,4-diaminobutane (compound 8) (44). A solution of
N-(7-Chloro-4-quinolyl)-N=-(NЉ-t-Boc-aminoethyl)-1,4-diamino-
butane (compound 7; 2.4 g, 6.2 mmol) and 3-iodo-1-propanol (0.6 g,
pH with propionate buffer. Ten-microliter volumes from the hemin stock
were then transferred to 96-well plates, followed by addition of propi-
onate buffer (180 ␮l per well) and previously sonicated lipid catalyst (10
3
.1 mmol) was refluxed in anhydrous CH CN for 8 h. The solvent was
3
␮l). Appropriate blanks and controls (e.g., no lipid catalyst, no drugs)
evaporated, and the resulting brownish yellow oil was taken up in
CH Cl (60 ml). This was extracted with saturated NaHCO , dried
were included in each plate. Plates were wrapped in plastic wrap and
incubated at 37°C with gentle shaking. Hz yield assays were terminated
after 16 h by addition of 100 ␮l of a solution of SDS dissolved in 0.1 M
bicarbonate buffer (pH 9.1) (final concentration/well of SDS, 2.5%, wt/
vol; 86.7 mM), and kinetic assays at multiple time points were quenched
in a similar fashion. The well contents were gently mixed and incubated at
room temperature for 10 min to allow undissolved crystals to settle. A
2
2
3
over anhydrous Na SO , and concentrated in vacuo. The crude oil was
2
4
purified via column chromatography (5% MeOH, CH Cl ), with a
2
2
gradual increase to 30% MeOH. The pure product eluted as a brownish
1
yellow oil (1.2 g, 2.7 mmol) in 86% yield. H NMR (400 MHz, CDCl )
3
␦
2
1
1.37 (s, 9H plus solvent peak overlap ϭ 15H), 1.57 (m, 2H), 1.81 (m,
H), 2.13 (m, 2H), 2.54 (m, 6H), 3.14 (m, 4H), 3.61 (m, 4H), 4.12 (bs,
H), 5.14 (bs, 1H), 5.25 (bs, 1H), 6.69 (d, 1H), 7.51 (d, 1H), 7.82
50-␮l aliquot from each well was then transferred to a second plate pre-
loaded with 200 ␮l/well of SDS solution (2.5%, wt/vol; 86.7 mM) in 0.1 M
bicarbonate buffer, and the absorbance at 405 nm was read with a 96-well-
plate-adapted ELx800 BioTek absorbance microplate reader.
Conversion of the absorbance values to the FPIX concentration (␮M)
remaining in the sample wells was done using a linear calibration curve
measured for each assay. FPIX stocks ranging in concentration from 0 to
1
3
(
s, 1H), 7.94 (s, 1H), 8.62 (d, 1H). C NMR (100 MHz, CDCl ) ␦
3
2
9
1
5.46, 25.74, 28.35, 30.91, 38.39, 43.35, 50.36, 51.82, 53.4, 57.48, 79.11,
8.30, 98.88, 116.72, 127.42, 127.84, 138.25, 140.73, 147.13, 155.02,
56.32.
(
iv) N-(7-Chloro-4-quinolyl)-N=-(N؆-aminoethyl)-N=-(3-hydroxy-
propyl)-1,4-diaminobutane (compound 9) (44). N-(7-Chloro-4-quino-
lyl)-N=-(NЉ-t-Boc-aminoethyl)-N=-(3-hydroxypropyl)-1,4-diamino-
butane (compound 9; 1.2 g, 2.7 mmol) was dissolved in anhydrous MeOH
1
9
6 ␮M were freshly prepared in 2.5% SDS–0.1 M bicarbonate buffer (pH
.1). Volumes of 250 ␮l were transferred to predesignated wells, the ab-
sorbance at 405 nm was read, and the data were fit to a straight line. Free
FPIX remaining in solution (inverse of Hz produced) was quantified us-
ing equation 1:
(
40 ml). 2 M HCl (14 ml, 28.0 mmol) was added, and the reaction was
allowed to stir at room temperature for 24 h. The solvent was evaporated,
and the crude product was taken up in CH Cl . This was extracted with 1
2
2
s
0
A
Ϫ A Ϫ C
M NaOH, and the combined organic layers were dried over anhydrous
X
405
405
͓
H ͔ ϭ
ϫ D
(1)
ͫ
ͬ
Na SO and concentrated in vacuo to afford the pure product (0.5 g, 1.4
␧405
2
4
1
s
4
0
405
X
mmol) in 53% yield. H NMR (400 MHz, CDCl ) ␦ 1.53 (m, 2H), 1.70 (m,
3
where [H ] is the concentration of FPIX (␮M) remaining, A and A
H), 1.85 (m, 2H), 2.42 (m, 4H), 2.66 (m, 4H), 3.22 (t, 2H), 3.52 (t, 2H), are the absorbance (405 nm) readings (averages from triplicate wells) of
05
2
3
1
4
1
.91 (t, 2H), 5.72 (t, 1H), 6.29 (d, 1H), 7.24 (d, 1H), 7.77 (d, 1H), 7.83 (d, the sample and blank, respectively, ε405 is the extinction coefficient for
1
3
H), 8.40 (d, 1H). C NMR (100 MHz, CDCl ) ␦ 25.13, 30.60, 39.51, FPIX, C is a constant obtained from the fit to the calibration curve, and D
3
8.55, 53.49, 54.26, 57.06, 57.62, 78.89, 98.60, 98.65, 114.16, 123.15, is the dilution factor.
27.03, 136.18, 139.03, 154.30, 156.05.
Antiplasmodial activity measurements. P. falciparum strains HB3
Effect of SDS on absorbance of FPIX. The effect of SDS on FPIX and Dd2 were obtained from the Malaria Research and Reference Reagent
absorbance was analyzed in two ways. First, a solution of FPIX (5 mM) Resource Center (Manassas, VA). Off-the-clot, heat-inactivated pooled
was prepared in 0.1 M NaOH and diluted to 10 ␮M in 0.1 M bicarbonate type O-positive human serum and O-positive human whole blood were
buffer (pH 9.1), and aliquots of an SDS stock solution (50%, wt/vol; 1.734 purchased from Biochemed Services (Winchester, VA). Custom 5%
M) were added to 1 ml of the FPIX solution in a stirred cuvette. Titration O –5% CO –90% N culturing gas blend was purchased from Robert’s
2
2
2
with SDS (to a final concentration of 0.5 to 5%, wt/vol) was monitored Oxygen (Rockville, MD).
358 aac.asm.org
Antimicrobial Agents and Chemotherapy

IC50 versus LD50 for CQ Analogues
All P. falciparum strains were maintained using the method of Trager
and Jensen (46), with minor modifications. Briefly, cultures were main-
tained under an atmosphere containing 5% O , 5% CO , 90% N gaseous
2
2
2
mix at 2% hematocrit and 1 to 2% parasitemia in RPMI 1640 supple-
mented with 10% type O-positive human serum, 25 mM HEPES (pH 7.4),
2
3 mM NaHCO , 11 mM glucose, 0.75 mM hypoxanthine, and 20 ␮g/liter
3
gentamicin with regular medium changes every 48 h. Antiplasmodial cy-
tocidal (LD ) and cytostatic (IC ) activities against the chloroquine-
5
0
50
sensitive (CQS) HB3 strain and CQR Dd2 strain were assessed. Cytostatic
antiplasmodial activities have previously been reported for some drugs
used in this study (24, 41–43), and additional activities were determined
as described previously (47, 48), with minor modifications.
The cytocidal assay utilizes a 6-h bolus dose with high concentrations
of drug, followed by washing drug away and growth in the absence of drug
for 48 h, while the cytostatic assay utilizes continuous growth for 48 h in
the constant presence of low concentrations of drug. Test compounds
were dissolved in deionized water or dimethyl sulfoxide (DMSO). Serial
drug dilutions were made using complete medium, and 100-␮l aliquots
were transferred to 96-well clear-bottom black plates. Following addition
of 100 ␮l of asynchronous or sorbitol-synchronized culture (1% final
parasitemia, 2% final hematocrit), plates were transferred to an airtight
chamber gassed with 5% O –5% CO –90% N and incubated at 37°C.
2
2
2
For the cytocidal assay, plates were incubated for 6 h, followed by
centrifugation with an Eppendorf 5804 centrifuge fitted with an A-2-
DPW rotor (Hauppauge, NY) at 700 ϫ g for 3 min. Drug-containing
medium was removed, and cell pellets were washed three times with 200
␮l of complete medium per wash, using the same centrifuge settings, and
resuspended in the same volume of medium. Washed plates along with
the cytostatic assay plates were incubated at 37°C for 48 h. After 48 h, 50 ␮l
of 10ϫ SYBR green I dye (diluted using complete medium from a
10,000ϫ DMSO stock) was added and the plates were incubated for an
additional 1 h at 37°C to allow DNA intercalation. Fluorescence was mea-
sured at 538 nm (485-nm excitation) using a Spectra GeminiEM plate
reader (Molecular Devices, Sunnyvale, CA) fitted with a 530-nm long-
pass filter. Linear standard curves of measured fluorescence versus known
parasitemia were prepared immediately prior to plate analysis. Back-
ground controls included fluorescence from uninfected red blood cells.
Data were analyzed using Microsoft Excel 2007 software, and IC50 and
LD50 values were obtained from sigmoidal curve fits to percent growth/
survival-versus-drug concentration data using SigmaPlot (version 11.0)
FIG 3 (A) SDS effects on the absorbance spectrum of FPIX. Increasing
amounts of SDS were titrated into a cuvette containing 10 ␮M FPIX while the
absorbance spectrum was recorded after each addition. Results are shown for
no SDS (red), 0.1% (3.47 mM) SDS (brown), 0.5% (17.3 mM) SDS (green),
software. Reported IC s are the average of three independent assays, with 1% (34.7 mM) SDS (black), 2.5% (86.7 mM) SDS (blue), and 5% (173 mM)
5
0
each assay conducted in triplicate (nine determinations total) and re- SDS (pink). (Inset) Peak height at 405 nm versus SDS concentration. a.u.,
ported Ϯ the standard error of the mean. LD50 values are the average of absorbance units. (B) Effect of SDS on the concentration-dependent absor-
bance (Abs.) of FPIX in the 96-well plate format. Results are shown for no SDS
two independent assays, with each assay conducted in triplicate (six de-
terminations total).
VAR calculations. Vacuolar accumulation ratio (VAR) calculations
were done using the Henderson-Hasselbalch equation (49, 50) (see equa-
tion 2 below) with the external pH at 7.33 and the DV pH at either 5.2 or
2
2
(
(
ꢀ, R ϭ 0.996), 0.1% (3.47 mM) SDS (Œ, R ϭ 0.988), 0.5% (17.3 mM) SDS
2 2
, R ϭ 0.992), 1% (34.7 mM) SDS (ꢁ, R ϭ 0.996), 2.5% (86.7 mM)
2
2
SDS (ᮀ, R ϭ 0.998), and 5% (173 mM) SDS (⌬, R ϭ 0.999). Increasing the
concentration of SDS increases the absorbance of FPIX to a maximum at
ϳ2.5% SDS. Each data point is an average of three independent measure-
ments, each performed in triplicate (9 determinations total).
5
.6 (45). Our calculations also assume that (i) charged (protonated) drugs
5
are Ͼ10 -fold less membrane permeant than neutral drugs and (ii) initial
rapid accumulation is not limited by binding to the drug target(s):
͓
drug͔
V
(Fig. 3A, arrow 1). We found that simple titration of the ␮-oxo
dimer with SDS results in a red shift of the FPIX Soret band from
͓
drug͔E
͑
pKa1 Ϫ pHV
͒
͑
pKa1 ϩ pKa2 Ϫ 2pHV
͒
͑ ͒
pKa1 ϩ pKa2 ϩ pKa3 Ϫ 3pHV
1
ϩ 10
ϩ 10
ϩ 10
389 nm to 405 nm (Fig. 3A, arrow 3), as well as a concentration-
ϭ
͑
pKa1 Ϫ pHE
͒
͑
pKa1 ϩ pKa2 Ϫ 2pHE
͒
͑ ͒
pKa1 ϩ pKa2 ϩ pKa3 Ϫ 3pHE
1
ϩ 10
ϩ 10
ϩ 10
dependent increase in absorbance. This is presumably because
where V denotes DV, E denotes external (blood), and pKa , pKa , and upon addition of SDS at concentrations above the critical micelle
1
2
pKa correspond to titratable N on the CQ analogues. DV-scaled in vivo
3
concentration, free ␮-oxo dimeric heme is converted to its mono-
meric form (52), generating the ␭max shift to 405 nm and a con-
activities for drugs were obtained by multiplying the raw antiplasmodial
IC s and LD s by the corresponding calculated VAR values.
Ϫ1
50
50
comitant increase in the extinction coefficient to 99,690 (M
Ϫ1
cm ). This effect is also easily measured in 96-well plate format
RESULTS
(
Fig. 3B), and the linear increase in absorbance then allows for
The broad FPIX absorbance peak observed in the absence of SDS, calibration of free FPIX at a known SDS concentration. That is,
characteristic of the ␮-oxo dimer (51), exhibits maximum ␭ conversion of FPIX t␱ ⌯z can be quantified via simple addition of
(␭max) at 389 nm (Fig. 3A, arrow 2), with a shoulder at 359 nm SDS, without plate-washing steps.
January 2013 Volume 57 Number 1
aac.asm.org 359

Gorka et al.
FIG 4 Optimization of lipid catalyst. Comparison of the Hz (␤-hematin)
catalytic efficiency of PS (L-␣-phosphatidyl-L-serine), SG (1-monostearin,
sMAG), DO (1,2-diolyl-rac-glycerol [18:1, cis-9]), DT (1,2-dioctanoyl-sn-
glycerol-3-PC), PG (1-monopalmitin, pMAG), DG (1,2-dipalmitoyl-sn-glyc-
erol-3-PC), PC (L-␣-phosphatidylcholine), and BD (blend, meaning an
equimolar mixture of PC, pMAG, and sMAG resulting in 1 mM/well final
concentration). FPIX (100 ␮M) was incubated in 1 M propionate buffer (at the
different pH values) at 37°C in the presence of 1 mM the corresponding lipid.
The amount of Hz formed in the corresponding lipid wells (average of tripli-
cate wells) was quantified relative to that for a lipid-free control after a 16-h
incubation period. The 100% Hz yield was calculated via equation 1, with the
sample absorbance being that of 100 ␮M FPIX.
To develop an assay that takes advantage of this observation
and that also mimics formation of Hz in vivo at physiological
temperature, pH, and ionic strength, we screened the catalytic FIG 5 pH-dependent kinetics for the production of Hz (␤-hematin) in the
presence of PC (ꢀ), pMAG (Œ), or sMAG () or the absence of a catalyst (⌬).
abilities of various lipids that are suggested from recent reports to
reside within the DV (11, 28). We systematically compared the
catalytic efficiency of PC to that of other lipids and mono- or
Sonicated lipids were incubated at 37°C with 100 ␮M FPIX in 200 ␮l of pro-
pionate buffer for 16 h at pH 5.2 (A) and pH 5.6 (B). Curve fitting was per-
formed with SigmaPlot (version 10.0) software using the equation y ϭ a(1 Ϫ
x
diacyl glycerols (Fig. 4). At physiological temperature, catalysis by b ). (Insets) The corresponding initial rate plots from time zero (t ) to 4 h (t )
0
4
(
Table 1). Inset data are fit to linear regressions, and the initial rates were
PC was comparable to if not better than that found for all other
lipids tested, including a blend of PC, pMAG, and sMAG (Fig. 4,
BD). We also found that lipid-catalyzed formation of Hz is pH
dependent, with the strongest pH dependence found for PS and
the weakest found for PC and the PC-pMAG-sMAG blend. More
acidic aqueous conditions favor formation of the heme head-to-
tail dimer (20); thus, acceleration of Hz formation at lower pH is
expected.
We selected PC as the catalyst for additional work because it
performed similarly to the blend yet is less expensive and more
easily handled. The optimal FPIX concentration was determined
to be 100 ␮M, since this provides a wide dynamic range (Ͼ0.6
absorbance unit), as it is converted to Hz over approximately 16 h
extracted from the slopes. Each data point is an average of three independent
measurements, each performed in triplicate (9 determinations total).
rates of Hz formation and noted that at pH 5.2, PC catalysis was
1.5-, 2-, and 5-fold faster than that with pMAG, sMAG, and the
control (no lipid), respectively, and that the rate for PC-catalyzed
Hz formation in this assay was very similar to that defined for the
rate of Hz growth in live parasites (11).
TABLE 1 Initial rates of production of Hz in the presence of PC,
pMAG, and sMAG and in the absence of lipid catalysts
(see below). Also, under control assay conditions, conversion of
a
Ϫ
Ϫ8
Initial rate (M s 1 [10 ])
1
00 ␮M FPIX to ⌯z was near 100% (see below).
We next compared the kinetics ␱f Hz formation at 37°C with Catalyst
pH 5.2
pH 5.6
PC, pMAG, or sMAG as catalysts and at two different pHs that
correspond to extremes in DV pH that have been measured for PC
various strains of P. falciparum (45) (Fig. 5; Table 1). Again, PC pMAG
was found to be the most efficient catalyst at either pH, and Hz sMAG
No lipid
0.67
3.61
2.39
1.78
0.57
1.77
1.51
1.66
a
formation appeared to plateau at about 16 h. We calculated initial
Initial rates were calculated within the first 4 h of the reaction (see insets in Fig. 5).
360 aac.asm.org
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IC50 versus LD50 for CQ Analogues
vacuolar accumulation (see Materials and Methods). However,
this did not improve the correlation between either IC50 or LD50
and Hz inhibition (data not show).
LD50 versus IC50 data highlight interesting trends for the cyto-
cidal and cytostatic activities observed in the correlation analysis
(
Fig. 7). For CQ, CQR strain Dd2 was ϳ10-fold resistant by IC50
but ϳ140-fold resistant by LD50 (48) (see Table S1 in the supple-
mental material). In contrast, AQ has low-nM LD s, with no
50
significant differences for HB3 versus Dd2 (48). Interestingly, for
compounds 1 and 7, Dd2 is hypersensitive by IC50 (more sensitive
than CQS strain HB3) but is either slightly resistant relative to
HB3 (compound 1) or has sensitivity similar to that of HB3 (com-
pound 7) when potency is quantified by LD . Dd2 is hypersensi-
50
tive to compounds 6 and 9 by LD50 but is resistant to compounds
FIG 6 Effect of pH on the Hz inhibition (␤-hematin inhibitory activity 2 to 5 and 8. Interestingly, some compounds with high IC s (low
50
[
(
(
BHIA]) IC50 of known antimalarial drugs: CQ (ꢀ), AQ (Œ), QN (), and QD
⌬). Experiments were performed at 37°C in a 16-h assay. Each data point
Ϯ SD) is the average of three independent measurements, each performed in
cytostatic activity) are more active by LD50 (high cytocidal po-
tency) (compounds 7 to 9). Overall, then, the good correlation
between Hz inhibition and IC , with a lack of a correlation be-
triplicate (9 determinations total) at the corresponding pH.
50
tween Hz inhibition and LD50 for the same drugs, spans a range of
CQ compound behavior for CQS versus CQR strains.
DISCUSSION
Using this standardized assay, we quantified inhibition of Hz
formation by common antimalarials (CQ, QN, QD, and AQ) at We report a more physiologically relevant high-throughput assay
several pHs. Overall, similar to other studies (19, 22, 25), we found for quantifying inhibition of Hz formation and test how cytostatic
that potencies were AQ Ͼ CQ Ϸ QD Ͼ QN across the range of (IC50) versus cytocidal (LD50) activities of CQ and CQ analogues
pHs (Fig. 6). We noted that different absolute IC s were obtained correlate with Hz inhibition. Consistent with most data from
5
0
at the same pH when the identity of the lipid catalyst was changed other Hz formation assays, we found that Hz inhibition measured
Table 2) but that the trends for the drugs remained similar. For under physiological conditions decreases for all drugs examined
(
CQ and AQ, the differences observed when PC versus pMAG cat- upon decreasing pH. Presumably, more acidic conditions require
alysts were used were relatively small (2- to 3-fold); however, in- a higher drug concentration to inhibit crystal growth because
terestingly, QN and QD IC s were more perturbed by altering the crystallization is accelerated and also because the solubility of pre-
5
0
lipid composition. This is consistent with the differences in CQ crystalline heme intermediates to which drugs bind is decreased at
versus QN lipid partitioning behavior noted earlier (52).
these pHs. We found that the efficiency of PC in catalyzing Hz
It has previously been suggested that Hz inhibition and anti- formation is comparable to if not better than the efficiency of
plasmodial potency are well correlated (19). However, another catalysis by monoacylglycerols, diacylglycerols, and other lipids.
study observed that upon correction for different levels of DV All lipids that we tested are found in DV lipid nanospheres har-
accumulation, the linear correlation existed only for CQS strains boring nascent Hz crystals, but it is currently not known specifi-
(
21). In both of these studies, the nonphysiological Hz formation cally which of the lipids accelerates the Hz formation that presum-
conditions and the wide structural diversity of the examined drugs ably occurs at these nanospheres (11, 28). Our data suggest that all
complicate interpretation. We thus tested for a possible correla- known components of these nanospheres are excellent catalysts.
tion between inhibition and antiplasmodial activity using a series
We used this assay to test previously hypothesized correlations
of CQ analogues that are structurally similar yet span a wide range between Hz inhibition and the antiplasmodial IC50 in order to
of antiplasmodial IC s and LD s (Fig. 1; see Table S1 in the ascertain if more close-to-physiological conditions for Hz forma-
5
0
50
supplemental material) (24, 41–43). These analogues also span the tion modified those conclusions. Dorn et al. (19) were the first to
effective monobasic, dibasic, and tribasic character and thus di- directly test the hypothesis that antiplasmodial activity was corre-
verge in calculated VARs (see Materials and Methods). When Hz lated with Hz inhibition by examining a small set of common
inhibition for these compounds was plotted against the antiplas- antimalarials (including quinoline methanols, 4-aminoquino-
modial IC50 determined versus live merozoite culture, we ob- lines, acridines, and nonquinolines). Although Cohen (53) and
served a good correlation when antiplasmodial activity for either
2
2
CQS (R ϭ 0.653) or CQR (R ϭ 0.639) strains was tabulated (Fig.
A and B). However, importantly, when LD50 was plotted versus
Hz inhibition, these correlations vanished (Fig. 7C and D).
As described earlier (24), replacement of the anilinal N by S or
O for some of these compounds significantly lowers the quinolinal
TABLE 2 IC50 for Hz inhibition at pH 5.2 for common antimalarials in
the presence of PC, pMAG, or sMAG
7
a
BHIA IC50 (␮M)
Compound
PC
pMAG
sMAG
N pK (to values near those seen for QN or QD; see reference24).
a
CQ
AQ
QN
67.7
25.4
237
111
22.4
11.7
59.8
12.6
34.9
16.5
131
In vivo antiplasmodial IC s are presumably defined, in part, by
5
0
the fold accumulation of the drug at the site of Hz formation (i.e.,
the acidic DV interior). We therefore scaled antiplasmodial IC s QD
36.2
50
and LD s by multiplying by the calculated VARs for each mole-
a
5
0
PC, pMAG, or sMAG were present at 1 mM. IC50s were determined using a 16-h
cule, such that the in vivo IC s were normalized to predicted endpoint assay at 37°C. BHIA, ␤-hematin inhibitory activity.
5
0
January 2013 Volume 57 Number 1
aac.asm.org 361

Gorka et al.
FIG 7 Correlation plots between the Hz inhibition (␤-hematin inhibitory activity [BHIA]) IC50 and antiplasmodial activities against HB3 (A, C) or Dd2 (B, D)
for a series of CQ analogues. Best-fit plots of the data were generated by the least-squares method using SigmaPlot software. (A) Plot of BHIA IC versus
5
0
2
2
antiplasmodial IC50 for CQS HB3 parasites (R ϭ 0.653); (B) plot of BHIA IC versus antiplasmodial IC50 for CQR Dd2 parasites (R ϭ 0.639); (C) plot of BHIA
IC50 versus antiplasmodial LD50 for HB3 parasites (R ϭ 0.015); (D) plot of BHIA IC versus antiplasmodial LD50 for Dd2 parasites (R ϭ 0.057). IC and LD50
50
2
2
50
50
values are those found in Table S1 in the supplemental material.
Macomber and Spintz (54) proposed that heme was a target for
A complete understanding of the correlation between antiplas-
quinoline antimalarial drugs more than 30 years before this study, modial IC50 activity and Hz inhibition requires more detailed
results from Dorn et al. (19) have been crucial for exploring the chemical information on the nature of solid-state aggregates of
molecular pharmacology of antimalarial drugs and have been heme and quinoline compounds. Earlier studies clearly showed
highly influential in guiding additional antimalarial drug develop- that the solubilities of monomeric and dimeric forms of FPIX
ment. Using a CQS strain (NF54), these authors observed a linear are dramatically affected by the presence of quinolines (51, 56),
correlation between the potency of Hz inhibition and antiplasmo- that CQ and other quinoline antimalarials bind with different
dial IC50 for common antimalarials, which are thus now known as affinities to monomeric versus dimeric FPIX (14, 17, 51), and
“heme-acting” antimalarial drugs. Hawley et al. (21) further in- that the solubility of drug-heme complexes in hydrophobic
vestigated this concept using CQS (3D7) and CQR (K1) strains phase may be more relevant to Hz inhibition potency (11, 52).
and a similar series of quinoline methanols, 4-aminoquinolines, Taken together, these data show that quinoline drug-heme in-
and acridines. In contrast to Dorn and coworkers (19), these au- teractions are significantly more complex than simple aqueous
thors observed no apparent correlation between the two activities equilibria predict.
when comparing raw IC50 data for either CQS or CQR strains.
Regardless, the principle conclusion of Dorn et al. (19) has
However, after VAR correction, Hawley and coworkers (21) did continued to strengthen the notion that the site of quinoline an-
observe a correlation, but only for the CQS strain. A third study by timalarial drug action must be the DV, where the drug then binds
Kaschula et al. (38) also observed a correlation between the two to free heme to prevent Hz crystallization. Therefore, altered DV
activities, but again, only when the antiplasmodial IC50 was scaled biochemistry that reduces drug-heme interaction must be the
to the relative VAR. In this study, the authors utilized a series of cause of resistance to these drugs, a prediction that has been vali-
2
-carbon 4-aminoquinoline derivatives with variable functional- dated by studies on the function of mutant P. falciparum chloro-
ities at position 7. quine resistance transporter (PfCRT; a DV membrane protein),
There are logical explanations for these differing results. First, which causes an elevated CQ IC50 in P. falciparum malaria (57).
the use of a nonhomologous series of compounds (e.g., quinolines Data in the present paper reveal a good correlation between
versus acridines) that have very different bioavailability profiles, as antiplasmodial IC s and Hz inhibition across a series of CQ ana-
50
well as the nonphysiological conditions of the Hz formation assays logues with a wide range of IC s (Fig. 7), similar to the conclu-
50
that were used, makes interpretation more complex. There are sions of Dorn et al. (19). Using Hz inhibition quantified under
multiple Hz precursors (FPIX monomer, ␮-oxo dimer, and head- physiological conditions with lipid catalyst, our data also suggest
to-tail dimer) against which this range of pharmacophores inter- that the correlation is not necessarily improved when estimated
acts (13, 14, 17, 51, 52, 55), and the relative effectiveness of mo- VARs or other pharmacokinetic parameters (e.g., pKa, octanol/
nomeric heme-drug versus dimeric heme-drug complexes for water partitioning, and water diffusion; see Table S1 in the sup-
inhibiting Hz formation likely differs.
plemental material) are applied to the data set (data not shown;
362 aac.asm.org
Antimicrobial Agents and Chemotherapy

IC50 versus LD50 for CQ Analogues
see Materials and Methods and Table S1 in the supplemental ma- ACKNOWLEDGMENTS
terial). Regardless, although the IC50s of our set of CQ analogues
This work was supported by NIH grants AI045957 (to P.D.R.) and
span a very wide range, they are not necessarily representative of AI060792 (to A.C.D.D. and P.D.R. and to C. Wolf, Georgetown Univer-
all CQ analogues studied to date, and correlation between anti- sity).
plasmodial IC50 and Hz inhibition IC50 for quinoline antimalari-
als may be dependent (at least in part) upon the specific drugs that
are assessed (see the accompanying paper [40]).
We thank Changan Xie for insightful discussions and the C. Wolf
laboratory at the Georgetown University Department of Chemistry for
providing additional aliquots of compounds 1 to 5.
Critically, however, and in distinct contrast to the correlation
with the IC , there is a complete lack of correlation between the
Hz inhibition IC50 and antiplasmodial LD50 for the same series of
CQ analogues (Fig. 7C and D). This is similar to conclusions
reached previously with a smaller set of quinoline methanol
compounds, including 9-epimers of quinine and quinidine
REFERENCES
5
0
1. World Health Organization. 2010. World malaria report 2010. World
Health Organization, Geneva, Switzerland. http://www.who.int/malaria
/publications/atoz/9789241564106/en/index.html.
2
. Cheeseman IH, Miller BA, Nair S, Nkhoma S, Tan A, Tan JC, Al Saai
S, Phyo AP, Moo CL, Lwin KM, McGready R, Ashley E, Imwong M,
Stepniewska K, Yi P, Dondorp AM, Mayxay M, Newton PN, White NJ,
Nosten F, Ferdig MT, Anderson TJ. 2012. A major genome region
underlying artemisinin resistance in malaria. Science 336:79–82.
(
see the accompanying paper [40]). Our test series of CQ ana-
logues were chosen because they exhibit a wide range of cytostatic
and cytocidal activities (see Results and Table S1 in the supple-
mental material) and also show a variety of other interesting
trends, as noted in Results. Strain Dd2 resistance to CQ is 10-fold
3. Roepe PD. 2009. Molecular and physiologic basis of quinoline drug re-
sistance in Plasmodium falciparum malaria. Future Microbiol. 4:441–455.
4. Sa JM, Chong JL, Wellems TE. 2011. Malaria drug resistance: new
observations and developments. Essays Biochem. 51:137–160.
by IC50 but 140-fold by LD , suggesting that a significantly
50
5. Barnes KI, Watkins WM, White NJ. 2008. Antimalarial dosing regimens
different pharmacology may be relevant to understanding the
different modes of action for CQ (48). AQ does not exhibit
significant differences in IC50 and LD50 activity for HB3 versus
Dd2 and is quite potent (low nM) by both antiplasmodial mea-
surements (see Table S1 in the supplemental material). This
suggests to us that the cytocidal targets for CQ versus AQ may
differ and/or that the mechanism of CQ cytocidal resistance is
distinct from that for AQ.
With these results, we suggest that three critical principles are
becoming increasingly apparent: (i) potent IC50 activity can but
does not always indicate potent LD50 activity for quinoline anti-
malarials, (ii) resistance to cytostatic drug effects does not always
correlate with resistance to cytocidal drug effects, and (iii) for CQ
and drug resistance. Trends Parasitol. 24:127–134.
6. Banerjee R, Liu J, Beatty W, Pelosof L, Klemba M, Goldberg DE. 2002.
Four plasmepsins are active in the Plasmodium falciparum food vacuole,
including a protease with an active-site histidine. Proc. Natl. Acad. Sci.
U. S. A. 99:990–995.
. Gamboa de Domínguez ND, Rosenthal PJ. 1996. Cysteine proteinase
inhibitors block early steps in hemoglobin degradation by cultured ma-
laria parasites. Blood 87:4448–4454.
. Yoshida T, Migita TC. 2000. Mechanism of heme degradation by heme
oxygenase. J. Inorg. Biochem. 82:33–41.
. Egan TJ, Combrinck JM, Egan J, Hearne GR, Marques HM, Ntenteni S,
Sewell BT, Smith PJ, Taylor D, van Schalkwyk DA, Walden JC. 2002.
Fate of haem iron in the malaria parasite Plasmodium falciparum.
Biochem. J. 365:343–347.
7
8
9
1
0. Pagola S, Stephens PW, Bohle DS, Kosar AD, Madsen SK. 2000. The
structure of malaria pigment beta-haematin. Nature 404:307–310.
and CQ analogues, the ability to inhibit Hz formation correlates 11. Pisciotta JM, Coppens I, Tripathi AK, Scholl PF, Shuman J, Bajad S,
strongly with IC50 potency but not with LD50 potency. The large
differences between IC50 and LD50 measures of activity against
HB3 and Dd2 parasites suggest that the molecular mechanisms of
resistance to the cytostatic and cytocidal effects of quinoline anti-
Shulaev V, Sullivan DJ, Jr. 2007. The role of neutral lipid nanospheres in
Plasmodium falciparum haem crystallization. Biochem. J. 402:197–204.
2. Tripathi AK, Garg SK, Tekwani BL. 2002. A physiochemical mechanism
of hemozoin (␤-hematin) synthesis by malaria parasite. Biochem. Bio-
phys. Res. Commun. 290:595–601.
1
malarial drugs likely differ (see also reference58). For CQ, cyto- 13. de Dios AC, Casabianca LB, Kosar A, Roepe PD. 2004. Structure of the
amodiaquine-FPIX mu-oxo dimer solution complex at atomic resolution.
Inorg. Chem. 43:8078–8084.
static resistance (elevated IC ) is mediated by PfCRT and/or P.
5
0
falciparum multidrug resistance protein (PfMDR1) mutations
that alter the electrochemical potential-driven DV transport of the
protonated drug (57, 59–63) and, hence, access to heme. How-
ever, mounting experimental evidence (48, 58) suggests the pos-
sibility of additional (non-DV) cytocidal targets for this class of
drugs, which then predicts the possibility of additional cytocidal
resistance mechanisms that are mechanistically distinct from cy-
tostatic resistance mechanisms (64).
In sum, we have used physiological assay conditions and anal-
ysis of a homologous series of CQ analogues that possess a wide
range of pharmacokinetic properties and antiplasmodial activities
to test the correlation between antiplasmodial activity and inhibi-
tion of Hz. All compounds were screened side by side under the
same conditions, using an assay incorporating physiological tem-
perature, ionic strength, and lipid catalyst. The results, coupled
with other recent data (48, 58), strongly support the idea that Hz
inhibition is the predominant target for the cytostatic activity of
CQ and related compounds but not the predominant target for
cytocidal activity.
1
4. de Dios AC, Tycko R, Ursos LMB, Roepe PD. 2003. NMR studies of
chloroquine-ferriprotoporphyrin IX complex. J. Phys. Chem. A 107:
5821–5825.
15. Egan TJ, Ncokazi KK. 2005. Quinolines decrease the rate of ␤-hematin
formation. J. Inorg. Biochem. 99:1532–1539.
1
6. Egan TJ, Ross DC, Adams PA. 1994. Quinoline anti-malarial drugs
inhibit spontaneous formation of beta-haematin (malaria pigment). FEBS
Lett. 352:54–57.
7. Leed A, DuBay K, Sears D, de Dios AC, Roepe PD. 2002. Solution
structures of antimalarial drug-heme complexes. Biochemistry 41:10245–
1
1
1
10255.
8. Sullivan DJ, Jr, Gluzman IY, Russell DG, Goldberg DE. 1996. On the
molecular mechanism of chloroquine’s antimalarial action. Proc. Natl.
Acad. Sci. U. S. A. 93:11865–11870.
9. Dorn A, Vippagunta SR, Matile H, Jaquet C, Vennerstrom JL, Ridley
RG. 1998. An assessment of drug-haematin binding as a mechanism for
inhibition of haematin polymerisation by quinoline antimalarials.
Biochem. Pharmacol. 55:727–736.
0. Egan TJ, Mavuso WW, Ncokazi KK. 2001. The mechanism of beta-
hematin formation in acetate solution. Parallels between hemozoin for-
mation and biomineralization processes. Biochemistry 40:204–213.
1. Hawley SR, Bray PG, Mungthin M, Atkinson JD, O’Neill PM, Ward SA.
1998. Relationship between antimalarial drug activity, accumulation, and
2
2
January 2013 Volume 57 Number 1
aac.asm.org 363

Gorka et al.
inhibition of heme polymerization in Plasmodium falciparum in vitro.
Antimicrob. Agents Chemother. 42:682–686.
chloroquine analogues carrying a multifunctional linear side chain.
Bioorg. Med. Chem. 17:6560–6566.
2
2
2
2. Huy NT, Uyen DT, Maeda A, Trang DT, Oida T, Harada S, Kamei K.
43. Yearick K, Ekoue-Kovi K, Iwaniuk DP, Natarajan JK, Alumasa J, de
Dios AC, Roepe PD, Wolf C. 2008. Overcoming drug resistance to
heme-targeted antimalarials by systematic side chain variation of
7-chloro-4-aminoquinolines. J. Med. Chem. 51:1995–1998.
2
007. Simple colorimetric inhibition assay of heme crystallization for
high-throughput screening of antimalarial compounds. Antimicrob.
Agents Chemother. 51:350–353.
3. Kurosawa Y, Dorn A, Kitsuji-Shirane M, Shimada H, Satoh T, Matile H, 44. Lekostaj JK, Natarajan JK, Paguio MF, Wolf C, Roepe PD. 2008.
Hofheinz W, Masciadri R, Kansy M, Ridley RG. 2000. Hematin polymer-
ization assay as a high-throughput screen for identification of new antimalar-
ial pharmacophores. Antimicrob. Agents Chemother. 44:2638–2644.
Photoaffinity labeling of the Plasmodium falciparum chloroquine resis-
tance transporter with a novel perfluorophenylazido chloroquine. Bio-
chemistry 47:10394–10406.
4. Natarajan JK, Alumasa JN, Yearick K, Ekoue-Kovi KA, Casabianca LB, 45. Bennett TN, Kosar AD, Ursos LM, Dzekunov S, Singh-Sidhu AB,
de Dios AC, Wolf C, Roepe PD. 2008. 4-N-, 4-S-, and 4-O-chloroquine
analogues: influence of side chain length and quinolyl nitrogen pKa on
activity vs chloroquine resistant malaria. J. Med. Chem. 51:3466–3479.
5. Ncokazi KK, Egan TJ. 2005. A colorimetric high-throughput beta-
hematin inhibition screening assay for use in the search for antimalarial
compounds. Anal. Biochem. 338:306–319.
6. Parapini S, Basilico N, Pasini E, Egan TJ, Olliaro P, Taramelli D, Monti
D. 2000. Standardization of the physicochemical parameters to assess in
vitro the beta-hematin inhibitory activity of antimalarial drugs. Exp. Para-
sitol. 96:249–256.
Fidock DA, Roepe PD. 2004. Drug resistance-associated pfCRT muta-
tions confer decreased Plasmodium falciparum digestive vacuolar pH.
Mol. Biochem. Parasitol. 133:99–114.
6. Trager W, Jensen JB. 1976. Human malaria parasites in continuous
culture. Science 193:673–675.
7. Bennett TN, Paguio M, Gligorijevic B, Seudieu C, Kosar AD, Davidson E,
Roepe PD. 2004. Novel, rapid, and inexpensive cell-based quantification of
antimalarial drug efficacy. Antimicrob. Agents Chemother. 48:1807–1810.
8. Paguio MF, Bogle KL, Roepe PD. 2011. Plasmodium falciparum resis-
tance to cytocidal versus cytostatic effects of chloroquine. Mol. Biochem.
Parasitol. 178:1–6.
9. Hawley SR, Bray PG, O’Neil PM, Park BK, Ward SA. 1996. The role of
drug accumulation in 4-aminoquinoline antimalarial potency. Biochem.
Pharmacol. 52:723–733.
2
2
4
4
4
4
5
5
5
5
2
7. Chong CR, Sullivan DJ, Jr. 2003. Inhibition of heme crystal growth by
antimalarials and other compounds: implications for drug discovery.
Biochem. Pharmacol. 66:2201–2212.
2
8. Jackson KE, Klonis N, Ferguson DJ, Adisa A, Dogovski C, Tilley L.
2
004. Food vacuole-associated lipid bodies and heterogeneous lipid envi-
ronments in the malaria parasite, Plasmodium falciparum. Mol. Microbiol.
4:109–122.
0. Krogstad DJ, Schlesinger PH. 1998. A perspective on antimalarial action:
effects of weak bases on Plasmodium falciparum. Biochem. Pharmacol.
5
3
5:793–798.
2
9. Sullivan DJ, Jr, Gluzman IY, Goldberg DE. 1996. Plasmodium hemozoin
formation mediated by histidine-rich proteins. Science 271:219–222.
0. Egan TJ, Chen JY, de Villies KA, Mabotha TE, Naidoo JJ, Ncokazi KK,
Langford McNaughton SJ, Pandiancherii S, Wood BR. 2006. Hemozoin
1. Casabianca LB, An D, Natarajan JK, Alumasa JN, Roepe PD, Wolf C, de
Dios AC. 2008. Quinine and chloroquine differentially perturb heme
monomer-dimer equilibrium. Inorg. Chem. 47:6077–6081.
2. Casabianca LB, Kallgren JB, Natarajan JK, Alumasa JN, Roepe PD,
Wolf C, de Dios AC. 2009. Antimalarial drugs and heme in detergent
micelles: an NMR study. J. Inorg. Biochem. 103:745–748.
3. Cohen SN, Phifer KO, Yielding KL. 1964. Complex formation between
chloroquine and ferrihaemic acid in vitro, and its effect on the antimalarial
action of chloroquine. Nature 202:805–806.
4. Macomber PB, Spintz H. 1967. Morphological effects of chloroquine on
Plasmodium berghei in mice. Nature 214:937–939.
5. Alumasa JN, Gorka AP, Casabianca LB, Comstock E, de Dios AC,
Roepe PD. 2011. The hydroxyl functionality and a rigid proximal N are
required for forming a novel non-covalent quinine-heme complex. J. In-
org. Biochem. 105:467–475.
6. Ursos LM, DuBay KF, Roepe PD. 2001. Antimalarial drugs influence the
pH dependent solubility of heme via apparent nucleation phenomena.
Mol. Biochem. Parasitol. 112:11–17.
7. Roepe PD. 2011. PfCRT-mediated drug transport in malarial parasites.
Biochemistry 50:163–171.
8. Cabrera M, Paguio MF, Xie C, Roepe PD. 2009. Reduced digestive
vacuolar accumulation of chloroquine is not linked to resistance to chlo-
roquine toxicity. Biochemistry 48:11152–11154.
9. Cooper RA, Ferdig MT, Su XZ, Ursos LM, Mu J, Nomura T, Fujioka H,
Fidock DA, Roepe PD, Wellems TE. 2002. Alternative mutations at
position 76 of the vacuolar transmembrane protein PfCRT are associated
with chloroquine resistance and unique stereospecific quinine and quini-
dine responses in Plasmodium falciparum. Mol. Pharmacol. 61:35–42.
0. Cooper RA, Lane KD, Deng B, Mu J, Patel JJ, Wellems TE, Su X, Ferdig
MT. 2007. Mutations in transmembrane domains 1, 4, and 9 of the Plas-
modium falciparum chloroquine resistance transporter alter susceptibility
to chloroquine, quinine, and quinidine. Mol. Microbiol. 63:270–282. (Er-
ratum, Mol. Microbiol. 64:1139–1148.)
3
(beta-hematin) biomineralization occurs by self-assembly near the lipid/
water interface. FEBS Lett. 580:5105–5110.
3
3
3
3
3
3
1. Fitch CD, Cai GZ, Chen YF, Shoemaker JD. 1999. Involvement of lipids
in ferriprotoporphyrin IX polymerization in malaria. Biochim. Biophys.
Acta 1454:31–37.
2. Huy NT, Uyen DT, Sasai M, Trang DT, Shiono T, Harada S, Kamei K.
5
2
006. A simple and rapid colorimetric method to measure hemozoin crys-
tal growth in vitro. Anal. Biochem. 354:305–307.
3. Pandey AV, Joshi SK, Tekwani BL, Chauhan VS. 1999. A colorimetric
assay for heme in biological samples using 96-well plates. Anal. Biochem.
5
2
68:159–161.
4. Pandey AV, Singh N, Tekwani BL, Puri SK, Chauhan VS. 1999. Assay of
beta-hematin formation by malaria parasite. J. Pharm. Biomed. Anal. 20:
5
2
03–207.
5. Trang DT, Huy NT, Uyen DT, Sasai M, Shiono T, Harada S, Kamei K.
006. Inhibition assay of beta-hematin formation initiated by lecithin for
5
2
screening new antimalarial drugs. Anal. Biochem. 349:292–296.
6. Tripathi AK, Gupta A, Garg SK, Tekwani BL. 2001. In vitro beta-
hematin formation assays with plasma of mice infected with Plasmodium
yoelii and other parasite preparations: comparative inhibition with quin-
oline and endoperoxide antimalarials. Life Sci. 69:2725–2733.
7. Tripathi AK, Khan SI, Walker LA, Tekwani BL. 2004. Spectrophoto-
metric determination of de novo hemozoin/beta-hematin formation in an
in vitro assay. Anal. Biochem. 325:85–91.
8. Kaschula CH, Egan TJ, Hunter R, Basilico N, Parapini S, Taramelli D,
Pasini DE, Monti D. 2002. Structure-activity relationships in 4-amino-
quinoline antiplasmodials. The role of the group at the 7-position. J. Med.
Chem. 45:3531–3539.
9. Dinio T, Gorka AP, McGinniss A, Roepe PD, Morgan JB. 2012. Inves-
tigating the activity of quinine analogues versus chloroquine resistant
Plasmodium falciparum. Bioorg. Med. Chem. 20:3292–3297.
0. Gorka AP, Sherlach KS, de Dios AC, Roepe PD. 2013. Relative to
quinine and quinidine, their 9-epimers exhibit decreased cytostatic activ-
5
5
3
3
6
3
61. Ferdig MT, Cooper RA, Mu J, Deng B, Joy DA, Su XZ, Wellems TE.
2004. Dissecting the loci of low-level quinine resistance in malaria para-
sites. Mol. Microbiol. 52:985–997.
4
ity and altered heme binding but similar cytocidal activity versus Plasmo- 62. Sidhu AB, Valderramos SG, Fidock DA. 2005. pfmdr1 mutations con-
dium falciparum. Antimicrob. Agents Chemother. 57:365–374.
tribute to quinine resistance and enhance mefloquine and artemisinin
4
4
1. Ekoue-Kovi K, Yearick K, Iwaniuk DP, Natarajan JK, Alumasa J, de
sensitivity in Plasmodium falciparum. Mol. Microbiol. 57:913–926.
Dios AC, Roepe PD, Wolf C. 2009. Synthesis and antimalarial activity of 63. Sidhu AB, Verdier-Pinard D, Fidock DA. 2002. Chloroquine resistance
new 4-amino-7-chloroquinolyl amides, sulfonamides, ureas and thio-
ureas. Bioorg. Med. Chem. 17:270–283.
2. Iwaniuk DP, Whetmore ED, Rosa N, Ekoue-Kovi K, Alumasa J, de Dios
AC, Roepe PD, Wolf C. 2009. Synthesis and antimalarial activity of new
in Plasmodium falciparum malaria parasites conferred by pfcrt mutations.
Science 298:210–213.
64. Sinai AP, Roepe PD. 2012. Autophagy in apicomplexa: a life sustaining
death mechanism? Trends Parasitol. 28:358–364.
364 aac.asm.org
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