Sphingolipid analogues inhibit development of malaria parasites

Article abstract of DOI:10.1021/ml2002136

Plasmodium-infected erythrocytes have been shown to employ sphingolipids from both endogenous metabolism as well as existing host pools. Therapeutic agents that limit these supplies have thus emerged as intriguing, mechanistically distinct putative targets for the treatment of malaria infections. In an initial screen of our library of sphingolipid pathway modulators for efficacy against two strains of the predominant human malaria species Plasmodium falciparum and Plasmodium knowlesi, a series of orally available, 1-deoxysphingoid bases were found to possess promising in vitro antimalarial activity. To better understand the structural requirements that are necessary for this observed activity, a second series of modified analogues were prepared and evaluated. Initial pharmacokinetic assessments of key analogues were investigated to evaluate plasma and red blood cell concentrations in vivo.

Full text of DOI:10.1021/ml2002136

Letter
pubs.acs.org/acsmedchemlett
Sphingolipid Analogues Inhibit Development of Malaria Parasites
Esmeralda V. S. Meyer,† Jason J. Holt,§ Kathryn R. Girard,† Mark T. Ballie,‡ Anatoliy S. Bushnev,‡
Stacey Lapp,† David S. Menaldino,§ Richard F. Arrendale,§ G. Prabhakar Reddy,§ Taylor J. Evers,§
Randy B. Howard,§ Deborah G. Culver,§ Dennis C. Liotta,‡,§ Mary R. Galinski,†,∥
and Michael G. Natchus*,§
†Emory Vaccine Center and Yerkes National Primate Research Center, Emory University, Atlanta, Georgia 30329, United States
‡Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, United States
§Emory Institute for Drug Discovery (EIDD), 1515 Dickey Drive, Atlanta, Georgia 30322, United States
∥Emory University School of Medicine, Department of Medicine, Division of Infectious Diseases, Emory University, Atlanta, Georgia
30322, United States
S
* Supporting Information
ABSTRACT: Plasmodium-infected erythrocytes have been shown to
employ sphingolipids from both endogenous metabolism as well as existing
host pools. Therapeutic agents that limit these supplies have thus emerged as
intriguing, mechanistically distinct putative targets for the treatment of
malaria infections. In an initial screen of our library of sphingolipid pathway
modulators for efficacy against two strains of the predominant human
malaria species Plasmodium falciparum and Plasmodium knowlesi, a series of orally available, 1-deoxysphingoid bases were found to
possess promising in vitro antimalarial activity. To better understand the structural requirements that are necessary for this
observed activity, a second series of modified analogues were prepared and evaluated. Initial pharmacokinetic assessments of key
analogues were investigated to evaluate plasma and red blood cell concentrations in vivo.
KEYWORDS: Enigmols, sphingolipids, malaria, drugs, antimalarials, Plasmodium
suggest that the inhibitory effect begins in the ring stage of
the parasite's development.
ovel antimalarials are especially needed in the face of
Nwidespread drug resistance against Plasmodium falcipa-
alciparum and to provide alternative compounds for inclusion
in future combination therapies.1 Data have also emerged
demonstrating that the sphingolipid pool is an important factor
in modulating vital eukaryotic cellular functions, including
those of the protozoan genus, Plasmodium. In malaria
infections, membrane formation is critical for the growth and
development of the Plasmodium parasite inside host eryth-
rocytes. New membraneous structures are formed as the
parasite invades these host red blood cells (RBCs) with the
formation of a parasitophorous vacuole and then throughout
the RBC cytoplasm to create trafficking networks, critical for
the import and export of nutrients and waste products as the
parasite grows and multiplies through its development stages.2
The Plasmodium sphingolipid metabolic pathway is known to
have activity of several enzymes (e.g., sphingomyelin synthase,
glucosylceramide synthase, and two sphingomyelinases),3−6
and compounds such as fumonisin B7 or 1-phenyl-2-
palmitoylamino-3-morpholino-1-propanol (PPMP)5,8 have
been shown to interfere with the sphingolipid metabolism of
P. falciparum (see the Supporting Information for the structures
of these and other compounds that have been investigated in
the context of their effect on P. falciparum). Here, we show that
selected sphingoid base analogues inhibit the normal growth
and development of both P. falciparum- and Plasmodium
knowlesi-infected erythrocytes, and morphological studies
Since the mid-1990s, we have been designing sphingolipid
analogues to modulate the biosynthesis of sphingolipids, such
as sphingosine, to suppress cellular proliferation for use as
anticancer agents with a higher safety margin than those that
are currently available. Toward this end, we have compiled a
rationally designed library of synthetic analogues and, in doing
so, developed several synthetic routes toward this compound
class.9−12 Recently, other groups have also shown interest in
this area and have published additional synthetic routes toward
this class of compounds.13−18 Of particular interest to us are the
1-deoxy-5-hydroxy-sphingoid base analogues, since they retain
many of the physical characteristics of the natural sphingolipids
but lack a C-1 primary hydroxyl group. We hypothesized that
by trans-locating the C-1 hydroxyl group to the C-5 position,
we would maintain similar compound hydrophobicity and
enzymatic recognition while eliminating the possibility for
phosphorylation of the C-1 hydroxyl group by sphingosine
kinase (SK). This is desirable since the phosphate intermediates
formed by SK are subject to catabolic degradation and also
possess undesirable pro-mitogenic and antiapoptotic properties.
Received: September 1, 2011
Accepted: December 6, 2011
Published: December 6, 2011
© 2011 American Chemical Society
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Letter
Given the significant role(s) that sphingolipids may play in
the formation of membrane structures of the malaria parasite
growing in RBCs, we hypothesized that our existing lead
enigmol12 1 and its analogues may also have unique potential as
novel antimalarial agents. Enigmol was shown to specifically
interrupt the sphingolipid pathway in a pleiotropic manner19
and thus had the potential to impede development of the
parasite in the infected RBC. We selected enigmol and an initial
set of existing novel sphingolipid analogs to study the potential
of such compounds as anti-malarial therapies. The syntheses of
enigmol, and its analogs appearing in Table 1 were performed
[3H]-labeled hypoxanthine uptake method was utilized to assay
our analogues for their ability to inhibit the in vitro growth and
replication of malaria parasites in RBCs (see the Supporting
Information).22 Preliminary dose−response assays were con-
ducted using the W2 and D6 strains of P. falciparum, with
known relative chloroquine (CQ) resistance and sensitivity
values, respectively, in such assays.23 The simian malaria
parasite P. knowlesi has also been recognized as a human
pathogen of public health concern in South East Asia,24 and it
can be cultured effectively in vitro.25 This species was therefore
included to probe the potential of multispecies efficacy within
this compound class.
a
Table 1 shows comparative inhibitory concentration (IC50)
values for P. falciparum W2 and D6 strains and P. knowlesi when
tested for their sensitivity to enigmol and 10 selected analogues.
Our initial results showed that most analogues tested exhibited
antimalarial activity on both W2 and D6 strains at less than 13
μM. There was an observable structure−activity relationship
(SAR) between the compounds, ranging over roughly 1 order
of magnitude. This SAR was quantitatively similar for both P.
falciparum strains. Enigmol was found to have an IC50 between
7 and 12 μM, which is similar to the level of antiproliferative
activity that it displayed in various cancer cell lines. This is in
turn sufficient to result in in vivo efficacy in human cancer
xenograft cancer models. N-Methylation tended to increase
potency as compared to the parent compounds. N-Methyl-
enigmol (5) was particularly potent against all three malaria
parasites with potencies in the low micromolar range, and this
is within an order of magnitude in potency relative to the
known positive control, chloroquine, in the W2 strain. N-
Methyl analogues 3 and 9 also displayed increased potency,
albeit modest, as compared to the parent compounds; however,
this potency advantage was lost with the addition of a second
N-methyl group as observed with N,N-dimethylenigmol
analogue 6. Pyrrolidine analogue 7 constrains C1 and the C2
amine functionality into a ring and demonstrates that this
relatively severe modification is well tolerated. Fluorination of
the side chain, such as with analogue 8 and its N-methyl
equivalent, 9, is also well tolerated. N-Acylation of enigmol
(10) or the N-palmitoyl analogue (11), with ceramide-like
functionality, displayed no activity. Interestingly, in all of these
cases, the analogues were most effective against P. knowlesi and
the P. falciparum W2 strain (PfW2) (known for its relative
resistance to chloroquine), supporting our supposition that the
sphingolipid analogues may be functioning through mecha-
nisms different from those that impart chloroquine resistance.
Taken together, these data also support our hypothesis that
sphingolipid analogues may function by distinctive inhibitory
mechanism(s) providing potentially multiple drug targets for
future consideration. Finally, as an initial assessment of the
viability of these compounds as human therapies, we tested the
cytotoxicity of enigmol in human foreskin fibroblasts (HFF-1)
cells. We used the standard sulforhodamine B assay with a
density of 20,000 cells per plate and found that it was
noncytotoxic after 72 h at 10 μM incubation, but cytotoxic at
50 μM, thus providing a narrow but sufficient safety index for
an acute therapy such as the one we are targeting.
Table 1. First Generation Sphingolipid Analogue SAR Data
a
Pf, P. falciparum; Pk, P. knowlesi; NE, no effect; and NT, not tested.
In our preliminary examinations, morphological changes
were detected in the PfW2 strain after incubation of the
cultures with N-methylenigmol (5). The effect was evident
between 28 and 48 h of incubation, with a quantitatively high
percentage of various aberrant or stunted forms of ring-stage
and trophozoite-stage parasites found on Giemsa-stained thin
The error is represented as the standard deviation. Bold number,
average of two experiments.
as previously described (see Supporting Information for specific
approaches and experimentals).11,12,20,21 The highly reliable
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blood smears (Figure 1). These findings can be contrasted with
the normal growth, development, and maturation of the
Figure 1. Compound 5 (NME = N-methylenigmol) inhibits the
normal development of P. falciparum (W2 strain). IRBC samples taken
28 and 48 h postincubation with 5 were compared to untreated
parasites (RPMI). The arrows point to stunted ring-stage parasites at
28 h and to an iRBC-containing two trophozoites with an abnormal
vacuolated appearance at 48 h.
Figure 2. Enigmol shows plasma accumulation after five daily doses
and 3−4-fold higher levels in RBCs.
acute antimalarial treatments. Importantly, no signs or
symptoms of toxicity have so far been observed in any of our
in vivo studies.
parasites without the drug. By the 48 h time point, the
parasitemia increased from 1 to 5.8
0.5% with healthy
As we were preparing a second generation of analogues, it
was necessary to conjugate the compounds to bovine serum
albumin (BSA) to increase their solubility as described
previously26 for testing in the [3H]-labeled hypoxanthine
uptake assay. The results for enigmol and N-methylenigmol
shown in Table 2 indicate that the formation of BSA complexes
with our sphingolipid analogues provides for slightly greater
efficacy in the assay, presumably due to the resolution of
solubility challenges or aggregation barriers.
The design of the second generation of analogues was driven
in large part by the hypothesis that the primary amine at C2
plays a key role in shaping the metabolism and tissue
distribution properties of the analogues in vivo. Additional
steric bulk at C2 could potentially reduce in vivo N-acylation by
ceramide synthase and thus increase plasma concentrations of
the free amine. Analogues were therefore prepared that were
modified at C2 with a set of dimethyl and cyclopropyl
analogues that would eliminate the chirality of the center.
Analogues of this type were accessed via method B (Supporting
Information), and a specific example for analogues 16, 17, and
18 is presented in Scheme 1. The oxime analogue 22 was
available via reaction of 16 with HONH2HCl. The related
cyclopropyl analogues were prepared similarly using method B
and starting with cyclopropyl methylketone. Unfortunately,
elimination of chirality at C2 had a detrimental effect on the
stereoselectivity of the aldol reaction in both cases. However,
because our initial results (Table 1) seemed to show that the
absolute stereochemistry played a minimal role, we decided to
test the racemic mixtures. Optimization of the synthesis to yield
enantiopure materials is currently in progress.
appearing parasites in the control samples, as expected, while it
remained at 1.3 0.3% and with aberrant parasite forms in the
sample incubated with N-methylenigmol (Figure 1). The
relative percentages of rings, trophozoites, and schizonts in
the cultures with and without the 5 compound were 41.9, 39.2,
and 18.9% versus 87.9, 4.2, and 8.05, respectively. These data
reflect the fact that the parasites that were incubated with N-
methylenigmol did not progress with normal development,
while the untreated parasites progressed through schizogony
with a high percentage of newly invaded RBCs and normal
development of new young ring-stage parasites.
Initial pharmacokinetic studies previously reported by our
group12 suggest that enigmol and N-methylenigmol possess
attractive pharmacologic properties that provide sustained
plasma and tissue levels in the micromolar range. We are
therefore encouraged that compounds from this class could
potentially provide antimalarial efficacy in vivo, even given the
observed micromolar potency levels. Both compounds
displayed very large volumes of distribution, suggesting
substantial tissue accumulation. We believe that this might
offer a possible explanation for the fact that we observe
consistent pharmacological efficacy in vivo in mouse human
tumor xenograft studies with enigmol despite the fact that the
observed plasma levels from oral dosing are lower than the
cellular IC50.12 As an initial test of this hypothesis, mice were
dosed with enigmol once per day orally at 30 mg/kg, and drug
levels were followed in both plasma and RBCs on day 1 (data
not shown) and day 5 of dosing (Figure 2). We found that
plasma levels increased by about 30% from accumulation and
that drug concentrations in the RBCs were roughly 3-fold
higher than plasma levels at any given time, thus supporting the
notion that enigmol partitions into membranous tissues. At the
high dose (30 mg/kg), drug levels in RBCs approached the
IC50 of enigmol and were sustained in the micromolar range for
nearly 24 h, while plasma levels remained mostly in the
nanomolar range. Accordingly, we were encouraged that a
rational drug design effort could deliver a second generation of
agents with improved potency and/or higher distribution
properties into RBCs that would potentially provide effective,
The second generation of compounds was tested after being
complexed with BSA, and the observed in vivo potencies were
generally within the range that was observed with enigmol and
N-methylenigmol using the same method. In general, the
incorporation of an additional substituent at C2 was well
tolerated and exerted a minimal effect on potency. Interestingly,
conversion of C3 to an SP2-hybridized ketone moiety (16) was
also well tolerated and provided a slight potency advantage over
the SP3 analogues 17 and 18; however, conversion of this
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ACS Medicinal Chemistry Letters
Letter
evidence that further drug design and SAR investigations may
lead to increasingly potent compounds in this series.
Table 2. Second Generation Sphingolipid Analogue SAR
Data*
In this report, we have demonstrated that sphingolipid
pathway modulators of the type represented by enigmol and its
analogues can inhibit the growth and replication of multiple
species of Plasmodium in RBCs in standardized in vitro assays.
While further research in this area is required, data to date
suggest that the mechanism(s) of action may be unique from
any of the commonly employed antimalarials. Thus, these
compounds may represent a potential new class of therapies
that may prove invaluable for drug resistant malaria strains
either as single agents or in combination with others.
Importantly, the impressive pharmacokinetic and tissue
distribution profile of enigmol and N-methylenigmol gives us
confidence that we will be able to identify a compelling
compound from this family of molecules that will prove to be
safe, if in fact it is shown to be an effective treatment for
malaria. As a next step, additional research is critical to test
these compounds in vivo, to gain a deeper understanding of
their mechanism(s) of action, and to develop new generation
structures with increased potency.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic experimental details, analytical data of compounds,
and biological assay protocols. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 404-727-9157. E-mail: mnatchu@emory.edu.
■
Author Contributions
The manuscript was written through contributions of E.M.,
J.J.H., D.S.M., R.B.H., M.R.G., and M.G.N. All authors have
given approval to the final version of the manuscript.
*
All samples except chloroquine and mefloquine were tested as 1:1
molar complexes with BSA. The error is represented as the standard
deviation. Single experiment in triplicate wells.
a
Funding
We gratefully acknowledge financial support from The Emory
Institute for Drug Discovery, Emory University.
Scheme 1. Synthetic Methods for the Preparation of
Enigmol and Its Analogues
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The consulting input and assistance of John W. Barnwell, Ketan
Desai, and James Sikorski are gratefully acknowledged. We are
also grateful to Suzanne Mays for input on the design of the
biological testing protocols.
ABBREVIATIONS
■
RBC, red blood cell; IC, inhibitory concentration; BSA, bovine
serum albumin; PPMP, phenyl-2-palmitoylamino-3-morpholi-
no-1-propanol; SK, sphingosine kinase; PfW2, P. falciparum W2
strain; PfD6, P. falciparum D6 strain; DIP-CI, (−)-B-
Chlorodiisopinoctimpheylborane; DMEA, Dimethylethyl
amine; NaN3, sodium azide; NaBH4, sodium borohydride;
NBS, N-bromosuccinimide
center to the more metabolically stable oxime 22 reversed this
potency advantage. As with enigmol, N-methylation in this
series of compounds tends to boost the potency of the
analogues by roughly 1 order of magnitude. In the case of
compound 33, we observed an IC50 of 250 nM, which provides
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