Relative to quinine and quinidine, their 9-epimers exhibit decreased cytostatic activity and altered heme binding but similar cytocidal activity versus Plasmodium falciparum

Article abstract of DOI:10.1128/AAC.01234-12

The 9-epimers of quinine (QN) and quinidine (QD) are known to exhibit poor cytostatic potency against P. falciparum (Karle JM, Karle IL, Gerena L, Milhous WK, Antimicrob. Agents Chemother. 36:1538-1544, 1992). We synthesized 9-epi-QN (eQN) and 9-epi-QD (eQD) via Mitsunobu esterification-saponification and evaluated both cytostatic and cytocidal antimalarial activities. Relative to the cytostatic activity of QN and QD, we observed a large decrease in cytostatic activity (higher 50% inhibitory concentration [IC₅₀s]) against QN-sensitive strain HB3, QN-resistant strain Dd2, and QN-hypersensitive strain K76I, consistent with previous work. However, we observed relatively small changes in cytocidal activity (the 50% lethal dose), similar to observations with chloroquine (CQ) analogues with a wide range of IC₅₀s (see the accompanying paper [A. P. Gorka, J. N. Alumasa, K. S. Sherlach, L. M. Jacobs, K. B. Nickley, J. P. Brower, A. C. de Dios, and P. D. Roepe, Antimicrob. Agents Chemother. 57:356-364, 2013]). Compared to QN and QD, the 9-epimers had significantly reduced hemozoin inhibition efficiency and did not affect pH-dependent aggregation of ferriprotoporphyrin IX (FPIX) heme. Magnetic susceptibility measurements showed that the 9-epimers perturb FPIX monomer-dimer equilibrium in favor of monomer, and UV-visible (VIS) titrations showed that eQN and eQD bind monomer with similar affinity relative to QN and QD. However, unique ring proton shifts in the presence of zinc(II) protoporphyrin IX (ZnPIX) indicate that binding of the 9-epimers to monomeric heme is via a distinct geometry. We isolated eQN- and eQD-FPIX complexes formed under aqueous conditions and analyzed them by mass, fluorescence, and UVVIS spectroscopies. The 9-epimers produced low-fluorescent adducts with a 2:1 stoichiometry (drug to FPIX) which did not survive electrospray ionization, in contrast to QN and QD complexes. The data offer important insight into the relevance of heme interactions as a drug target for cytostatic versus cytocidal dosages of quinoline antimalarial drugs and further elucidate a surprising structural diversity of quinoline antimalarial drug-heme complexes. Copyright

Full text of DOI:10.1128/AAC.01234-12

Relative to Quinine and Quinidine, Their 9-Epimers Exhibit
Decreased Cytostatic Activity and Altered Heme Binding but Similar
Cytocidal Activity versus Plasmodium falciparum
Alexander P. Gorka,a,b,c Katy S. Sherlach,a,b,c Angel C. de Dios,c Paul D. Roepea,b,c
Department of Biochemistry and Molecular & Cellular Biology,a Center for Infectious Disease,b and Department of Chemistry,c Georgetown University, Washington, DC,
USA
The 9-epimers of quinine (QN) and quinidine (QD) are known to exhibit poor cytostatic potency against P. falciparum (Karle
JM, Karle IL, Gerena L, Milhous WK, Antimicrob. Agents Chemother. 36:1538–1544, 1992). We synthesized 9-epi-QN (eQN) and
9-epi-QD (eQD) via Mitsunobu esterification-saponification and evaluated both cytostatic and cytocidal antimalarial activities.
Relative to the cytostatic activity of QN and QD, we observed a large decrease in cytostatic activity (higher 50% inhibitory con-
centration [IC50s]) against QN-sensitive strain HB3, QN-resistant strain Dd2, and QN-hypersensitive strain K76I, consistent
with previous work. However, we observed relatively small changes in cytocidal activity (the 50% lethal dose), similar to observa-
tions with chloroquine (CQ) analogues with a wide range of IC50s (see the accompanying paper [A. P. Gorka, J. N. Alumasa, K. S.
Sherlach, L. M. Jacobs, K. B. Nickley, J. P. Brower, A. C. de Dios, and P. D. Roepe, Antimicrob. Agents Chemother. 57:356–364,
2013]). Compared to QN and QD, the 9-epimers had significantly reduced hemozoin inhibition efficiency and did not affect pH-
dependent aggregation of ferriprotoporphyrin IX (FPIX) heme. Magnetic susceptibility measurements showed that the
9-epimers perturb FPIX monomer-dimer equilibrium in favor of monomer, and UV-visible (VIS) titrations showed that eQN
and eQD bind monomer with similar affinity relative to QN and QD. However, unique ring proton shifts in the presence of
zinc(II) protoporphyrin IX (ZnPIX) indicate that binding of the 9-epimers to monomeric heme is via a distinct geometry. We
isolated eQN- and eQD-FPIX complexes formed under aqueous conditions and analyzed them by mass, fluorescence, and UV-
VIS spectroscopies. The 9-epimers produced low-fluorescent adducts with a 2:1 stoichiometry (drug to FPIX) which did not sur-
vive electrospray ionization, in contrast to QN and QD complexes. The data offer important insight into the relevance of heme
interactions as a drug target for cytostatic versus cytocidal dosages of quinoline antimalarial drugs and further elucidate a sur-
prising structural diversity of quinoline antimalarial drug-heme complexes.
alaria remains a very serious threat to global health, affecting vacuole (DV) of the parasite, by inhibiting the crystallization of
M
hundreds of millions of people and killing approximately 1
toxic ferriprotoporphyrin IX (FPIX) heme to nontoxic hemozoin
via binding to the precrystalline monomeric and/or dimeric form
of heme (6–10). Resistance to QN is multifactorial and in various
studies has been linked to mutations and/or overexpression of
several genes, including those encoding the P. falciparum chloro-
quine resistance transporter (PfCRT) (11, 12), multidrug resis-
tance protein (PfMDR1) (13), and Naϩ/Hϩ exchanger (PfNHE)
(14). One theory for why QN remains active against some CQR
phenotypes is that certain mutant PfCRT isoforms that confer
reduced CQ accumulation via increased PfCRT-mediated trans-
port of CQ do not accommodate increased transport of QN, even
though CQ and QN are structurally similar (15–17). We have
recently discovered that the mechanism of resistance to the cyto-
cidal (cell-killing) activity of CQ is likely distinct from the mech-
anism of resistance to the cytostatic (growth-inhibitory) activity
million annually (1). Five species of Plasmodium cause disease in
humans, with Plasmodium falciparum being the most lethal (2).
Given the growing spread of resistance to current antimalarial
drugs and the lack of an effective vaccine (1–4), development of
novel, cost-effective, and efficacious drugs is of utmost impor-
tance. A vital component to new drug development is elucidating
the molecular mechanism of effective antimalarial drugs as well as
the mechanism(s) of parasite drug resistance. Further improve-
ment to quinoline-based antimalarials has historically focused on
modification of the 4- and 8-aminoquinolines, whereas synthesis
of quinine (QN) derivatives has largely been avoided due, in part,
to toxicity concerns and complexities in chemical synthesis (5).
Yet QN, a quinoline methanol natural product from the bark
of the Cinchona tree (4), has been used as an effective antimalarial
drug for centuries, and QN resistance (QNR) remains relatively
low. Correspondingly, QN is currently a WHO-recommended
therapy for some chloroquine (CQ)-resistant (CQR) and artemis-
inin (ART)-resistant P. falciparum infections. Despite long-term
use and important activity against drug-resistant malaria, the mo-
lecular mechanism of action of QN has not yet been fully eluci-
dated. Understanding similarities versus differences relative to
CQ would assist with the additional development of effective
quinoline antimalarial drugs.
Received 15 June 2012 Returned for modification 27 September 2012
Accepted 26 October 2012
Published ahead of print 31 October 2012
Address correspondence to Paul D. Roepe, roepep@georgetown.edu.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AAC.01234-12.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.01234-12
Similar to CQ and other quinoline-based antimalarial drugs,
QN is believed to inhibit heme detoxification within the digestive
January 2013 Volume 57 Number 1
Antimicrobial Agents and Chemotherapy p. 365–374
aac.asm.org 365

Gorka et al.
of CQ (16, 18, 19; see also the accompanying paper [20]). Other formed in aqueous solution and determined their stoichiometry
than one recent report (19), there has been no quantification of and fluorescence properties. In sum, the data elucidate key differ-
the 50% inhibitory concentration (IC50) (cytostatic) versus the ences in heme interactions for the isomeric pairs, suggest a novel
50% lethal dose (LD50) (cytocidal) for QN and its stereoisomers; model for eQN and eQD complexation with heme, and shed light
therefore, it is currently not known how these parameters differ on the relevance of heme interactions for the cytostatic versus
for various strains and isolates of P. falciparum.
cytocidal activities of the Cinchona alkaloids.
The behavior of QN stereoisomers is particularly interesting
and is in theory a quite useful tool for elucidating QN pharmacol-
ogy. Karle and coworkers reported that while QN (8S,9R) and its
MATERIALS AND METHODS
Materials and chemicals. Routine chemicals, media, and solvents were
8,9-diastereomer quinidine (QD) (8R,9S) potently inhibit the reagent grade or better, purchased from Sigma-Aldrich (St. Louis, MO) or
Fisher Scientific (Newark, DE), and used without further purification,
unless otherwise noted. Sterile polystyrene 96-well tissue culture plates
and other laboratory plastics were purchased from Fisher Scientific (New-
ark, DE). 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). D2O, dimethyl sulfoxide d6 (DMSO-d6), and CDCl3 were pur-
chased from Cambridge Isotope Laboratories Inc. (Andover, MA). Zin-
c(II) protoporphyrin IX (ZnPIX) was purchased from Frontier Scientific
(Logan, UT). SYBR green I nucleic acid dye was purchased from Invitro-
growth of malarial parasites, 9-epi-QN (eQN) (8S,9S) and
9-epi-QD (eQD) (8R,9R) do not, showing strongly elevated IC50s
against CQ-sensitive (CQS) strain D6 and CQR strain W2 (21).
Curiously, W2 was found to be approximately 3-fold more sensi-
tive to eQN and eQD than D6. Albeit under nonphysiological
conditions, Egan et al. demonstrated that the 9-epimers inhibit
hemozoin formation less well in vitro than QN and QD (9). These
findings, along with structural analysis of the compounds, led
Karle et al. to hypothesize that the altered orientation of intermo-
lecular hydrogen bonds with cellular receptor sites for the threo gen (Eugene, OR).
Antimalarial drugs QN monohydrochloride dihydrate, QD hydro-
alkaloids eQN and eQD relative to that of their erythro counter-
parts, QN and QD (21), reduces Cinchona alkaloid potency. Re-
cent work (6, 8, 10, 11, 13–17, 22) has suggested that the cellular
receptor sites alluded to by Karle et al. may be one or more forms
of free FPIX heme within the DV. Warhurst and coworkers inves-
tigated the physicochemical properties of the erythro versus threo
isomers and calculated that the latter have an elevated aliphatic N
pKa (9.5 versus 8.6 for the erythro isomer) and a reduced log D at
pH 7.4, predicting decreased membrane transfer (22). Thus, in-
chloride monohydrate, CQ diphosphate, and amodiaquine (AQ) dihy-
drochloride dihydrate were purchased from Sigma-Aldrich (St. Louis,
MO). eQN and eQD were synthesized as described below. Synthesis of
cinchonine analog 2 (CN-2) CN-4, QD analog 2 (QD-2), QN analog 8
(QN-8), and QN-12 was reported previously (23). The structures of all
drugs used in this study are shown in Fig. 1.
General methods. Flash chromatography was performed on Kieselgel
60 (particle size, 0.032 to 0.063 mm). Thin-layer chromatography (TLC)
analyses were performed on Selecto Scientific flexible TLC plates (silica gel
trinsic differences in the antiplasmodial activities of the isomer 60, F 254, 200 ␮m). NMR spectra were obtained on a 400-MHz (1H) and
100-MHz (13C) Varian Fourier transform (FT)-NMR spectrometer
pairs may result from (i) a different chemical interaction(s) with
(Santa Clara, CA) using CDCl3 as the solvent, unless otherwise indicated,
heme, (ii) a decreased ability of eQN and eQD to partition from
and using tetramethylsilane (TMS) as the external standard. Mass spec-
aqueous to lipid phase (i.e., to cross membranes) (22), or (iii)
troscopic (MS) measurements were performed in methanol (MeOH) or
perhaps altered binding to other (nonheme) cellular receptors. In
acetonitrile (ACN) on a Varian 500 mass spectrometer equipped with an
parasites that are resistant to quinoline drugs, mutations in DV
electrospray ionization (ESI) source. The electrospray needle was oper-
membrane transporters with stereospecific drug interactions (13,
15–17) might be predicted to further affect 9-epimer activity for
these QNR or CQR strains.
ated at 5 kV using N2 as both the nebulizing gas (35 lb/in2) and the drying
gas (350°C, 10 lb/in2). Sample was delivered using a syringe pump em-
ploying a gas-tight 1-ml syringe at a constant flow rate of 200 ␮l/min.
Optical rotation (␣) was measured at 589 nm (sodium D line) in ethanol
To further distinguish between these possibilities, we hypoth-
esized, based on a recent model for QN-heme adducts (6), that it (EtOH; 1-mg/ml solution) on a Rudolph Instruments DigiPol-DP781-
TDV polarimeter (Denville, NJ) at room temperature using a 1-dm cell.
Melting point analysis was performed on a Barnstead electrothermal
manual MelTemp apparatus.
Synthesis of eQN and eQD. (S)-(6-Methoxyquinolin-4-yl) [(2S,4S,8R)-
8-vinylquinuclidin-2-yl]methanol (eQN) and (R)-(6-methoxyquinolin-4-
yl) [(2R,4S,8R)-8-vinylquinuclidin-2-yl]methanol (eQD) were prepared by
one-pot Mitsunobu esterification-saponification (24). A stirred solution of
QN or QD (500 mg, 1.5 mmol, 1.0 equivalent), triphenylphosphine (Ph3P;
525 mg, 2.0 mmol, 1.3 equivalents), and p-nitrobenzoic acid (PNBA; 284 mg,
1.7 mmol, 1.1 equivalents) in anhydrous tetrahydrofuran (THF; 15 ml) was
is possible that the lack of hemozoin inhibition by eQN and eQD
is due to altered monomeric heme binding arising from the
change in configuration at the 9-position carbon. Consistent with
solution and solid-state nuclear magnetic resonance (NMR) data,
Alumasa and coworkers recently suggested that QN forms a highly
fluorescent 1:1 complex with FPIX through a dative FeOO inter-
action which is stabilized by an intramolecular five-membered
ring formed via hydrogen bonding between the hydroxyl proton
and quinuclidine nitrogen (6). Alumasa et al. also showed that, in
solution, QN and QD bind to monomeric FPIX with high affinity cooled to 0°C in an ice water bath, and diisopropyl azodicarboxylate (DIAD,
0.33 ml, 1.7 mmol, 1.1 equivalents) was added dropwise. The resulting mix-
ture was stirred at 0°C for 20 min, allowed to warm gradually to room tem-
perature, and stirred for an additional 3 h. After cooling to 0°C, lithium hy-
droxide (LiOH; 1 M, 10 ml) and MeOH (2 ml) were added, the solution was
gradually warmed to room temperature again, and the reaction mixture was
stirred for 12 h more. Organic solvents were removed in vacuo, and the resi-
due was quenched with water (10 ml) and extracted with dichloromethane
(CH2Cl2; 50 ml). The organic phase was separated, washed with saturated
brine, and dried over anhydrous sodium sulfate, and the solvent was removed
in vacuo to afford the crude product as a yellow oil. This was further purified
and perturb the FPIX monomer-dimer equilibrium in favor of
monomer (6).
We performed similar in vivo and in vitro experiments with the
isomeric pairs to further elucidate Cinchona alkaloid antiplasmo-
dial activity and resistance. We also measured the cytostatic (IC50)
and cytocidal (LD50) activities for QN-susceptible (QNS) versus
QNR strains, hemozoin inhibitory activities, heme binding affin-
ity, magnetic moment for drug-heme solutions, drug effects on
pH-dependent heme aggregation, and heme ring current effects
on bound drug. We isolated eQN- and eQD-heme complexes by flash chromatography over silica using an initial CHCl3-diethyl ether (3:1)
366 aac.asm.org
Antimicrobial Agents and Chemotherapy

9-Epimers of QN and QD
FIG 1 Structures of drugs used in this study. Positions 8 and 9 on the QN pharmacophore are highlighted. EtO indicates that an ethyl ester resides at that carbon
(ϪOC2H5).
mobile phase followed by CHCl3-MeOH-triethylamine (40:1:4) for product 20 ␮g/liter gentamicin with regular medium changes every 48 h. Antiplas-
elution. Thepureproductwasobtainedasanoff-whitesolidin28%(eQN)or modial cytocidal and cytostatic activity was assessed against the CQS HB3
31% (eQD) yield.
strain, CQR Dd2 strain, and CQR K76I line (11, 12), as previously de-
eQN. 1H NMR (CDCl3; 400 MHz; m, multiplet; s, singlet; d, doublet; scribed (19, 26), with minor modifications. The cytocidal assay utilizes a
dd, doublet of a doublet) ␦ 1.00 (m, 1H), 1.26 (m, 1H), 1.51 (m, 1H), 1.70 6-h bolus dose with high concentrations of drug, followed by washing
(m, 3H), 2.40 (s, 1H), 2.89 (m, 2H), 3.38 (m, 3H), 3.95 (s, 3H), 5.00 (m, drug away and growth in the absence of drug for 48 h, while the cytostatic
2H), 5.16 (d, J ϭ 10 Hz, 1H), 5.72 (m, 1H), 7.37 (dd, J ϭ 2.8 Hz, J ϭ 9.2 assay utilizes continuous growth for 48 h in the constant presence of low
Hz, 1H), 7.40 (d, J ϭ 4.4 Hz, 1H), 7.68 (d, J ϭ 2.8 Hz, 1H), 8.02 (d, J ϭ 9.2 concentrations of drug. Test compounds were dissolved in deionized wa-
Hz, 1H), 8.72 (d, J ϭ 4.4 Hz, 1H); 13C NMR (CDCl3, 100 MHz) ␦ 24.7, ter, 50% EtOH, or DMSO. Serial drug dilutions were made using com-
27.0, 27.1, 29.6, 39.1, 40.9, 55.3, 55.7, 61.6, 70.7, 102.5, 115.3, 120.1, 121.5, plete medium, and 100-␮l aliquots were transferred to 96-well clear-bot-
128.0, 131.6, 140.3, 143.9, 144.8, 147.5, 157.6; melting point, 183 to 185°C; tom black plates. Following addition of 100 ␮l of asynchronous or
[␣]22 (EtOH) ϩ23 (c 1.0); MS (ESI) m/z calculated for C20H24N2O2: sorbitol-synchronized culture (1% final parasitemia, 2% final hemato-
D
324.18. Found (M ϩ H)ϩ: 325.1.
crit), plates were transferred to an airtight chamber gassed with 5%
eQD. 1H NMR (CDCl3, 400 MHz) ␦ 1.04 (m, 1H), 1.32 (m, 1H), 1.60 CO2–5% O2–90% N2 and incubated at 37°C.
(m, 2H), 1.72 (s, 1H), 2.35 (m, 1H), 3.02 (m, 6H), 3.93 (s, 3H), 5.13 (m,
For the cytocidal assay, plates were incubated for 6 h, followed by
3H), 5.89 (m, 1H), 7.36 (dd, J ϭ 2.8 Hz, J ϭ 9.2 Hz, 1H), 7.48 (d, J ϭ 4.4 centrifugation with an Eppendorf 5804 centrifuge fitted with an A-2-
Hz, 1H), 7.58 (d, J ϭ 2.4 Hz, 1H), 8.02 (d, J ϭ 9.2 Hz, 1H), 8.74 (d, J ϭ 4.4 DPW rotor (Hauppauge, NY) at 700 ϫ g for 3 min. Drug-containing
Hz, 1H); 13C NMR (CDCl3, 100 MHz) ␦ 23.9, 26.4, 27.3, 38.7, 46.8, 49.1, medium was removed, and cell pellets were washed three times with 200
55.4, 62.4, 69.8, 101.9, 115.0, 120.0, 121.7, 128.0, 131.6, 139.8, 144.7, ␮l of complete medium per wash, using the same centrifuge settings, and
144.8, 147.5, 157.5; melting point, 185 to 187°C; [␣]22 (EtOH) ϩ75 (c resuspended in the same volume of medium. Washed plates along with
D
1.0); MS ESI m/z calculated for C20H24N2O2: 324.18. Found (M ϩ H)ϩ: the cytostatic assay plates were incubated at 37°C for 48 h. After 48 h, 50 ␮l
325.0.
of 10ϫ SYBR green I dye (diluted using complete medium from a
Antiplasmodial activity measurements. P. falciparum strains HB3 10,000ϫ DMSO stock) was added and plates were incubated for an addi-
and Dd2 were obtained from the Malaria Research and Reference Reagent tional 1 h at 37°C to allow DNA intercalation. Fluorescence was measured
Resource Center (Manassas, VA). P. falciparum strain K76I was a gener- at 538 nm (485-nm excitation) using a Spectra GeminiEM plate reader
ous gift of Roland Cooper (Dominican University of California, San Ra- (Molecular Devices, Sunnyvale, CA) fitted with a 530-nm long-pass filter.
fael, CA). Off-the-clot, heat-inactivated pooled type O-positive human Linear standard curves of measured fluorescence versus known para-
serum and type O-positive human whole blood were purchased from sitemia were prepared immediately prior to plate analysis. Background
Biochemed Services (Winchester, VA). Custom 5% O2–5% CO2–90% controls included fluorescence from uninfected red blood cells. Data were
N2 culturing gas blend was purchased from Robert’s Oxygen (Rock- analyzed using the Microsoft Excel 2007 program, and IC50 and LD50
ville, MD).
values were obtained from sigmoidal curve fits to percent growth/surviv-
All P. falciparum strains were maintained using the method of Trager al-versus-drug concentration data using SigmaPlot (version 11.0) soft-
and Jensen (25) with minor modifications. Briefly, cultures were main- ware. Reported values are the averages of three independent assays, with
tained under an atmosphere containing 5% CO2, 5% O2, and 90% N2 each assay conducted in triplicate (nine determinations total) and re-
gaseous mix at 2% hematocrit and 1 to 2% parasitemia in RPMI 1640 ported Ϯ the standard error of the mean (SEM).
supplemented with 10% type O-positive human serum, 25 mM HEPES
␤-Hematin crystallization inhibition. ␤-Hematin crystal growth in-
(pH 7.4), 23 mM NaHCO3, 11 mM glucose, 0.75 mM hypoxanthine, and hibition under physiological conditions in the presence of lipid catalyst
January 2013 Volume 57 Number 1
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Gorka et al.
was assessed using a modified 96-well-plate high-throughput assay (see 3 min using an Eppendorf 5415 D microcentrifuge (Hauppauge, NY). The
the accompanying paper [20]). Briefly, the assay uses physiological tem- supernatant was removed and added to a 96-well plate containing 24 mM
perature and lipid catalyst and relies on the differential solubility proper-
ties of crystalline and noncrystalline forms of FPIX in 2.5% SDS (86.7
mM) and alkaline bicarbonate buffer (0.1 M, pH 9.1) to quantify crystal-
lized heme. Hemin was dissolved in 0.1 M NaOH to 2 mM, 10 ␮l was
transferred to each well of a 96-well plate, and propionate buffer (180 ␮l at
the desired pH) plus 10 ␮l of sonicated phosphatidylcholine suspension
(10 mg/ml) were then added (final FPIX heme concentration, 100 ␮M).
Drugs at different concentrations were then added, the contents of the
plates were mixed, and the plates were wrapped in plastic wrap and incu-
bated at 37°C for 16 h. The assay was terminated by the addition of 100 ␮l
of a solution of SDS dissolved in 0.1 M bicarbonate buffer (pH 9.1; final
SDS concentration per well, 86.7 mM). The well contents were gently
mixed, and the plate was incubated at room temperature for 10 min to
allow undissolved hemozoin crystals to settle. A 50-␮l aliquot from each
well was then transferred to a second plate preloaded with 200 ␮l of SDS
solution (86.7 mM) in 0.1 M bicarbonate buffer (pH 9.1). The absorbance
of noncrystallized heme was recorded at 405 nm using a 96-well-plate-
adapted ELx800 BioTek absorbance microplate reader (Winooski, VT).
Standard curves of known heme concentration in the same solvent were
generated for each assay and fit with a linear regression. Free heme re-
maining in solution (the inverse of the amount of hemozoin produced)
was quantified using equation 1:
MES-Tris buffer–0.1 M NaCl (pH 6.6), resulting in a 1:4 (vol/vol) dilu-
tion. A standard curve of known heme concentration was generated for
each assay. The absorbance at 405 nm was measured using a 96-well-plate-
adapted ELx800 BioTek absorbance microplate reader (Winooski, VT).
Data were analyzed using the Microsoft Excel 2007 program, and the pH
at half FPIX solubility (pH1/2) was extracted from sigmoidal curve fits to
FPIX concentration-versus-pH data using SigmaPlot (version 11.0) soft-
ware. Values are averages of three replicates, each performed in triplicate
(nine determinations total), and are reported Ϯ SEM.
Formation and analyses of aqueous drug-heme complexes. Drug-
heme complexes formed under aqueous conditions were prepared as pre-
viously described for similar experiments (6). Hemin was dissolved in 0.1
M NaOH to give a 2 mM stock, and drug (100 mM in deionized water) was
titrated into this solution while monitoring the pH. At a pH of ϳ8.5,
copious heme was observed to precipitate, leaving a colorless solution of
residual QN. The precipitate was isolated via vacuum filtration, washed
with 0.1 M phosphate buffer (pH 7.0) to remove uncomplexed drug and
heme, and dried under high vacuum to afford the complex as a dull green
or dull gray powder.
Mass spectrometry analyses using a Varian 500 MS were performed by
dissolving the precipitate in ACN. Fluorescence spectra were recorded
from 5 ␮M samples dissolved in 1:1 MeOH–0.2 M HEPES (pH 7.2) using
a Photon Technology International QuantaMaster 40 fluorometer (Bir-
mingham, NJ) at 371 nm (334 nm excitation) using 1- and 2-nm slit
widths. Spectra were processed using the Felix GX (version 4.0.3) and the
Microsoft Excel 2007 programs.
Adduct stoichiometry was measured by dissolving each complex in
100% MeOH (which completely dissociates the complex), 40% (vol/vol)
DMSO–0.2 M HEPES, pH 7.2 (which completely dissociates the com-
plex), or 100% ACN (in which the complex remains intact) and quanti-
fying the concentration of drug and FPIX via UV-VIS at 325 and 402 nm,
respectively. Calibration curves of drug and monomeric FPIX were pre-
pared by serial dilution of a 10 mM stock in DMSO in 40% (vol/vol)
DMSO–0.2 M HEPES (pH 7.2).
Drug self-association studies. The propensity for eQN and eQD to
self-associate relative to QN and QD was measured in order to further
probe heme interactions (30). Drug stocks were prepared in CD3OD to a
final concentration of 1 or 50 mM. A 1-ml aliquot of each stock was
transferred to a 5-mm (outer diameter) NMR tube, and NMR measure-
ments were made at 298 K on a Varian FT-NMR spectrometer operating
at a proton frequency of 400 MHz. Chemical shifts were referenced to the
residual methyl signal of CH3OH at 3.29 ppm. Spectra were processed
using MestReC (version 4.8.1.1) software (Santiago de Compostela,
Spain), and chemical shift differences were calculated by subtracting the
value for each resonance measured at 50 mM from the corresponding
value measured at 1 mM.
Magnetic susceptibility measurements. Magnetic susceptibility mea-
surements were performed as previously described (27) in 40% DMSO–
0.1 M phosphate buffer (pH 7.0). Both hemin and drug stock solutions
(20 mM) were prepared in 100% DMSO-d6. Test samples (40% [vol/vol]
DMSO–0.1 M phosphate buffer at pH 7.0) were prepared by adding 200
␮l of hemin and the corresponding test compound into a 1.5-ml micro-
centrifuge tube, followed by addition of 600 ␮l of 0.1 M phosphate buffer
to give a 1:1 solution of both components at 4 mM. The pH of the resulting
samples was taken to be the pH of the buffered aqueous medium mea-
sured at 25°C using an Accumet Basic AB15 pH meter. The samples were
transferred into 5-mm NMR tubes fitted with coaxial inserts containing
the test compound in a similar solvent system. Measurements were made
with a Varian FT-NMR spectrometer with a proton frequency of 400
MHz, and data were analyzed using MestReC (version 4.8.6) software.
Magnetic susceptibility was determined using the Evans method (31) em-
As405 Ϫ A0405 Ϫ C
͑
͒
␹
H
ϭ
ϫ D
(1)
͓ ͔
ͭ
ͮ
␧
405
where [HX] is the concentration of heme (␮M) remaining, A4s 05 and A0405
are the absorbances at 405 nm (average from triplicate wells) of the sample
and blank, respectively, ε405 is the extinction coefficient for heme, C is a
constant obtained from the fit to the calibration curve, and D is the dilu-
tion factor. Data were analyzed using the Microsoft Excel 2007 program,
and ␤-hematin IC50s were obtained from sigmoidal curve fits to percent
crystal growth inhibition-versus-drug concentration data using the Sig-
maPlot (version 11.0) software. Reported values are the averages of three
independent assays, with each assay conducted in triplicate (nine deter-
minations total) and reported Ϯ SEM.
Heme affinity measurements. Drug-heme affinity measurements
were performed by monitoring the changes in the absorbance of the Soret
band of heme in the presence of increasing concentrations of drug. Hemin
was dissolved in DMSO or 0.1 M NaOH to 5 mM, followed by serial
dilution to 5 ␮M in 40% DMSO–0.2 M HEPES (pH 7.4) for measuring
binding to monomer (27). Drug solutions were prepared by dissolving the
compound in DMSO and diluting to 3 mM in 40% DMSO–0.02 M
HEPES (pH 7.4). A cuvette containing 3 ml of freshly prepared heme (5
␮M) was titrated with increasing concentrations of drug (final concentra-
tion, 0 to 210 ␮M), the sample was mixed following each addition, and the
absorbance of heme at 402 nm was recorded using an Agilent 8453 UV-
visible (VIS) spectrophotometer (Santa Clara, CA). Solvent dilution con-
trols were similarly performed (final volume dilution, 6.54%) using the
relevant drug-free medium. Spectral and data analyses were performed
using Kaleida Graph and SigmaPlot (version 11.0) software. Nonlinear
least-squares curve fitting of the raw data was done using the Levenberg-
Marquardt algorithm (7, 28) (initial affinity coefficient [Ka] input, 0.01
␮MϪ1) and Kas were computed.
Heme aggregation studies. The effect of various drugs on the pH-
dependent solubility of FPIX was measured as previously performed (29),
with minor modification. Briefly, drug stocks were prepared in 24 mM
morpholineethanesulfonic acid (MES)-Tris buffer–0.1 M NaCl (pH 6.6)
to a concentration of 1 mM. Hemin was dissolved in 0.1 M NaOH to 18
mM. Test solutions were prepared by combining 20 ␮l of drug, 10 ␮l of
FPIX, and 980 ␮l of 24 mM MES-Tris buffer–0.1 M NaCl at the desired
pH, for final drug and FPIX concentrations of 20 ␮M and 178 ␮M, re-
spectively. Samples were mixed and incubated at room temperature for 30 ploying equation 2, which is appropriate for a superconducting magnet at
min. Aggregated FPIX was pelleted by centrifugation at 1.61 ϫ 104 ϫ g for 298 K:
368 aac.asm.org
Antimicrobial Agents and Chemotherapy

9-Epimers of QN and QD
to afford eQN and eQD, respectively (Fig. 2), and purified (see
Material and Methods).
Antiplasmodial activity. Cytostatic and cytocidal antiplasmo-
dial activities against HB3 (QNS), Dd2 (QNR), and K76I (QN-
hypersensitive) strains of P. falciparum were measured using the
SYBR green I assay (26) (Table 1). Previously, strain K76I was
found to be similarly CQR and QD resistant (QDR) relative to
Dd2 (as defined by IC50 data) yet hypersensitive to QN, owing to a
PfCRT mutation at position 76 (Dd2 harbors a K76T mutation)
(11, 12). Similar to previous work (21), we found that eQN has an
IC50 ϳ60-fold higher than that of QN against HB3 and ϳ10-fold
higher than that of QN against Dd2. The IC50 of eQN for K76I was
ϳ70-fold higher than that of QN, behavior that is more similar to
that against HB3 than to that against Dd2. For eQD, the effects
were more pronounced, with IC50 activities ϳ190-fold and ϳ25-
fold higher than those of QD against HB3 and Dd2, respectively.
For K76I, eQD had an IC50 ϳ10-fold higher than that of QD,
behavior that is now more similar to that against Dd2 than to that
against HB3 (e.g., the converse of the trend for eQN versus QN).
Curiously, and as also found previously (21), Dd2 showed mild
hypersensitivity to both eQN and eQD, and we found that relative
to the sensitivity of HB3, strain K76I was even more hypersensitive
FIG 2 Synthesis of eQN and eQD via a one-pot Mitsunobu esterification-
saponification of QN and QD (24).
Ϫ3⌬v
␹m ϭ
ϩ ␹
(2)
D
4␲c
where ␹ is the molar susceptibility of the paramagnetic substance (in
m
cm3/mol), ⌬␯ is the chemical shift difference (in ppm) between a refer-
ence proton in the sample and that in a solution lacking the paramagnetic to both 9-epimers than Dd2.
compound, c is the concentration of FPIX (in mol/ml), and ␹ is the
Importantly, however, these trends did not hold when potency
was tabulated via cytocidal activity (LD50; cf. Table 1). eQN
showed LD50s ϳ1.8-fold and ϳ1.2-fold higher than those of QN
against HB3 and Dd2, respectively. eQD LD50s were ϳ40-fold and
ϳ1.2-fold higher than those of QD against HB3 and Dd2, respec-
tively. Put another way, for any individual strain, the altered ste-
reochemistry of the 9-epimers vastly decreased the cytostatic po-
tency (changes IC50) but (with one important exception for the
IC50 of eQD versus that of QD against HB3) only mildly altered
the cytocidal potency (LD50). One interpretation is that the mo-
lecular targets for Cinchona alkaloid cytostatic and cytocidal ac-
tivities must differ. Also, on the basis of the patterns for the QNR
strains versus those for the hypersensitive strains, it also seems
likely that mechanisms of cytostatic versus cytocidal resistance
must differ.
D
diamagnetic susceptibility of heme (6.9 ϫ 10Ϫ4 cgs units). Solvent sus-
ceptibility correction and the solution-solvent density differences are ig-
nored. The molar susceptibility was converted to magnetic moment (␮)
using equation 3, where T is the temperature (K):
␮ ϭ 2.8
␹m T
(3)
͙
Solution NMR experiments with ZnPIX. Solution NMR experiments
using ZnPIX were conducted as previously described (6), with minor
modification. ZnPIX and drug stock solutions were prepared at 10 mM in
DMSO-d6. Drug-heme solutions (2:1; 3.334 and 1.667 mM, respectively)
were then prepared and titrated to pH 7.0. Four hundred microliters of
this solution was transferred to a microcentrifuge tube, followed by addi-
tion of 600 ␮l of D2O. Thus, test samples contained a 2:1 drug-to-ZnPIX
ratio in 40% DMSO-d6–D2O. These solutions were transferred to 5-mm
(outer diameter) NMR tubes for analysis, one-dimensional proton NMR
spectra were recorded and analyzed as stated above, and chemical shifts
were referenced to the residual DMSO peak.
Consistent with these conclusions, when defined by IC50, strain
K76I exhibited rather significant (Ն8-fold) hypersensitivity to
eQN and QN relative to that of HB3 but similar (Ͻ2-fold) differ-
RESULTS
Synthesis of eQN and eQD. Copious amounts of eQN and eQD ences in LD50s for the same drugs relative to those for HB3 (Table
were prepared via a one-pot Mitsunobu esterification-saponifica- 1). The QD-eQD pair displays a different trend. Relative to HB3,
tion reaction, as described previously (24). QN and QD were con- strain K76I showed mild (ϳ3.5-fold) eQD hypersensitivity, as de-
verted to the p-nitrobenzoate ester using p-nitrobenzoic acid and fined by IC50, but 3-fold greater resistance, as defined by LD50.
triphenylphosphine (oxidized to triphenylphosphine oxide via Strain K76I was 5-fold more resistant to QD than HB3 via IC50 but
DIAD), followed by in situ saponification with lithium hydroxide, an incredible 200-fold more resistant than HB3 via LD50. See also
TABLE 1 Antiplasmodial IC50s and LD50s for QNS strain HB3, QNR strain Dd2, and the K76I straind
Experimental IC50 (nM)a
Experimental LD50 (␮M)a
Alkaloid
HB3
Dd2
Rfb
K76I
Rfc
HB3
Dd2
Rfb
K76I
Rfc
QN
eQN
QD
107. 2 (12.4)
6,588.5 (358.2)
41.9 (6.0)
265.6 (14.6)
2,237.6 (76.4)
147.2 (3.1)
2.5
0.3
3.5
0.5
12.1 (0.9)
0.1
0.1
5.0
0.3
9.11 (0.4)
16.1 (1.6)
0.4 (0.1)
36.3 (0.9)
42.4 (0.5)
33.6 (0.1)
39.8 (0.9)
4.0
2.6
77.7
2.3
17.3 (0.9)
15.0 (1.0)
105.6 (4.4)
49.6 (3.3)
2.0
1.0
244.3
3.0
838.9 (64.5)
209.5 (2.3)
2,175.6 (125.2)
eQD
7,893.6 (46.8)
3,588.3 (194.1)
17.6 (1.4)
a Experimental IC50 and LD50 values are averages of three independent measurements (9 replicates total), with SEMs reported in parentheses.
b Rf ϭ Dd2 value/HB3 value.
c Rf ϭ K76I value/HB3 value.
d The K76I strain is hypersensitive to QN but resistant to QD (11, 12, 15).
January 2013 Volume 57 Number 1
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Gorka et al.
TABLE 2 ␤-Hematin inhibitory IC50 data at pH 5.2 and 5.6a
␤-Hematin IC50 (␮M)
Alkaloid
pH 5.2
pH 5.6
QN
eQN
QD
255.4 (22.7)
Ͼ1,000
176.2 (4.9)
Ͼ1,000
42.5 (8.3)
Ͼ3,000
27.2 (2.7)
Ͼ3,000
eQD
a Less than 10% inhibition is seen for the 9-epimers at 1 mM, leading to an estimated
IC50 above several mM. Experimental IC50 and values are averages of three independent
measurements (9 replicates total), with SEMs reported in parentheses.
FIG 4 Drug-heme binding curves. Affinity was measured by titrating increas-
ing concentrations of QN (ꢀ), eQN (Œ), QD (ꢀ), and eQD (⌬) into a solution
of monomeric FPIX, followed by nonlinear least-squares analysis (Levenberg-
Marquardt algorithm [7, 28]) to determine Ka (Table 3; see Materials and
Methods). a.u., absorbance units.
references 11, 12, 15, 17, and 19 for selected quinoline activity data
versus these strains.
Hemozoin inhibition. As mentioned above, inhibition of par-
asite hemozoin formation is believed to be the principal basis of
antiplasmodial activity for quinoline drugs like the Cinchona al-
kaloids. However, this theory is based entirely on cytostatic activ-
ity (IC50) data, since, with one exception (19), in vitro antiplasmo-
dial activity is always routinely quantified via IC50. While we
indeed found that QN and QD actively inhibit the formation of
hemozoin under physiological conditions in the low- to mid-␮M
range at both pH 5.2 and 5.6, eQN and eQD do not, with inhibi-
tory IC50s being above several mM (Table 2). We use measure-
ment at both pH 5.2 and 5.6 to mimic variable physiological con-
ditions, since different DV pHs for sensitive (HB3) versus resistant
(Dd2) strains have been measured in some studies (32). These
data suggest that the loss of cytostatic activity for the 9-epimers is
due to an inability to prevent hemozoin formation, as also sug-
gested by Egan et al. (9).
We next plotted the hemozoin (␤-hematin) inhibitory IC50
(␤-hematin inhibitory activity [BHIA]) against a series of QN
analogues with variable cytostatic potency (23) to test for possible
correlations (Fig. 3). The antiplasmodial IC50 was indeed mildly
correlated with the BHIA IC50 across the series (R2 Ͼ 0.54). How-
ever, interestingly, the correlation vanished when LD50 data were
plotted (R2 Ͻ 0.01; Fig. 3, caption). Consistent with the data in
Table 1, this further suggests that Cinchona alkaloid targets for
cytostatic versus cytocidal activities differ.
formed under physiologically relevant aqueous conditions. How-
ever, drug-heme titrations are performed in 40% aqueous DMSO
due to the decreased intensity of the Soret band, less-than-strict
adherence to Beer’s law, insolubility, and potential for heme
dimerization and aggregation outside these conditions (7, 27, 33).
We found that eQN binds monomer with a similar affinity as QN;
however, eQD binds with a ϳ5-fold lower Ka than QD (Fig. 4;
Table 3).
Heme aggregation effects. The effect of various drugs on the
pH-dependent solubility of monomeric FPIX was measured by
varying the pH versus a constant FPIX concentration (178 ␮M)
and drug concentration (20 ␮M) (see reference 29 for a discussion
of these aggregation phenomena and their presumed role in he-
mozoin formation). We found that eQN and eQD did not affect
the pH-dependent solubility of FPIX, whereas QN, QD, CQ, and
AQ produced a marked effect over a relatively narrow pH range
(Fig. 5A; see also reference29). This effect is immediately apparent
when the midpoints of each solubility curve are quantified
(Fig. 5B).
Drug-heme adducts. We next isolated the eQN-, eQD-, QN-,
and QD-monomeric FPIX complexes formed under aqueous con-
ditions using methods published previously (6) and characterized
their properties. Qualitatively, we found that the 9-epimer ad-
ducts are weaker than those formed with QN and QD, as evi-
denced by the ease of resolubilization in neutral buffer following
precipitation and the fact that yields were significantly lower than
those for QN and QD under the same conditions. The eQN- and
eQD-FPIX adducts were also ϳ20- and 10-fold less fluorescent,
respectively, than the QN- and QD-FPIX adducts (Fig. 6; Table 4).
Heme binding affinity. As described below, the 9-epimers
promote stabilization of monomeric heme (see “FPIX magnetic
moment” below). Ideally, experiments to determine the affinity of
the isomeric pairs for binding monomeric FPIX would be per-
TABLE 3 Drug-heme affinity coefficients for binding monomeric FPIXb
Experimental Ka for monomer
Alkaloid
(MϪ1 [104])a
R2
QN
eQN
QD
0.75 (0.07)
0.61 (0.11)
4.40 (0.04)
0.94 (0.10)
1.00
1.00
0.99
1.00
eQD
FIG 3 Antiplasmodial activity of a series of QN analogues (23). (A) BHIA IC50
versus antiplasmodial IC50; (B) BHIA IC50 versus antiplasmodial LD50. The
IC50s shown are averages of three independent measurements, each done in
triplicate (9 replicates total) as reported previously (23). LD50 values are aver-
ages Ϯ SEMs of three replicates and are as follows: CN-2, 2,337.9 Ϯ 4.3 nM;
CN-4, 2,360.6 Ϯ 13.4 nM; QD-2, 2,234.1 Ϯ 7.4 nM; QN-8, 2,251.4 Ϯ 28.4 nM;
QN-12, 2,346.5 Ϯ 13.2 nM.
a Experimental Ka values are averages of three independent measurements (9 replicates
total), with SEMs reported in parentheses.
b Drug-heme affinity coefficients for binding monomeric FPIX were obtained via
nonlinear least-squares analysis of binding data using the Levenberg-Marquardt
algorithm, which is described elsewhere (7, 28). See the caption to Fig. 4 for binding
data.
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9-Epimers of QN and QD
TABLE 4 Drug-monomeric heme adduct properties
Fluorescence
maximum (103)a
Adduct
␭
␭
stoichiometryb ESI molecular ion(s)
ex
em
Species
FPIX
QN-FPIX 360
eQN-FPIX 17.6
QD-FPIX 255
eQD-FPIX 21.7
(334 nm) (371 nm) for drug-FPIX observed (m/z)c
1.13
1.15
363
17.8
256
616
0.9 (0.1)
2.0 (0.3)
1.2 (0.1)
2.4 (0.1)
325, 616, 938, 940, 941
325, 616
325, 616, 938, 940, 941
325, 616
FIG 5 Drug effects on pH-dependent heme aggregation. (A) Concentration of
free (nonaggregated) FPIX remaining in solution versus pH in the presence of
no drug (ꢀ), CQ (Œ), QN (ꢀ), QD (⌬), AQ (ꢁ), eQN (ᮀ), and eQD (}); (B)
the midpoint (pH1/2) of each curve was calculated via sigmoidal regression.
21.9
a Data correspond to those in Fig. 6. ␭ex and ␭em, excitation and emission ␭’s maxima,
respectively.
b Values are averages of measurements performed in methanol, acetonitrile, and 40%
(vol/vol) DMSO–0.2 M HEPES (pH 7.2), with SEMs reported in parentheses.
c m/z 325, (M ϩ H)ϩ for QN or QD free base; m/z 616, (M)ϩ for FPIX; m/z 938, 940,
and 941, (M Ϫ 2H)ϩ, (M)ϩ, and (M ϩ H)ϩ, respectively, for C54H55FeN6O6,
corresponding to a 1:1 drug-heme complex. Peaks at m/z 498, 771, and 860 were also
observed and were possible FPIX and adduct fragments. See also Fig. S1 in the
supplemental material.
ESI-MS analysis of the QN and QD complexes gave rise to peaks at
m/z 325 and 616, corresponding to free QN Hϩ/QD Hϩ and free
FPIX monomer, respectively, and clear major peaks at m/z 938,
940, and 941 (Table 4; see Fig. S1 in the supplemental material).
These findings coincide with the calculated m/z values of (M Ϫ
2H)ϩ equal to 938.6, (M)ϩ equal to 940.4, and (M ϩ H)ϩ equal to
941.4 for C54H55FeN6O6, corresponding to 1:1 drug-FPIX com-
plexes (6). The 9-epimer adducts, however, did not survive elec-
trospray ionization, as evidenced by peaks at m/z 325 and 616 only
(Table 4; see Fig. S1 in the supplemental material), which again
suggests that these are drug-heme complexes weaker than those
formed by QN and QD (6).
We measured the drug-FPIX stoichiometry for each of the four
complexes via UV-VIS titration (see Materials and Methods).
Each adduct was dissolved in 100% methanol (which completely
dissociates the complex), 40% (vol/vol) DMSO-HEPES (which
completely dissociates the complex), or 100% acetonitrile (in
which the complex remains intact), and three separate measure-
ments for the concentration of drug and FPIX in each sample were
performed at 325 and 402 nm, respectively. Interestingly, in spite
of their weak nature, the results show that eQN and eQD adducts
have a 2:1 stoichiometry (drug-FPIX), while QN and QD form 1:1
adducts (Table 4) (6).
propensity for dimerization might explain the 2:1 drug-heme stoi-
chiometry for the epimer-heme adducts. Chemical shifts of the
quinoline protons were recorded at 1 mM and 50 mM concentra-
tions, since if significant dimerization occurs, a chemical shift dif-
ference of Ն0.1 ppm is observed between each resonance at 1 and
50 mM (see reference 30 for a detailed description). The largest
difference measured for the isomer pairs was 0.02 ppm, with most
differences being Ͻ0.01 ppm (see Table S1 in the supplemental
material). Importantly, then, chemical shift differences were
nearly identical for each drug, indicating no difference in the
dimerization behavior of the 9-epimers and that of QN and QD.
FPIX magnetic moment (␮). We measured the magnetic sus-
ceptibility of FPIX (4 mM) in the presence of each isomer (4 mM)
in 40% DMSO-phosphate buffer (100 mM) at pH 7.0. These mea-
surements distinguish between the monomeric and ␮-oxo di-
meric FPIX species due to the strong antiferromagnetic coupling
between the two high-spin (spin [S] ϭ 5/2) ferric ions afforded by
the oxide bridge in the latter (27). Theoretically, the ␮-oxo dimer
Drug self-association. The propensity for eQN and eQD to
self-associate, meaning that they form dimers through ␲-␲ inter-
actions between the quinoline rings, was compared to that previ-
ously measured for QN and QD (30), to test whether an increased
exhibits a magnetic moment per iron of 1.7 ␮ (the theoretical
B
value for S ϭ 1/2), whereas the monomer gives 5.9 ␮B (the theo-
retical value for S ϭ 5/2). Our results (Table 5) showed that FPIX
has a magnetic moment of 5.33 ␮B in the presence of eQN and 5.45
TABLE 5 Changes in chemical shift and ␮ for ZnPIX and FPIX,
respectively, in the presence of drugd
Change in chemical shifta (ppm) for the following
protonb:
c
Compound
2
3
5
6
7
8
FPIX ␮
QN
eQN
QD
eQD
CQ
0.02
0.02
0.02
0.03
0.04
0.32
0.03
0.40
Ϫ0.08
0.03
Ϫ0.11
0.04
0.02 0.01
0.10 0.44
0.02 0.01
0.07 0.51
5.33
5.33
6.00
5.53
Ϫ0.19 Ϫ0.34 Ϫ0.10 Ϫ0.96
Ϫ0.28 2.41
FIG 6 Fluorescence excitation and emission spectra for 5 ␮M QN-FPIX ad-
duct (gray dotted line, top), QD-FPIX adduct (gray dash-dot-dot line, second
from top), QD (gray continuous line, third from top), QN (gray dashed line,
fourth from top), eQD (black dash-dot line, fifth from top), eQN (black dotted
line, sixth from top), eQN-FPIX adduct (black dash-dot-dot line, seventh
from top), eQD-FPIX adduct (black continuous line, eighth from top), and
FPIX (black dashed line, bottom) performed in 1:1 methanol–0.2 M HEPES
(pH 7.2) at an excitation ␭ of 334 nm and an emission ␭ of 371 nm.
a The chemical shift difference between the corresponding proton in the presence and
absence of heme (ZnPIX in 40% DMSO-d6–D2-O, pH 7.0). The negative sign indicates
an upfield shift relative to the pure drug.
b Quinoline protons are numbered according to the IUPAC numbering system of the
corresponding carbon atoms (see Table S1 in the supplemental material).
c Measured in solution for FPIX in the presence of the corresponding drug.
d See Materials and Methods.
January 2013 Volume 57 Number 1
aac.asm.org 371

Gorka et al.
␮ in the presence of eQD. This is in close agreement with the these isomers suggest (see also reference19) that the molecular
B
measured magnetic moment in the presence of QN and QD and mechanisms of resistance to the cytostatic and cytocidal effects of
characteristic of the high-spin monomeric species. In contrast, the quinoline antimalarial drugs likely differ, which is consistent with
presence of CQ results in a magnetic moment of 2.41 ␮B, which is conclusions from previous drug transport analyses (18). Cyto-
characteristic of the low-spin dimeric species. Thus, similar to QN static resistance to QN (i.e., elevated IC50) is believed to be medi-
and QD, the 9-epimers stabilize monomeric heme but not dimeric ated at least in part by PfCRT and/or PfMDR1 mutations that alter
heme.
electrochemical potential-driven DV transport of the protonated
Solution NMR experiments with ZnPIX. Finally, NMR stud- drug (13, 14, 16, 17). A lack of eQN cytostatic resistance in Dd2
ies using nonparamagnetic ZnPIX were used to further probe (lower IC50 relative to that of HB3) is easily rationalized by the
drug-monomeric heme interactions (see reference6). Clear per- finding that stereochemically distinct eQN was not transported as
turbations in chemical shift for the quinoline protons in the pres- well as QN by the Dd2 mutant PfCRT, since substrate recognition
ence versus absence of ZnPIX were found (Table 5). The magni- by transporters is often quite stereoselective (16). This is entirely
tude and sign (upfield versus downfield) of these shifts illustrate consistent with key conclusions reached previously on the basis of
different distances between the quinoline and porphyrin ring sys- the IC50 behavior for strain K76I (11, 12, 15). Namely, this strain
tems (6). As expected, relatively large upfield (negative) shifts for harbors a mutant Dd2 PfCRT isoform, but with I replacing T at
CQ aromatic protons indicate a short (approximately 3- to 4-Å) position 76, which then produces stereochemically distinct re-
CQ-FPIX interplanar distance, as previously determined (6). QN sponses to quinoline drugs (e.g., QN versus QD; see references 11
and QD show very minor changes in chemical shift, suggesting a 12 for a complete discussion).
wider (6-Å) distance between the quinoline and porphyrin for
Second, we suggest that the highly altered cytostatic activities
these alkaloids, as previously determined (see references 6 and10). but more similar cytocidal activities of the 9-epimers relative to
In contrast to CQ, QN, and QD, however, eQN and eQD exhibit those of QN and QD indicate that the stereochemistry of the
quite large downfield (positive) shifts in quinoline protons 3, 7, 9-epimers negatively affects the interaction with the predominant
and 8, suggesting a quite short drug-heme distance in an arrange- cytostatic (IC50) target but does not necessarily affect the interac-
ment that allows significant deshielding of these specific quinoline tion with the predominant cytocidal (LD50) target(s). Consistent
nuclei. These distance constraints, combined with additional data with the hemozoin inhibition data presented here, as well as in
presented above, suggest a model for epimer binding to mono- other studies (8–10), we favor the interpretation that the predom-
meric heme that is described below.
inant IC50 target for quinoline antimalarials is one or more forms
of uncrystallized heme within the DV, specifically, monomeric
FPIX heme in the case of QN and QD (6). This is consistent with
DISCUSSION
Recent results from our laboratory have suggested the importance perturbations of monomer-dimer ratios, binding behavior, both
of distinguishing between cytostatic and cytocidal activity when the reduced cytostatic potencies and the reduced hemozoin inhi-
characterizing antimalarial drug potency (19; see the accompany- bition activity of the 9-epimers, and no effect on pH-dependent
ing paper [20]), and the present work further emphasizes this heme aggregation that is believed to facilitate hemozoin crystalli-
principle. Previously, we found that resistance to CQ is 10-fold zation (29). We also show that while the 9-epimers are capable of
higher for Dd2 than HB3 when measured by IC50 but 120-fold binding monomeric FPIX, they do so with altered stoichiometry.
higher for Dd2 than HB3 when measured by LD50, suggesting The BHIA IC50s versus antiplasmodial activities for a series of five
significantly altered pharmacology at the different dosages (19). structurally related QN analogs synthesized previously (23) shows
The result was less dramatic for QN and QD, which show 2- and a strong correlation with cytostatic potency but not cytocidal po-
7-fold increased resistance for Dd2 than HB3, respectively, when tency.
measured by IC50 and 4- and 60-fold increased resistance for Dd2
Third, these data suggest which DV species of heme are partic-
than HB3, respectively, when measured by LD50. However, when ularly relevant for forming hemozoin. At least three forms of free
combined with data obtained in this study, it has becoming in- FPIX heme exist in the DV: monomer, ␮-oxo dimer, and head-
creasingly clear that cytostatic and cytocidal targets for quinoline- to-tail dimer. An unresolved issue is which species is more directly
based antimalarial drugs, including Cinchona alkaloids, likely dif- relevant for hemozoin formation and, hence, which interactions
fer and that mechanisms of resistance to agents with cytostatic with which species might be the most important for quinoline
versus cytocidal activities likely differ (16).
drug cytostatic potency. The 9-epimers do not inhibit formation
Similar to Karle et al. (21), we observed vast differences in IC50 of hemozoin under physiological conditions, and although they
activity between QN and QD and their 9-epimers, with the promote the high-spin FPIX monomer in solution (Table 5) and
epimers showing significantly reduced potency (Table 1). In con- form complexes with FPIX that precipitate from aqueous solu-
trast, the activities measured by LD50 tell a different story. While tion, they do not do so with the same affinity, stoichiometry, and
eQN and eQD were less potent than QN and QD when measured fluorescence properties as the complexes formed with QN and
by IC50, for each strain, the fold differences in LD50 between the QD. The former are qualitatively weaker and less favored, as evi-
isomer pairs were far lower, and in some cases (e.g., strain K76I), denced by the ease of resolubilization in neutral buffer following
the 9-epimer LD50s were lower (they had higher cytocidal potency precipitation and significantly lower yield. Moreover, the
than QN and QD). We found that relative to strain HB3, strain 9-epimer complexes do not survive electrospray ionization, lead-
Dd2 was resistant to QN and hypersensitive to eQN when potency ing to sole peaks corresponding to free drug and FPIX in the mass
was defined via IC50 but that the strain was resistant to both when spectrum (Table 4; see Fig. S1 in the supplemental material). The
potency was defined by LD50. Overall, these observations and oth- 9-epimers also do not have a measureable effect on pH-dependent
ers lead to several major conclusions.
heme aggregation (Fig. 5). This is in agreement with the well-
First, the large differences in IC50s (but not LD50s) observed for accepted concept that 4-quinolinemethanols interact more
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9-Epimers of QN and QD
weakly with heme and are less potent inhibitors of hemozoin for-
mation than the 4-aminoquinolines. Strikingly, eQN and eQD
form solution adducts with monomeric FPIX at a ratio of 2:1
(drug-FPIX), while QN and QD form 1:1 complexes that are less
stable in solution. Taken together, these results suggest that heme
monomers are the chemical form of heme that is the most relevant
for hemozoin formation.
Fourth, spectroscopic data allow us to propose a logical model
for 9-epimer binding to free heme. Previously, Alumasa et al.
found that the QN-FPIX adduct was Ͼ5-fold more fluorescent
than QN (heme is not fluorescent at the wavelengths used) (6), a
result that we again observed for both QN- and QD-FPIX com-
plexes. This is likely due to transition dipole ordering in the com-
plex (6), the structure of which is proposed to form through a
noncovalent interaction between the heme iron center and the
hydroxyl oxygen of QN, stabilized by a hydrogen-bonded intra-
molecular 5-membered ring (Fig. 7, center). Given the stoichiom-
etry and fluorescence properties that we observed for the QD-
FPIX complex, it is expected to have a similar structure and
geometry. In contrast, the 9-epimer complexes are significantly
less fluorescent than free drug and over 10-fold less fluorescent
than the corresponding QN and QD adducts. NMR experiments
with ZnPIX also revealed that eQN and eQD exhibit heme inter-
actions in solution that are distinctly different from those of QN,
QD, and CQ (Table 5). The large downfield shifts observed for the
quinoline protons indicate a close quinoline-porphyrin distance
in an arrangement that allows deshielding of specific quinoline
nuclei (such as a face-to-edge ␲-␲ interaction in which the quin-
oline moieties are oriented along the sides of the porphyrin). QN
and QD show only minor shifts in the presence of ZnPIX, indicat-
ing a larger drug-heme distance and the FeOO binding model
reported previously (6). The large upfield shifts observed for CQ
quinoline protons reveal a close quinoline-porphyrin distance in
an arrangement that allows shielding of the nuclei (such as a ␲-␲
interaction in which the quinoline is oriented above the plane of
the porphyrin) (10).
Taken together, the 2:1 (drug-FPIX) stoichiometry, Evans
method data showing preferential binding to the monomer, a lack
of drug self-association, low adduct fluorescence, and ZnPIX data
suggest that eQN and eQD form complexes with monomeric FPIX
via an H structure, where the vertical lines represent the quinoline
moieties and the horizontal line represents FPIX (Fig. 7, top).
Static quenching resulting from formation of such a complex
would lead to the observed drop in fluorescence intensity. Binding
FIG 7 Proposed heme binding models. (Top) An eQN-monomer binding
model based on stoichiometry, fluorescence, Evans (31), self-association, and
ZnPIX data. The model suggests that eQN and eQD bind via face-to-edge ␲-␲
interactions between the least sterically hindered sides of the porphyrin and
quinoline moieties. Those protons showing the greatest downfield shift (Table
of the drugs through a face-to-edge ␲-␲ interaction with the steri- 5) are denoted with an asterisk and lie within the FPIX x-y plane. (Center)
Previously determined QN-monomer structure (6). (Bottom) Previously de-
termined QN–␮-oxo dimeric FPIX complex, which has been shown to form
via face-to-face ␲-␲ interactions, with the quinoline system oriented above the
vinyllic side of FPIX (10). QD complex structures are expected to be similar to
cally less hindered sides of the porphyrin would give rise to the
observed deshielding of the quinoline nuclei.
Caner et al. (34) suggest that the epi-isomers should be better
able to form an intramolecular H-bonded 5-membered ring re-
cently shown to be important for binding of QN to monomeric
heme (6). We previously argued that the stability of this ring pro-
moted binding to monomer, so if it is more stable for the epi-
the QN structures, and the eQD complex is expected to be similar to eQN-
FPIX.
isomers, the 2:1 H model for epi-isomer binding might appear to is a hindrance when binding to heme via this geometry. Thus, the
be paradoxical. Importantly however, for QN and QD, upon for- epimers must interact along the sides of the porphyrin. That is,
mation of the ring, the two bulky groups (quinoline and quinu- bulky group positioning due to the intrinsic stereochemistry of
clidine) are on the same side of the molecule, which is not pre- the drugs and the resulting steric arrangements of the heme inter-
ferred for free drug; however, this leaves one face completely free action drives differential complex formation.
for interacting with heme, and thus, the FeOO interaction is
The H model for 9-epimer binding is less stable than FeOO
formed (6). In the case of eQN and eQD, upon formation of the dative adduct structures for QN and QD with FPIX (Fig. 7, cen-
ring, the bulky groups are on opposite sides of the molecule, which ter), consistent with the weak ability of the 9-epimer adducts to
January 2013 Volume 57 Number 1
aac.asm.org 373

Gorka et al.
CRT protein determined the stereospecific activity of antimalarial Cin-
chona alkaloids. Antimicrob. Agents Chemother. 56:5356–5364.
13. Sidhu AB, Valderramos SG, Fidock DA. 2005. pfmdr1 mutations con-
tribute to quinine resistance and enhance mefloquine and artemisinin
sensitivity in Plasmodium falciparum. Mol. Microbiol. 57:913–926.
14. 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.
15. 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, 64:1139–1148.).
16. Roepe PD. 2011. PfCRT-mediated drug transport in malarial parasites.
Biochemistry 50:163–171.
17. Sidhu AB, Verdier-Pinard D, Fidock DA. 2002. Chloroquine resistance
in Plasmodium falciparum malaria parasites conferred by pfcrt mutations.
Science 298:210–213.
18. 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.
inhibit hemozoin formation. Similar to QN, the 9-epimers may
also form complexes with ␮-oxo dimeric heme (10) (Fig. 7, bot-
tom). Regardless, the antiplasmodial, hemozoin inhibition, heme
binding, heme aggregation, and Evans method data discussed
above suggest that, for the QN isomer pairs, interaction with mo-
nomeric FPIX (and, thus, the monomeric adduct structure) is
more important for hemozoin inhibition than interaction with
␮-oxo dimeric FPIX. However, taken in their entirety, our data
suggest that these structures are not as relevant for the cytocidal
(parasite cell-killing) activity of Cinchona alkaloids as they are for
cytostatic (growth-inhibition) activity. We suggest that for both
QN analogues and CQ analogues (see the accompanying paper
[20]), complete optimization of in vivo activity will require defi-
nition of their cytocidal targets, as well as the mechanism(s) of
cytocidal drug resistance.
ACKNOWLEDGMENTS
19. Paguio MF, Bogle KL, Roepe PD. 2011. Plasmodium falciparum resis-
tance to cytocidal versus cytostatic effects of chloroquine. Mol. Biochem.
Parasitol. 178:1–6.
20. Gorka AP, Alumasa JN, Sherlach KS, Jacobs LM, Nickley KB, Brower
JP, de Dios AC, Roepe PD. 2013. Cytostatic versus cytocidal activities of
chloroquine analogues and inhibition of hemozoin crystal growth. Anti-
microb. Agents Chemother. 57:356–364.
21. Karle JM, Karle IL, Gerena L, Milhous WK. 1992. Stereochemical
evaluation of the relative activities of the cinchona alkaloids against Plas-
modium falciparum. Antimicrob. Agents Chemother. 36:1538–1544.
22. Warhurst DC, Craig JC, Adagu IS, Meyer DJ, Lee SY. 2003. The
relationship of physico-chemical properties and structure to the differen-
tial antiplasmodial activity of the cinchona alkaloids. Malaria J. 2:26. doi:
10.1186/1475-2875-2-26.
23. Dinio T, Gorka AP, McGinniss A, Roepe PD, Morgan JB. 2012. Inves-
tigating the activity of quinine analogs versus chloroquine resistant Plas-
modium falciparum. Bioorg. Med. Chem. 20:3292–3297.
We thank Jeremy Morgan and Theresa Dinio (University of North Caro-
lina, Wilmington, NC) for providing QN structural analogs, Roland Coo-
per (Dominican University) for providing strain K76I, John Alumasa
(The Pennsylvania State University) for helpful advice, Ansley Brown
(University of South Florida Morsani College of Medicine) for technical
assistance, and the Georgetown University Chemistry Department for
synthetic chemistry support.
Financial support was received from NIH grant R0I AI056312.
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