The hydroxyl functionality and a rigid proximal N are required for forming a novel non-covalent quinine-heme complex

Article abstract of DOI:10.1016/j.jinorgbio.2010.08.011

Quinoline antimalarial drugs bind both monomeric and dimeric forms of free heme, with distinct preferences depending on the chemical environment. Under biological conditions, chloroquine (CQ) appears to prefer to bind to μ-oxo dimeric heme, while quinine (QN) preferentially binds monomer. To further explore this important distinction, we study three newly synthesized and several commercially available QN analogues lacking various functional groups. We find that removal of the QN hydroxyl lowers heme affinity, hemozoin (Hz) inhibition efficiency, and antiplasmodial activity. Elimination of the rigid quinuclidyl ring has similar effects, but elimination of either the vinyl or methoxy group does not. Replacing the quinuclidyl N with a less rigid tertiary aliphatic N only partially restores activity. To further study these trends, we probe drug-heme interactions via NMR studies with both Fe and Zn protoporphyrin IX (FPIX, ZnPIX) for QN, dehydroxyQN (DHQN), dequinuclidylQN (DQQN), and deamino-dequinuclidylQN (DADQQN). Magnetic susceptibility measurements in the presence of FPIX demonstrate that these compounds differentially perturb FPIX monomer-dimer equilibrium. We also isolate the QN-FPIX complex formed under mild aqueous conditions and analyze it by mass spectrometry, as well as fluorescence, vibrational, and solid-state NMR spectroscopies. The data elucidate key features of QN pharmacology and allow us to propose a refined model for the preferred binding of QN to monomeric FPIX under biologically relevant conditions. With this model in hand, we also propose how QN, CQ, and amodiaquine (AQ) differ in their ability to inhibit Hz formation.

Full text of DOI:10.1016/j.jinorgbio.2010.08.011

Journal of Inorganic Biochemistry 105 (2011) 467–475
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
The hydroxyl functionality and a rigid proximal N are required for forming a novel
non-covalent quinine-heme complex
John N. Alumasa a, Alexander P. Gorka a, Leah B. Casabianca d,1, Erica Comstock e,1
,
Angel C. de Dios a,c, Paul D. Roepe a,b,c,
⁎
a
Department of Chemistry, Georgetown University, 37th and O Streets, NW Washington, D.C. 20057, USA
b
Department of Biochemistry and Molecular Biology & Cellular Biology, Georgetown University, 37th and O Streets, NW Washington, D.C. 20057, USA
Center for Infectious Diseases, Georgetown University, 37th and O Streets, NW Washington, D.C. 20057, USA
Department of Chemistry, University of Illinois at Chicago, Illinois, USA
c
d
e
Department of Chemistry, University of North Carolina at Chapel Hill, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 May 2010
Received in revised form 17 August 2010
Accepted 18 August 2010
Available online 22 September 2010
Quinoline antimalarial drugs bind both monomeric and dimeric forms of free heme, with distinct preferences
depending on the chemical environment. Under biological conditions, chloroquine (CQ) appears to prefer to
bind to μ-oxo dimeric heme, while quinine (QN) preferentially binds monomer. To further explore this
important distinction, we study three newly synthesized and several commercially available QN analogues
lacking various functional groups. We find that removal of the QN hydroxyl lowers heme affinity, hemozoin
(Hz) inhibition efficiency, and antiplasmodial activity. Elimination of the rigid quinuclidyl ring has similar
effects, but elimination of either the vinyl or methoxy group does not. Replacing the quinuclidyl N with a less
rigid tertiary aliphatic N only partially restores activity. To further study these trends, we probe drug-heme
interactions via NMR studies with both Fe and Zn protoporphyrin IX (FPIX, ZnPIX) for QN, dehydroxyQN
(DHQN), dequinuclidylQN (DQQN), and deamino-dequinuclidylQN (DADQQN). Magnetic susceptibility
measurements in the presence of FPIX demonstrate that these compounds differentially perturb FPIX
monomer-dimer equilibrium. We also isolate the QN-FPIX complex formed under mild aqueous conditions
and analyze it by mass spectrometry, as well as fluorescence, vibrational, and solid-state NMR spec-
troscopies. The data elucidate key features of QN pharmacology and allow us to propose a refined model for
the preferred binding of QN to monomeric FPIX under biologically relevant conditions. With this model in
hand, we also propose how QN, CQ, and amodiaquine (AQ) differ in their ability to inhibit Hz formation.
© 2010 Elsevier Inc. All rights reserved.
Keywords:
Hemozoin
Antimalarial drugs
Ferriprotoporphyrin IX
1. Introduction
the 17th century [4] and remained a first line drug therapy vs. malaria
until the discovery and widespread introduction of chloroquine (CQ)
Malaria remains one of the world's most tragic infectious diseases,
afflicting hundreds of millions and killing millions worldwide annually
[1]. The lack of an effective vaccine coupled with the continued spread
and evolution of resistance to antimalarial drugs [1–3], makes
development of additional inexpensive, novel, and efficacious anti-
malarial drugs particularly urgent. To achieve this goal, a better
understanding of the molecular mechanism of action of effective
antimalarial drugs as well as the mechanism(s) of parasite antimalarial
drug resistance is needed.
[5]. Today, the World Health Organization (WHO) currently recom-
mends the use of QN in the absence of artemisin combination
therapies (ARTs) to treat CQ resistant (CQR) P. falciparum malaria. In
the laboratory, it is known that QN remains active vs. some CQR
strains of P. falciparum, but the molecular explanation for this is
unknown. This is in part due to the fact that, surprisingly, despite
extensive use of the drug, the molecular mechanism of action of QN
has not been fully elucidated. Most current research on quinoline-
based antimalarials has focused on 4-amino and 8-amino quinolines,
even though it is clear a QN pharmacophore has specific advantages.
Quinoline antimalarials are believed to target heme detoxification
within the digestive vacuole (DV) of the malaria parasite [6–9].
Studies directed at understanding this include genetic, cell biological,
and molecular pharmacological approaches. Genetically, QN resis-
tance (QNR) has been linked to key mutations and/or over-expression
of the P. falciparum chloroquine resistance transporter (PfCRT)
[10,11], the P. falciparum multidrug resistance protein (PfMDR1)
Quinolines are an extremely important class of antimalarial drugs.
The cinchona alkaloid quinine (QN) was effectively used as early as
⁎
Corresponding author. Department of Chemistry, Georgetown University, 37th and
O Streets, NW Washington, D.C. 20057, USA. Tel.: +1 202 687 7300; fax: +1 202 687
6209.
E-mail address: roepep@georgetown.edu (P.D. Roepe).
Current address.
1
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.08.011

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J.N. Alumasa et al. / Journal of Inorganic Biochemistry 105 (2011) 467–475
[12–15], and either mutation or increased activity of the P. falciparum
Na+/H+ exchanger (PfNHE) [16,17]. The existence of various
combinations of these multiple genetic events in various QNR strains,
leading to combinations of altered function in three membrane
proteins, presumably explains why levels of QNR are distributed
across a wide range (e.g. 2, 3, 4, 5,… 9-fold, etc.). In contrast, across all
strains that have been examined, P. falciparum CQR conferred
predominantly by mutations in a single gene (pfcrt) [11] is typically
characterized as a ≥10-fold increase in growth inhibition IC50, with no
increases seen between 1- and 10-fold. In the presence of PfCRT
mutations conferring this 10-fold jump in IC50, it is believed that
mutation and/or increased expression of PfMDR1 can then further
modulate quinoline drug resistance patterns.
In terms of molecular pharmacology, similar to CQ, QN is known to
inhibit the crystallization of toxic ferriprotoporphyrin IX (FPIX) heme
to nontoxic hemozoin (Hz) [6–9]. Additional data defining non-
covalent binding of QN to free μ-oxo dimeric heme [18] suggests
inhibition of Hz formation could be by non-covalent binding to pre-
crystalline dimeric forms of FPIX. However, several forms of FPIX exist
in biological environments, including reduced and oxidized monomer,
μ-oxo dimer, and tethered head-to-tail dimer [19]. QN binding to the
different forms is not fully defined, and it is not known which form(s)
are most relevant for QN-mediated inhibition of Hz formation. Recent
studies [20,21] suggest CQ prefers binding to dimeric forms of FPIX
while QN prefers binding to monomer, and that equilibria between the
various complexes are significantly influenced by partitioning be-
tween aqueous and hydrophobic (i.e. membrane lipid) phases.
Earlier evidence based on spectroscopic comparisons of QN
adducts to those of N-coordinated ligand complexes of tetraphenyl-
porphyrinato iron(III) and chloroiron(III) protoporphyrin IX dimethyl
ester appeared to eliminate possible Fe-quinolynyl N covalent
coordination for a QN-monomeric FPIX complex [22]. Based on 13C
NMR results, these authors hypothesized alkoxide coordination
between the hydroxyl group of QN and iron center of FPIX, but the
data did not allow for determination of QN-FPIX stoichiometry.
Constantinidis et al studied drug-heme interaction between QN and
urohemin I/uroporphyrin I [23], and their results were also consistent
with the QN hydroxyl acting as a covalent axial ligand to heme iron.
These data further suggested a 2:1 (urohemin:QN) binding stoichi-
ometry, consistent with QN as an axial ligand to the μ-oxo dimer form
of FPIX. Other studies have shown that stereochemical configuration
is crucial to the activity of QN-related alkaloids. For example, threo-epi
cinchona alkaloids are known to be significantly less active than the
erythro-alkaloids [24–26].
features necessary for interaction with FPIX and for biological activity.
We assess antiplasmodial and in vitro Hz inhibitory activities as well
as FPIX binding affinities for these compounds. We measure FPIX
magnetic moment in the presence of several analogs and compare
these results to earlier results for QN. We perform solution NMR
studies in the presence of ZnPIX to quantify effects of heme binding on
proton chemical shifts, and use these shifts to estimate distance
between drug and heme aromatic rings. We also report the first
isolation of a QN-FPIX complex from aqueous solution. Detailed
spectroscopic analyses of this complex shows that formation of a 1:1,
non-covalent complex between QN free base and monomeric FPIX
occurs in aqueous solution, and that this complex likely forms an
insoluble, non-crystalline aggregate in the DV that then stabilizes
weak QN-FPIX interaction. Collectively, our findings allow us to
propose a novel model for QN binding to monomeric FPIX, as well as
biologically relevant models that distinguish QN vs. CQ inhibition of
Hz formation.
2. Materials and methods
2.1. Materials
All reagents and solvents were purchased from Sigma-Aldrich (St.
Louis, MO) unless otherwise noted. Iodoethane was purchased from
Acros Organics (Geel, Belgium). Propionic acid, sodium propionate,
anhydrous sodium sulfate, clear polystyrene and sterile tissue culture
flat bottom 96-well plates were purchased from Fisher Scientific
(Newark, DE). Sodium dodecyl sulfate was purchased from Bio-Rad
laboratories (Hercules, CA). Hemin was purchased from Fluka (Buchs,
Switzerland). Zinc protoporphyrin IX was purchased from Frontier
Scientific (Logan, Utah). D2O and DMSO-d6 were purchased from
Cambridge Isotope Laboratories Inc. (Andover, MA).
2.2. Culturing
P. falciparum strains Dd2, GCO3, 7G8, and HB3 were obtained from
the Malaria Research and Reference Reagent Resource Center (Manassas,
VA). Off-the-clot, heat-inactivated pooled O+ human serum and O+
human whole blood were purchased from Biochemed Services
(Winchester, VA). SYBR Green I nucleic acid dye was purchased from
Invitrogen (Eugene, OR). Custom 5% O2/5% CO2/90% N2 culturing gas
blend was purchased from Robert's Oxygen (Rockville, MD). RPMI 1640,
hypoxanthine, HEPES, and Giemsa stain were from Sigma-Aldrich (St.
Louis, MO).
Using magnetic susceptibility measurements, we have recently
shown that unlike CQ, QN promotes formation of monomeric rather
than dimeric FPIX [21]. This suggests that structural differences
among quinoline antimalarials may result in different mechanisms of
Hz inhibition. If so, this might then form the basis for understanding
the lack of strict correspondence between levels of CQR and QNR
[16,17]. Egan and colleagues have recently obtained a very informa-
tive crystal structure of a halofantrine (HF)-FPIX complex that shows
alkoxide coordination to FPIX, but were unsuccessful in obtaining
diffraction quality QN-FPIX crystals [27]. Nonetheless, based on the
HF-FPIX structure and energy profile similarities between QN, QD and
HF, these workers propose that QN-FPIX interaction involves covalent
coordination between the hydroxyl moiety of QN and the Fe3+ center
of FPIX. This model is consistent with previous studies mentioned
above [22]. Thus, the majority of molecular studies have implicated
that the hydroxyl group of QN interacts with FPIX Fe3+ in some
fashion [22–27] but there are conflicting data and interpretation
regarding the covalent vs. non-covalent nature of that interaction, QN:
FPIX stoichiometry, and QN structural features that influence binding
to FPIX.
2.3. Test compounds
Quinine dihydrochloride was purchased from VWR International
(Westchester, PA). Cinchonine (CN) was purchased from Acros
Organics (Geel, Belgium), quinidine (QD) hydrochloride monohy-
drate, halofantrine hydrochloride, and hydroquinine (HQN) were
purchased from Sigma-Aldrich (St. Louis, MO). 2-((6-methoxyquino-
lin-4-yl)methyl)-8-vinylquinuclidine (DHQN, 2), 1-(6-methoxyqui-
nolin-4-yl)propan-1-ol (DADQQN, 5), and 2-(diethylamino)-1-(6-
methoxyquinolin-4-yl)ethanol (DQQN, 8) were synthesized as de-
scribed in the Methods section.
2.4. General methods
All commercially available reagents and solvents were used
without further purification. Flash chromatography was performed
on Kieselgel 60, particle size 0.032–0.063 mm. Thin layer chromatog-
raphy analyses were performed on Selecto Scientific flexible TLC
plates (silica gel 60 – F 254, 200 micron). NMR spectra were obtained
on a 400 MHz (1H NMR) and 100 MHz (13C NMR) Varian FT-NMR
spectrometer using CDCl3 as solvent unless otherwise indicated, and
In this study, we synthesize three novel QN analogs, and use these
together with commercially available analogs to define QN structural

J.N. Alumasa et al. / Journal of Inorganic Biochemistry 105 (2011) 467–475
469
using tetramethylsilane (TMS) as the external standard. Mass
spectroscopic measurements were performed in MeOH or ACN on a
Varian500 ESI Mass spectrometer.
2.5. Synthesis of 2-((6-methoxyquinolin-4-yl)methyl)-8-vinylquinuclidine
(2, Scheme 1)
QN was reacted with p-toluenesulfonyl chloride in the presence of
a mild base to give the tosyl-protected QN derivative 1 in 34% yield.
Treatment of 1 with lithium aluminum hydride (LiAlH4) in tetrahy-
drofuran (THF) at room temperature resulted in the reduction of the
hydroxyl functionality to give 2 (DHQN) as a yellowish oil in 27% yield
(see Supporting Information for details, Scheme 1).
Scheme 2. Synthesis of 1-(6-methoxyquinolin-4-yl)propan-1-ol (5, DADQQN) and 2-
(diethylamino)-1-(6-methoxyquinolin-4-yl)ethanol (8, DQQN): i) SeO2, 4:1 dioxane/
H2O, 90 °C, 15 h, 70%; ii) EtMgBr, THF, 0 °C, 6 h, 57% yield; iii) MeNO2, Et3N, MeOH,
−10 °C, 5 h, 66%; iv) LiAlH4, THF, 0 °C to rt, 1 h, 35%; (v) EtI, Et3N, DMF, rt, 24 h, 18%.
2.6. Synthesis of 1-(6-methoxyquinolin-4-yl)propan-1-ol (5, Scheme 2)
This was prepared through a Grignard reaction involving the
addition of ethylmagnesium bromide to a solution of 6-methoxyqui-
noline-4-carbaldehyde (4) at 0 °C with continuous stirring to give the
pure product 5 (DADQQN) in 57% yield (Scheme 2).
using complete media from a 10,000X DMSO stock) was added, the
plates incubated for an additional 1 h at 37 °C to allow DNA
intercalation, and fluorescence measured at 530 nm (490 nm excita-
tion) using a Spectra GeminiEM plate reader (Molecular Devices). Data
analysis was performed using Sigma Plot 10.0 software after down-
loading data in Excel format. For each assay, each drug dilution was
analyzed in triplicate, and the results from three separate assays
averaged ( SD). All drugs were tested against two quinine sensitive
(QNS), and two quinine resistant (QNR) strains of P. falciparum (GCO3
and HB3, 7G8 and Dd2, respectively).
2.7. Synthesis of 2-(diethylamino)-1-(6-methoxyquinolin-4-yl)ethanol
(8, Scheme 2)
6-Methoxy-4-methylquinoline (3) was oxidized with selenium
dioxide in a mixture of dioxane and water to give 6-methoxyquino-
line-4-carbaldehyde (4) in 70% yield. A Henry reaction with
nitromethane in the presence of triethylamine afforded nitroaldol 6
in 66% yield, which was subsequently reduced to amino alcohol 7 in
35% yield using LiAlH4. In the final step, 7 was reacted with two
equivalents of iodoethane in the presence of triethylamine to give QN
analogue 8 (DQQN) in 18% yield (see Supporting Information for
details, Scheme 2).
2.9. β-hematin growth inhibition studies
β-hematin growth inhibition was assessed using a recently
perfected 96-well plate high throughput assay [31]. Briefly, the
assay uses physiologic temperature and lipid catalyst, and relies on
the differential solubility properties of crystalline and non-crystalline
forms of FPIX in 2.5% SDS (86.7 mM) and alkaline bicarbonate buffer
(0.1 M, pH 9.1). Hemin was dissolved in 0.1 M NaOH to make a 2 mM
stock. 10 μL was then transferred to 96-well plate wells, followed by
propionate buffer (180 μL at desired pH) and 10 μL of sonicated
phosphatidyl choline suspension (10 μL) to a final heme concentra-
tion of 100 μM. Drugs were added at different concentrations, plates
were mixed, wrapped in plastic wrap, and incubated at 37 °C for 16 h.
The assay was terminated by adding 100 μL of a solution of SDS
dissolved in 0.1 M bicarbonate buffer (pH 9.1) {final concentration/
well of SDS 2.5% (w/v), 86.7 mM}. The well contents were gently
mixed and the plate incubated at room temperature for 10 min to
allow un-dissolved Hz crystals to settle. A 50 μL aliquot from each well
was then transferred to a second plate preloaded with 200 μL of SDS
solution (2.5% w/v, 86.7 mM) in 0.1 M bicarbonate buffer. Absorbance
of non-crystallized heme was recorded at 405 nm with a 96-well
plate-adapted ELx800 BioTek absorbance microplate reader. Percent
Hz formed was analyzed using Microsoft Excel (2007 Edition) and
Sigma Plot 10.0 software [31].
2.8. Antiplasmodial activity measurements
All P. falciparum strains were maintained using the method of Trager
and Jensen [28] with minor modifications. Briefly, cultures were
maintained under an atmosphere containing 5% CO2, 5% O2 & 90% N2
gaseous mix at 2% hematocrit and 1–2% parasitemia in RPMI 1640
supplemented with 10% O+ human serum, 25 mM 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid (HEPES; pH 7.4), 24 mM NaHCO3,
11 mM glucose, 0.75 mM hypoxanthine, and 20 μg/L gentamycin with
regular media changes every 48 h. The antiplasmodial activity was
assessed essentially as previously described [29] with minor modifica-
tions. Test compounds were diluted to make 2× stocks using complete
media under sterile conditions. 100 μL of these were then transferred
into 96-well plates followed by incubation at 37 °C. Sorbitol synchro-
nized cultures were utilized for the assays with N95% of the parasites in
the ring stage [30]. Cultures were diluted to give a working stock of 0.5%
parasitemia and 2% hematocrit and 100 μL was transferred to each drug-
preloaded well (final 1% hematocrit and 0.5% parasitemia). The plates
were transferred to an airtight chamber which was then gassed and
incubated at 37 °C. After 72 h, 50 μL of 10× SYBR Green I dye (diluted
2.10. Heme affinity measurements
Drug-heme affinity measurements were performed by monitoring
the changes in the absorbance of heme in the presence of increasing
concentrations of drug. Hemin was dissolved in DMSO to 5 mM, which
was then serially diluted to 5 μM in 40% DMSO/0.2 M acetate buffer,
pH 5.0. Drug solutions were prepared by dissolving the compound in
DMSO and diluting to 1.5 mM in 40% DMSO/0.02 M HEPES, pH 7.4. A
cuvette containing 1 mL of freshly prepared heme (5 μM) was titrated
with increasing drug (0–210 μM), the sample mixed following each
addition, and absorbance of heme at 402 nm recorded using an
Agilent UV–VIS spectrophotometer (final volume dilution 6.54%).
Solvent dilution controls were performed similarly and spectral and
Scheme 1. Synthesis of 2-((6-methoxyquinolin-4-yl)methyl)-8-vinylquinuclidine
(2, DHQN): i). TsCl, Et3N, CH2Cl2, 9 h, 0 °C to rt, 34% yield; ii). LiAlH4, THF, 12 h, rt, 27% yield.

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J.N. Alumasa et al. / Journal of Inorganic Biochemistry 105 (2011) 467–475
data analyses performed using Kaleida Graph and Sigma Plot 10.0
software. Nonlinear least squares curve fitting of the raw data was
done using the Levenberg–Marquardt algorithm (initial Ka
input=0.01 μM−1) and affinity coefficients (Ka) were computed
(see caption to Fig. 2).
Mass spectrometry analyses using a Varian 500 MS were
performed by dissolving the precipitate in ACN. IR analyses of heme,
QN, and the adduct were performed in KBr pellets on a Nicolet 380
FTIR spectrometer (Thermo-Electron Corporation). Briefly, dried solid
samples of either QN, heme, or the adduct were added to powdered
KBr (1:3 ratio sample to KBr), ground, and pressed. Fluorescence
spectra were recorded from 5 μM samples dissolved in 1:1 methanol/
0.2 M HEPES (pH 7.2) using a QM 2001-4 Quantum Master
Fluorometer (shown in Fig. S1 of “Supplemental Data”). Solid-state
13C CP-MAS NMR spectra were acquired on a Bruker Avance DRX-500
spectrometer operating at a 13C frequency of 125.76 MHz and using
XWIN-NMR version 2.1 software. Samples were packed into a 4-mm
rotor and spun at 9 kHz MAS at room temperature. For QN base, QN
monohydrochloride dihydrate, and QN dihydrochloride samples,
2048 scans were collected; for the QN-FPIX complex, 15,360 scans
were collected. The recycle delay was 3 s and the CP contact time was
1 ms. 45 kHz CP and 63 kHz TPPM decoupling were used. The spectral
width was 83,333 Hz. Exponential line broadening of 100 Hz was
applied to all spectra. Chemical shifts were externally referenced to
TMS using the CO signal of glycine at 176.4 ppm [33].
2.11. Magnetic susceptibility measurements
Magnetic susceptibility measurements were performed as previ-
ously described [21] in 40% (v/v) DMSO/100 mM phosphate buffer
(pH 7.0). Both hemin (Fluka) and drug stock solutions (20 mM) were
prepared in 100% DMSO-d6. Test samples (40% v/v DMSO/100 mM
phosphate buffer at pH 7.0) were prepared by adding 200 μL of hemin
and the corresponding test compound into a 1.5 mL microcentrifuge
tube followed by the addition of 600 μL of 100 mM 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 measured 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 Unity INOVA 500 MHz
NMR spectrometer with a proton frequency of 499.789 MHz and
data analyzed using the Varian VNMR version 5.1 software. Magnetic
susceptibility was determined using the Evans method [32] employing
the equation (Eq. (1)) appropriate for a superconducting magnet
at 298 K:
3. Results
We first designed and synthesized a series of QN analogs
containing the QN pharmacophore but lacking specific functional
groups (DHQN, DQQN, and DADQQN; Schemes 1 & 2, Fig. 1), and also
obtained two additional commercially available QN analogues (CN,
HQN) lacking additional groups. We calculated simple physicochem-
ical characteristics for these (Table 1) which indicate that, in most
cases, the structural alterations do not significantly perturb these
relative to QN. One exception is DADQQN, which is predicted to have
lower DV accumulation (lower VAR, cf. Table 1) due to the loss of the
basic terminal N. For the remaining analogs, both quinolyl N and
aliphatic N pKas were comparable to those for QN, making them
effectively monobasic under biological conditions. Correspondingly,
calculated vacuolar accumulation ratios (VAR) [34,35] for these
compounds were comparable.
Antiplasmodial activities for these compounds were measured vs.
two QNS (HB3, GCO3) and two QNR (Dd2, 7G8) strains of P. falciparum
using the SYBR Green I assay ([29], Table 2). Results show that QD and
CN are approximately 4–5 fold more active than QN, and that QN and
HQN had quite similar activity. DQQN, DADQQN, and DHQN were the
least active, exhibiting IC50s~7–100 fold higher than QN, respectively,
depending on the strain (Table 2). These results demonstrate that
elimination of the methoxy group improves the activity of QN such
that it is then similar to QD and that loss of the vinyl group has no
effect on QN antiplasmodial activity. In contrast, IC50s for DHQN and
DADQQN were, in general, N10 μM for both QNS & QNR strains,
indicating that loss of the hydroxyl moiety or the rigid quinuclidine
ring essentially destroys activity. Interestingly, replacement of a
tertiary aliphatic N at the same position as that found in the QN
quinuclidine ring (DQQN) partially restored activity.
As mentioned, inhibition of parasite Hz formation is hypothesized
to be the principle basis of antiplasmodial activity for the cinchona
alkaloids [6–9]. Thus, we investigated the ability of these compounds
to inhibit β-hematin (Hz) crystal growth in vitro (BHIA activity) in
order define the significance of QN functional groups in this process. A
similar trend was observed at both pH 5.2 and pH 5.6 [36] (Table 3).
We observed ≥5-fold difference between BHIA at pH 5.6 vs. pH 5.2 for
all compounds with the exception of CN, which was only 2-fold
different at the two pH, and DADQQN which had quite poor activity at
both. At pH 5.6, the BHIA IC50s of QD, QN, CN, and HQN were
conspicuously lower than DHQN, DADQQN, and DQQN IC50, and these
three analogs had even poorer IC50s (N2 mM) at pH 5.2. These results
demonstrate the importance of the hydroxyl and rigid quinuclidyl
moeity for β-hematin crystal growth inhibition by QN. However, we
xm = −3Δν= 4πc + xD
ð1Þ
where xm is the molar susceptibility of the paramagnetic substance in
cm3/mol, Δν is the chemical shift difference (in ppm) between a
reference proton in the sample and that in a solution lacking the
paramagnetic compound, c is the concentration of FPIX in mol/mL,
and xD is the diamagnetic susceptibility of heme (6.9×10−4 cgs
units). Solvent susceptibility corrections and the solution-solvent
density differences are ignored. The molar susceptibility was
converted to magnetic moment (μ) using Eq. (2) below, where T is
the temperature (K):
μ = 2:8√ðxmTÞ
ð2Þ
2.12. Solution NMR experiments using zinc (II) protoporphyrin IX
A 10 mM stock solution of ZnPIX was prepared in 0.05 M NaOH in
D2O, and 10 mM drug stock solutions were prepared by dissolving the
corresponding HCl salts in D2O. Equimolar drug-heme solutions
(1.667 mM each) were then prepared and titrated to pH 7.0. 600 μL of
this solution was transferred to a microcentrifuge tube (1.5 mL)
followed by the addition of 400 μL of DMSO-d6, that is test samples
contained a 1:1 drug to ZnPIX ratio at 500 μM. These solutions were
then transferred to a 5 mm NMR tube for analyses. 1D & 2D proton
NMR spectra were recorded and analyzed as stated above on the
500 MHz NMR spectrometer using tetramethylsilane as the external
reference.
2.13. Formation and analyses of aqueous QN-heme complex
A QN-heme complex formed under aqueous conditions was
prepared by first dissolving hemin in 0.1 M NaOH to give a 2 mM
stock solution. This solution was slowly titrated with increasing
volumes from a QN stock (100 mM in deionized water) while
monitoring the pH (see Results). At a molar ratio of 1:9 (heme to
QN) copious heme was observed to precipitate, leaving a clear
solution of QN (see Results). The resultant precipitate was isolated via
vacuum filtration and freeze-dried.

J.N. Alumasa et al. / Journal of Inorganic Biochemistry 105 (2011) 467–475
471
Fig. 1. Structures of compounds used in our analyses. CQ – chloroquine; AQ – amodiaquine; HF – halofantrine; QD – quinidine; QN – quinine; DHQN – dehydroxyquinine; CN – cinchonine;
HQN – hydroquinine; DQQN – dequinuclidylquinine; DADQQN – deamino-dequinuclidylquinine.
do not find a strong correlation when plotting antiplasmodial activity
vs. BHIA for these QN analogues (data not shown).
indicating, as expected, that these drugs strongly stabilize FPIX
monomer in solution. In contrast, DHQN, DQQN, and DADQQN
exhibited moments of 2.52, 2.89 and 2.83 μB, respectively. These are
similar, but not identical to, μB previously measured in the presence of
CQ [19] and are consistent with the presence of low spin μ-oxo
dimeric FPIX.
Additional NMR studies using non-paramagnetic ZnPIX were used
to further probe these drug-heme interactions. We measured
perturbations in chemical shift for the quinoline protons in the
presence vs. absence of ZnPIX, knowing that different values for these
shifts illustrate different distances between the quinoline and
porphyrin ring systems [18]. As expected [18], relatively large changes
in chemical shift for CQ aromatic ring protons 2, 3, 5, and 6 indicate a
short (approximately 3–4 Å, see [18]) CQ-FPIX interplanar distance,
whereas for QN the very small change for equivalent proton shifts
indicates a much wider (estimated 6 Å [18]) distance between the
rings (Table 4).
To further define the interaction between QN and FPIX, we
attempted to isolate the QN-FPIX complex formed under aqueous
conditions. One method we perfected, based on simple titration as
described in Methods, leads to precipitation of a greenish, highly
fluorescent (see Supplemental Fig. 1) precipitate from aqueous solution.
This precipitate was initially isolated at pH ~8.5 but can be obtained in
lower yield at pH 6.0–7.0. We analyzed the presumed QN-FPIX adduct
by electrospray ionization – mass spectrometry (ESI–MS) in positive ion
mode and obtained minor peaks at m/z 325.2 and 616.2, corresponding
to free QNH+ and free FPIX monomer, respectively, and clear major
peaks at m/z 938.5, 940.4, and 941.3 (Fig. 3). The major peaks coincide
with the calculated m/z values (M−2H)+ =938.58, (M)+=940.35,
We next tested if BHIA is related to affinity for monomeric heme
since we have recently concluded that QN, unlike CQ, promotes
formation of monomeric heme [21]. Although it would be ideal to
perform these experiments under physiologically relevant aqueous
conditions, drug-FPIX titrations were performed in 40% aqueous
DMSO since monomeric FPIX is unstable in aqueous solution [37]. The
results indicate significant differences in heme binding for the
compounds (Fig. 2), with HF, QD, and CN showing similar binding
that is also higher affinity than that for QN and HQN. DHQN, DADQQN,
and DQQN showed little to no heme binding under these conditions,
in general consistent with the above data showing reduced efficiency
of these three molecules in inhibiting β-hematin crystal growth.
We next measured the magnetic susceptibility of FPIX (4 mM) in
the presence of test compounds (4 mM) in 40% DMSO/phosphate
buffer (100 mM), pH 7.0. These measurements easily distinguish
between the μ-oxo bridged vs. monomeric FPIX species and have been
used previously to determine preference of a particular FPIX species
for different quinoline drugs [21]. Our results (Table 4, far right
column) clearly show significantly different perturbations in mono-
mer-dimer equilibria upon addition of QN vs. DHQN, DQQN, or
DADQQN, as determined by comparing heme μ measured in the
presence of these compounds. For QN, QD, and HF, a magnetic
moment of 5.45 μB was observed, which is close to the upper limiting
value of 5.6 μB corresponding to the high spin monomeric species [21],
Table 1
Calculated physicochemical properties for QN, QD, and QN analogs under investigation.
Cyt
CPD ID
MWa
LogPb
pKac
pKa1
pH7.2VARd
Table 2
(Oct/H2O)
pKa2
DVpH 5.2
DVpH 5.6
Antiplasmodial activities against two QNS strains (GCO3, HB3) and two QNR strains
(Dd2, 7G8).
QN
QD
324.42
324.81
308.42
294.39
274.36
217.26
326.43
2.66
3.06
3.65
2.63
2.45
2.34
2.90
5.69
5.49
5.69
5.10
5.75
5.78
5.72
9.16
9.09
9.17
8.75
9.55
–
393
392
393
374
437
85.2
85.1
85.3
81.9
92.4
2.43
88.8
a
Experimental IC50 (nM)
DHQN
CN
DQQN
DADQQN
HQN
CPD ID
HB3b
Dd2c
SId
GCO3b
7G8c
SIe
4.63
415
QN
QD
HF
DHQN
CN
DQQN
DADQQN
HQN
81 8.4
18 2.2
320 50
90 8.4
3.2 0.4
N10,000
82 8.1
2100 410
N10,000
410 57
4.0
5.0
2.0
–
99 13
350 39
110 2.3
1.8 0.3
N10,000
95 18
1600 150
N10,000
490 53
3.6
3.7
1.3
–
9.39
28 7.6
1.4 0.3
N10,000
19 3.4
2700 280
N10,000
110 14
1.6 0.7
N10,000
22 6.8
2600 58
9600 290
100 19
MW – Molecular weight; VAR – Vacuolar accumulation ratio.
a
Determined using ChemOffice Ultra 10.0.
3.8
0.83
–
5.1
0.6
–
b
LogP online calculator for calculation of molecular properties and drug-likeness
(http://www.molinspiration.com/cgi-bin/properties).
c
SPARC online calculator (http://ibmlc2.chem.uga.edu/sparc) pKa1 and pKa2
4.0
4.4
represent the pKas of the quinolyl and aliphatic nitrogen respectively.
d
Calculated using the Henderson-Hasselbalch equation (DV
&
Cytosolic pH as
aThe experimental IC50s are an average of three separate determinations ( SD); Strain
defined above; see also Natarajan JK, et al. J Med Chem. 2008, 51, 3466).
phenotype: bQNS, cQNR; SId – (IC50 Dd2/IC50 HB3), SIe – (IC50 7G8/IC50 GCO3).

472
J.N. Alumasa et al. / Journal of Inorganic Biochemistry 105 (2011) 467–475
Table 3
In vitro β-hematin growth inhibitory activities (BHIA).
a
BHIA IC50 (μM)
CPD
pH 5.2
pH 5.6
QN
QD
HF
DHQN
CN
DQQN
DADQQN
HQN
260 15
130 2.5
100 3.1
N2000
370 11
N2000
49 0.4
23 2.3
24 4.7
230 24
170 36
210 12
1900 53
44 1.6
N2000
280 9.0
a
The experimental IC50s are averages of two separate determinations each performed
in triplicate ( SE).
Fig. 3. Mass spectrum of the QN-FPIX adduct in ACN. The spectrum shows m/z peaks at
325.2 corresponding to (M+H)+ for QN free base, m/z 616.2 corresponding to (M)+ for
FPIX, and 938.5, 940.4, 941.3 which match the calculated m/z values (M−2H)+=938.58,
(M)+=940.35, and (M+H)+=941.36 for C54H55FeN6O6 that correspond to a 1:1 QN-
heme complex. The peaks at m/z 498.2, 771.6, and 859.8 are possible FPIX fragments.
IR spectral analyses of the adduct (Fig. 4A,B, top spectrum “W”)
revealed a sum of QN (“X”) and heme (“Y”) vibrational peaks, but the
conspicuous absence of peaks indicative of QNH+ (Fig. 4 “Z”),
including the strong N-H stretch centered near 2575 cm−1 (Fig. 4A),
as well as the prominent 1702 cm−1 heme propionate carbonyl
Fig. 2. Drug-heme binding curves. Affinity was measured by titrating increasing
concentrations of QN (▲), QD (□), HF (Δ), CN (●), HQN (○), DHQN (▼), DQQN (◊), or
DADQQN (■) dissolved in 40% DMSO/acetate buffer (0.2 M, apparent pH 5.0) into a
solution of heme (10 μM) followed by non-linear least squares curve fitting to
determine Ka. The affinity coefficients were determined to be 3.92×104 (R2 =0.99),
2.45×104 (R2 =0.99), 1.87×104 (R2 =1.00), 1.96×104 (R2 =1.00), and 2.18×104 M−1
(R2 =0.99) for HF, QN, QD, CN, and HQN, respectively. No statistically reliable
coefficients could be determined for DHQN, DQQN, and DADQQN.
and (M+H)+=941.36 for C54H55FeN6O6, corresponding to 1:1 QN-
FPIX complexes. One simple interpretation of the data is that the three
peaks correspond to complexes between QN free base and FPIX with
deprotonated propionate side chains, QN free base and protonated FPIX,
and QN free base and protonated FPIX but including 13C natural
abundance for the 54 carbons, respectively.
Table 4
Changes in chemical shifts and magnetic moments (μ).
Proton #a/change in chemical shiftb (ppm)
CPD ID
2
3
5
6
7
8
μc
CQ
AQ
DHQN
DQQN
DADQQN
QN
−0.20
−0.11
−0.23
−0.03
0.00
−0.03
−0.03
−
−0.09
−0.06
−0.44
−0.04
0.00
−0.04
−0.04
−
−0.20
−0.19
−0.40
−0.07
0.00
−0.03
−0.04
−
−0.18
–
−0.14
−0.15
−0.17
−0.03
0.00
−0.03
−0.03
−
2.14
4.48
2.52
2.89
2.83
5.45
6.01
5.87
−0.06
–
–
–
–
–
–
–
−0.13
−0.04
−0.01
−0.03
−0.03
−
QD
HF
a
Quinoline protons are numbered according to the IUPAC numbering system of the
corresponding carbon atoms (see Fig. 6).
Fig. 4. IR spectra for the QN-heme adduct (W, dotted line), QN free base (X, dashed
line), hematin (Y, continuous line), and QN hydrochloride salt (Z, dash-dot line). A) The
broad peak at 2687–2400 cm−1 in the QN hydrochloride spectrum that corresponds to
the N–H stretch of the protonated quinuclidyl nitrogen is absent in the QN free base,
heme, and the QN-FPIX adduct spectra; B) the strong peak at 1702 cm−1 in the heme
spectrum that corresponds to the carbonyl stretch of the heme carboxylic acids is
absent in the QN free base, QN hydrochloride, and QN-FPIX adduct spectra.
b
Chemical shift difference between the corresponding proton in the presence and
absence of heme (ZnPIX in 40% DMSO-d6/D2O pH 7.0). The negative sign indicates an
up-field shift relative to the pure drug.
c
Measured magnetic moments for FPIX in 40% DMSO-d6/Phosphate buffer (0.1 M)
pH 7.0 in the presence of the corresponding drug.

J.N. Alumasa et al. / Journal of Inorganic Biochemistry 105 (2011) 467–475
473
A
B
QN-Heme Adduct
250
200
150
100
50
0
-50
250
200
150
100
50
0
-50
ppm
ppm
C
D
250
200
150
100
50
0
-50
250
200
150
100
50
0
-50
ppm
ppm
Fig. 5. Solid state 13C CP-MAS NMR spectra for QN free base (A), QN-FPIX adduct (B), QN hydrochloride salt (C), and QN dihydrochloride salt (D). The corresponding chemical shifts
for QN free base: δ 18.8, 28.8, 43.0, 53.5, 59.0, 70.8, 100.1, 113.8, 125.7, 131.4, 143.7,153.1,158.4; QN-FPIX adduct: 19.6, 28.5, 42.8, 58.9, 71.0, 101.1, 118.6, 131.5, 147.2, 158.4; QN
hydrochloride salt: 20.0, 27.9, 39.0, 45.1, 57.3, 62.1, 66.7, 86.4, 100.0, 118.5, 131.5, 144.3, 159.3; QN dihydrochloride salt: 19.6, 24.6, 31.2, 40.1, 45.5, 58.4, 65.2, 76.3, 104.5, 108.3,
117.2, 124.1, 143.2, 152.7, 159.1.
stretch (Fig. 4B). Thus, surprisingly, the isolated complex is most likely
QN free base complexed with fully ionized monomeric FPIX.
Solid state 13C NMR analyses of the precipitated adduct (Fig. 5B)
shows clear aromatic and aliphatic QN peaks and an obvious lack of
contact shifts previously observed for similar CQ-FPIX precipitates
[38]. In particular, there is only a 3 ppm shift in the peak at ~70 ppm,
which corresponds to the carbon bearing the QN hydroxyl group.
These data clearly indicate a non-covalent interaction between QN
and FPIX. Consistent with IR analysis, the pattern of adduct peaks is
most similar to the spectrum for QN free base (Fig. 5A), but differs
significantly relative to the spectra for the mono- and di-protonated
salts (Fig. 5C,D, respectively).
data, but includes important new information. The complex we isolate
does involve FPIX monomer, not dimer, has a 1:1 stoichiometry similar to
the conclusions of [22,27], and does involve interaction between the QN
hydroxyl O and the Fe center of FPIX, which is also consistent with [22–
27]. However, the data in this paper clearly shows that in aqueous
environments the interaction is non-covalent, eliminating models
wherein QN-FPIX interaction is stabilized by Fe–O or Fe–N covalent
(dative covalent) bonds. Also, the data in this paper show (surprisingly)
Thus, in aqueous solution, QN appears to preferentially bind to
ionized monomeric FPIX in the free base form. As shown in Fig. 6, this
complex must be non-covalent, with ring systems approximately 6 Å
apart. To further stabilize such a complex, and consistent with the
critical nature of cinchona alkaloid stereochemistry as well as our
results with QN analogues in this paper, we propose that a strong
hydrogen bond between the QN hydroxyl H and the quinuclidinyl N is
essential. Presumably due to interaction of the hydroxyl O with heme
Fe, the more polarized O–H bond of QN enhances this hydrogen
bonding.
Fig. 6. Proposed QN-FPIX adduct structure. The model involves coordination between
the –OH group of QN and Fe of FPIX, aided by the formation of a strong hydrogen bond
between the –OH proton and the quinuclidyl nitrogen, resulting in the formation of a
five-membered ring. Note also that in this model the quinoline ring is tilted away from
porphyrin ring system resulting in minimal π–π interactions. Further, the methoxy
group of QN is oriented in such a way as to be spatially close to the vinyl group as
observed in NOESY experiments with ZnPIX (data not shown).
4. Discussion
The data in this paper strongly support a model for QN-FPIX binding
depicted in Fig. 6. This model is consistent with some previously published

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J.N. Alumasa et al. / Journal of Inorganic Biochemistry 105 (2011) 467–475
that QN prefers to associate with ionized FPIX in the free base form. This
preference is likely a consequence of the stabilizing hydroxyl H/
quinuclidine N hydrogen bond geometry that we propose (Fig. 6).
Both CQ and QN are potent antiplasmodial drugs, both bind to heme
and inhibit Hz formation, but clearly their heme binding preferences
differ in interesting and important ways. In aqueous solution, CQ clearly
prefers binding to μ-oxo dimeric FPIX, whereas QN prefers binding to
monomeric FPIX. The QN:FPIX monomer species forms under aqueous
conditions, but is clearly more stable in either aggregate form (e.g. data
in this paper) or as a complex partitioned into the hydrophobic phase
[20]. Two points from the recent literature further illuminate the
pharmacologic significance of these conclusions. First, lower pH that
promotes aggregation of μ-oxo dimeric heme also appears to accelerate
Hz formation [21,31,39], as does the presence of lipid [31,39]. Lipid
nanospheres are found in the P. falciparum DV and nascent Hz crystals
appear to be associated with these. Thus, it is reasonable to propose that,
biologically, QN is most potent in preventing Hz formation as a free base
QN-monomeric FPIX complex either imbedded in lipid or existing as an
amorphous solid state aggregate stabilized by low DV pH. In constrast,
even though CQ is capable of interacting with monomeric FPIX via a
dative Fe-quinolinal N bond under specific conditions [38], it much
more strongly prefers to bind to μ-oxo dimer in aqueous solution.
Interestingly, data in Table 4, along with previous results [40], suggests
that AQ, which is slightly more potent than either QN or CQ, may inhibit
Hz formation by a combination of these two effects.
aggregated state or, alternatively, proceeds to HT dimer via dilute
monomeric FPIX. It is also possible that a non covalent π–π stabilized
heme dimer is a third intermediate in conversion to HT dimer, as has
been proposed by two other groups [41,42].
As diagramed in Fig. 7B-D, an alternate “linear” model is that μ-oxo
dimer FPIX is an obligate precursor to HT dimer. We suggest direct
conversion from μ-oxo to HT dimers within an acid and lipid stabilized
heme aggregate is the physiologically preferred route, as is consistent
with the known pH of the parasite DV [36], the presence of DV lipid
nanospheres [39], and the pH-dependent aqueous vs. lipid partitioning
behavior of FPIX [20,21]. Via the cyclic model (Fig. 7A) CQ and QN would
have opposing effects on stabilizing μ-oxo dimer vs. monomer,
respectively, but either of these effects could also drive equilibrium
away from HT dimer and hence inhibit conversion to Hz, if HT dimer
forms equally well from either monomer or μ-oxo dimer. However, we
argue that this predicts QN-CQsynergy that tothe best of our knowledge
has not ever been observed. Via the second model (Fig. 7B,C) the two
drugs simply operate at different points in linear conversion of
monomeric FPIX to Hz that must proceed in some fashion via μ-oxo
dimer. CQ again stabilizes the μ-oxo dimer, slowing conversion to HT
dimer and Hz, similar to earlier proposals (e.g. see [7]). In contrast, QN
slows conversion of monomer to μ-oxo, but not μ-oxo to HT. This model
is easily modified to include a π–π stabilized heme dimer [41,42]
between monomer and μ-oxo dimer, wherein CQ still shifts equilibrium
towards the μ-oxo dimer and QN shifts equilibrium towards the
monomer, with both binding phenomena preventing formation of a
non-covalent π–π stabilized heme dimer.
As shown in Table 4, AQ complexation with FPIX yields an
intermediate FPIX magnetic moment (4.48), as well as intermediate
effects on changes in the chemical shifts of AQ protons (e.g. −0.11 for the
proton at position 2, relative to −0.20 for CQ and −0.03 for QN). These
data, along with recalling that AQ has slightly higher potency in parasite
proliferation assays relative to either CQ or QN, lead us to suggest that AQ
is capable of both types of HT conversion inhibition. Meaning, we predict
that AQ: μ-oxo dimer complexes and acid aggregated AQ : monomer
complexes are both relevant for AQ inhibition of Hz formation. The
overall point is that any expectation that different quinoline antimalarial
drugs all show antimalarial activity via essentially the same molecular
mechanism is a simplification that neglects distinct molecular prefer-
ences for differentFPIXchemical forms. A caveat of this conclusion is that
different patterns of resistance to these drugs could be due, at least in
part, to the parasite manipulating bioavailability of different FPIX
chemical forms. This can be done rather easily in solution via subtle
changes in pH, ionic environment, or lipid composition.
These features can be incorporated into two simple models for
how the formation of Hz from free monomeric FPIX is inhibited by
different quinoline antimalarials (Fig. 7). The principle difference is
that in the first “cyclic” model (Fig. 7A) Hz is envisioned to be formed
from either monomeric FPIX or μ-oxo dimeric FPIX. It is well known
that acid aggregation of μ-oxo dimer accelerates formation of Hz
[6–9,39] but it is not known if μ-oxo dimer directly converts to head-
to-tail dimer (“HT dimer”, the unit cell of crystalline Hz) within the
Lastly, strong fluorescence observed for the QN-FPIX adduct (Fig.
S1) is an interesting property for QN-heme complexes not previously
observed to our knowledge. It is at least 7-fold higher than QN
fluorescence (heme is not fluorescent at this wavelength) and is likely
due to transition dipole ordering in the complex. We suggest this
fluorescence represents a convenient marker for further optimization
of quinoline drug activity.
Abbreviations
AQ
Amodiaquine
BHIA
CN
CQ
CQR
CQS
β-hematin Inhibitory Activity
Cinchonine
Chloroquine
Chloroquine Resistant
Chloroquine Sensitive
Fig. 7. The effect of CQ, AQ, & QN on the hemozoin (Hz) crystallization process. A)
Possible cyclic pathway for Hz crystallization. Both the monomer and μ-oxo dimer are
envisioned to be possible direct precursors to the head-to-tail (HT) dimer which then
crystallizes to Hz. Introduction of CQ shifts equilibrium away from both the HT dimer
and the monomer by promoting μ-oxo dimer. QN shifts equilibrium away from the HT
and μ-oxo dimers by promoting formation of the monomer. AQ interacts with both
monomer and μ-oxo dimer providing a possible rationale for this drugs' higher potency
in Hz inhibition relative to CQ/QN. B–D) Alternate linear pathway for Hz crystallization
envisioning that μ-oxo dimer is the direct precursor to HT dimer. B) Introduction of CQ
to the linear pathway shifts equilibria towards the μ-oxo dimer. C) QN shifts
equilibrium towards the monomer limiting conversion to μ-oxo dimer. D) AQ stabilizes
both monomer and dimer, which synergistically shifts equilibrium away from HT
dimer.
DADQQN Deamino-dequinuclidylquinine
DHQN
DQQN
DV
Dehydroxyquinine
Dequinuclidylquinine
Digestive Vacuole
DVpH
Steady state digestive vacuolar pH
ESI-MS electrospray ionization mass spectrometry
FPIX Iron Protoporphyrin IX

J.N. Alumasa et al. / Journal of Inorganic Biochemistry 105 (2011) 467–475
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Hb
HEPES
HF
Hz
PfCRT
Zinc Protoporphyrin IX
Hemoglobin
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
Halofantrine
Hemozoin
Plasmodium falciparum chloroquine resistance transporter
PfMDR Plasmodium falciparum multi drug resistance protein 1
PfNHE
QD
Plasmodium falciparum sodium proton exchange
Quinidine
Quinine
Quinine Resistant
Quinine Sensitive
QN
QNR
QNS
THF
VAR
tetrahydrofuran
Vacuolar Accumulation Ratio
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Acknowledgements
We thank Drs. Bahram Moasser and Shibu Abraham (Georgetown)
for the insightful discussions regarding the mass spectrometry results,
Dr. Dan McEleheny (UIC) for help with the solid-state NMR, and
Daniel P. Iwaniuk and Dr. Christian Wolf for their advice and help with
quinine analogue synthesis. L.B.C. gratefully acknowledges the
support of the AAUW Postdoctoral Fellowship.
Supported by NIH grants AI045957 (PDR) and AI060792 (AdD,
PDR and Christian Wolf, Georgetown University).
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