Biological Studies and Target Engagement of the 2- C -Methyl- d -Erythritol 4-Phosphate Cytidylyltransferase (IspD)-Targeting Antimalarial Agent (1 R,3 S)-MMV008138 and Analogs

Article abstract of DOI:10.1021/acsinfecdis.7b00159

Malaria continues to be one of the deadliest diseases worldwide, and the emergence of drug resistance parasites is a constant threat. Plasmodium parasites utilize the methylerythritol phosphate (MEP) pathway to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are essential for parasite growth. Previously, we and others identified that the Malaria Box compound MMV008138 targets the apicoplast and that parasite growth inhibition by this compound can be reversed by supplementation of IPP. Further work has revealed that MMV008138 targets the enzyme 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD) in the MEP pathway, which converts MEP and cytidine triphosphate (CTP) to cytidinediphosphate methylerythritol (CDP-ME) and pyrophosphate. In this work, we sought to gain insight into the structure-activity relationships by probing the ability of MMV008138 analogs to inhibit PfIspD recombinant enzyme. Here, we report PfIspD inhibition data for fosmidomycin (FOS) and 19 previously disclosed analogs and report parasite growth and PfIspD inhibition data for 27 new analogs of MMV008138. In addition, we show that MMV008138 does not target the recently characterized human IspD, reinforcing MMV008138 as a prototype of a new class of species-selective IspD-targeting antimalarial agents.

Full text of DOI:10.1021/acsinfecdis.7b00159

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Article
Biological studies and target-engagement of the 2-C-
methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD)-
targeting antimalarial agent (1R,3S)-MMV008138 and analogs
Maryam Ghavami, Emilio Fernando Merino, ZhongKe Yao, Rubayet Elahi, Morgan
Simpson, Maria Fernandez-Murga, Joshua Hayden Butler, Michael Casasanta,
Priscilla Krai, Maxim Totrov, Daniel J. Slade, Paul R. Carlier, and Maria Belen Cassera
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00159 • Publication Date (Web): 26 Oct 2017
Downloaded from http://pubs.acs.org on October 31, 2017
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Biological studies and target-engagement of the 2-C-methyl-D-erythritol 4-phosphate
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cytidylyltransferase (IspD)-targeting antimalarial agent (1R,3S)-MMV008138 and analogs
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Maryam Ghavami,† Emilio F. Merino,‡, ZhongꢀKe Yao,† Rubayet Elahi,‡ Morgan E. Simpson,‡
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Maria L. FernándezꢀMurga,‡ Joshua H. Butler,§ Michael A. Casasanta,‡ Priscilla M. Krai,‡ Maxim
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M. Totrov,¶ Daniel J. Slade,‡ Paul R. Carlier,*,† and Maria Belen Cassera*,‡,
†Department of Chemistry and Virginia Tech Center for Drug Discovery, Hahn Hall South, 800
West Campus Drive, Virginia Tech, Blacksburg, Virginia 24061, United States
‡Department of Biochemistry and Virginia Tech Center for Drug Discovery, Engel Hall, 340 West
Campus Drive, Virginia Tech, Blacksburg, Virginia 24061, United States
§Department of Biochemistry and Molecular Biology, and Center for Tropical and Emerging
Global Diseases (CTEGD), 120 Green St., University of Georgia, Athens, Georgia 30602,
United States
¶Molsoft LLC, 11199 Sorrento Valley Road, San Diego, California 92121, United States
*Correspondence:
Maria Belen Cassera, maria.cassera@uga.edu, Tel. 706ꢀ542ꢀ5192, FAX 706ꢀ542ꢀ1738
Paul R. Carlier, pcarlier@vt.edu, Tel. 540ꢀ231ꢀ9219, FAX 540ꢀ231ꢀ3255
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Malaria continues to be one of the deadliest diseases worldwide, and the emergence of drug
resistance parasites is a constant threat. Plasmodium parasites utilize the methylerythritol
phosphate (MEP) pathway to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl
pyrophosphate (DMAPP), which are essential for parasite growth. Previously, we and others
identified that the Malaria Box compound MMV008138 targets the apicoplast, and that parasite
growth inhibition by this compound can be reversed by supplementation of IPP. Further work
has revealed that MMV008138 targets the enzyme IspD in the MEP pathway, which converts
MEP and CTP to cytidine diphosphate methylerythritol (CDPꢀME) and pyrophosphate. In this
work, we sought to gain insight into the structureꢀactivity relationships by probing the ability of
MMV008138 analogs to inhibit PfIspD recombinant enzyme. Here, we report PfIspD inhibition
data for fosmidomycin (FOS) and 19 previously disclosed analogs, and report parasite growth
and PfIspD inhibition data for 27 new analogs of MMV008138. In addition, we show that
MMV008138 does not target the recently characterized human IspD, reinforcing MMV008138 as
a prototype of a new class of speciesꢀselective IspDꢀtargeting antimalarial agents.
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Key words: Plasmodium, malaria, MEP pathway, MMV008138, IspD, structure-activity studies
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Human malaria is caused by five species of Plasmodium. Around 250 million cases of
malaria occur every year with 95% of the infections caused by Plasmodium falciparum and
Plasmodium vivax.1 Malaria continues to be one of the deadliest diseases worldwide, and the
continued emergence of drug resistance is a constant threat. Therefore, efforts to identify and
characterize new leads for development of antimalarial drugs with different mechanisms of
action are still needed. In order to catalyze the development of new antimalarials, the Medicines
for Malaria Venture (MMV) and SCYNEXIS assembled the Malaria Box,2 an open access library
composed of 400 compounds originally identified by phenotypic screening of nearly 6,000,000
compounds from the research libraries of Saint Jude Children’s Research Hospital, Novartis,
and GlaxoSmithKline.3 Over 290 assays had been performed to screen the Malaria Box and
substantial information about these compounds is now available.4
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Malaria parasites contain a vestigial plastid called the apicoplast, which performs vital
functions such as the biosynthesis of isoprenoid precursors, fatty acids, and part of the heme.5ꢀ6
Plasmodium parasites utilize the methylerythritol phosphate (MEP) pathway to synthesize
isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are
essential for parasite growth.7ꢀ8 This pathway is absent in humans, who rely on the mevalonate
pathway instead. Recently, it has been suggested that the MEP pathway and the biosynthesis
of the isoprenoid precursors IPP and DMAPP represent the sole essential function of this
organelle during asexual intraerythrocytic development of the parasites9 as well as during
gametocytogenesis.10 The strongest support for this proposal stems from the observation that
both loss of the apicoplast function as well as loss of the organelle can be chemically rescued
by supplementing the growth medium with IPP. Therefore, the growth inhibitory effect of drugs
that directly target biosynthesis of isoprenoid precursors or indirectly disrupt their biosynthesis
by interfering with processes essential for apicoplast biogenesis, such as apicoplast DNA
replication, transcription, and protein translation, may be reverted by IPP supplementation.9 As a
result, the reversal of growth inhibition by IPP supplementation can be used as a phenotypic
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screening diagnostic to identify compounds that target the apicoplast, thus, identifying their
mechanism of action and narrowing their potential molecular targets.9 Previously, we identified
the drugꢀlike Malaria Box compound MMV008138 (Figure 1) using this method, and proposed
that the molecular target may be within the MEP pathway, especially because we noted that
MMV008138 did not present a delayed death phenotype and the apicoplast was not lost in the
presence of IPP, similar to fosmidomycin (FOS).11 Further analyses revealed that the active
stereoisomer of this molecule is (1R,3S)ꢀconfigured,12ꢀ13 and that its MEP pathway target is the
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cytidylyltransferase IspD (E.C.2.7.7.60) which converts 2ꢀCꢀmethylꢀDꢀerythritol 4ꢀphosphate
(MEP) and CTP to cytidine diphosphate methylerythritol (CDPꢀME) and pyrophosphate.12, 14
One of the most striking features of (1R,3S)ꢀMMV008138 (henceforth 1a, Scheme 1) is
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its speciesꢀselectivity toward the malarial IspD.12,
Though 1a was reported to inhibit P.
falciparum IspD with Ki values in the 7ꢀ13 nM range, it did not inhibit Arabidopsis thaliania,
Mycobacterium tuberculosis, or Escherichia coli IspDs at concentrations up to 10 ꢁM.12, 14 This
E. coli IspD (EcIspD) insensitivity is especially important since the MEP pathway is present in
the human microbiome.15 Therefore, unlike antibiotics such as doxycycline (DOX) that kill
malaria parasites by interfering with apicoplast’s genome expression,16 or FOS that inhibits 1ꢀ
deoxyꢀDꢀxylulose 5ꢀphosphate reductoisomerase (DXR) in the MEP pathway also affecting the
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gut microbiome,8,
compound 1a may not affect beneficial gut bacteria. Moreover,
doxycycline is used for prophylaxis in endemic areas with multidrugꢀresistance and in
combination therapies; however, it is not recommended for pregnant women and children under
the age of 8.20 Therefore, antimicrobial agents that are parasiteꢀspecific are ideally suited for the
development of malaria chemopreventive agents that are not toxic to pregnant women and
children and do not impact the human intestinal microbiome. Indeed, we previously confirmed
that 1a had no effect on E. coli growth at concentrations up to 500 ꢁM.13
Subsequent to our discovery of 1a as a MEPꢀpathway inhibitor,11 we prepared 34 close
structural analogs designed to probe P. falciparum growth inhibition structureꢀactivity
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relationships (SAR).13 We determined that in addition to (1R,3S)ꢀconfiguration, potent growth
inhibition required 2,4ꢀdisubstitution of the Dꢀring, featuring at least one electronꢀwithdrawing
substituent. For example, whereas 1a inhibited P. falciparum (Dd2 strain) growth with an IC50
value of 250 ± 50 nM, neither 1b (bearing an unsubstituted phenyl ring) nor 1u (bearing 2’,4’ꢀ
dimethyl substitution) inhibited P. falciparum growth at 10,000 nM (Figure 1). However, 2’ꢀchloro
substituted analog 1c showed weak growth inhibition, and 1e (2’ꢀchloroꢀ4’ꢀmethyl substituted)
nearly recapitulated the potency of 1a. The carboxy substituent at C3 of the C ring also proved
to be essential; replacement with CO2Me (3a) or H ((±)ꢀ11a) abrogated growth inhibition
potency. However, weak growth inhibition was restored with the first amide analog 6a, and
methyl amide derivative 7a proved equipotent to 1a.
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In the present work, we sought to gain insight into these structureꢀactivity relationships
by probing the ability of these compounds and new analogs to inhibit PfIspD recombinant
enzyme, the proposed parasite target of 1a. In this paper, we report PfIspD inhibition data for
FOS and 19 previously disclosed analogs, and report growth inhibition and PfIspD inhibition
data for 27 new analogs of 1a. As we will show below, these analogs show an excellent
correlation between P. falciparum Dd2 strain growth inhibition and PfIspD inhibition, confirming
the proposed antimalarial mode of action. In addition, we expanded the biological studies to
assess cytotoxicity and stage specificity profile of 1a.
RESULTS AND DISCUSSION
Structure-activity relationships. We first measured the kinetic parameters of recombinant
IspD (KmMEP = 12.0 ± 2.5 µM, kcatMEP = 7.6 ± 0.6 sꢀ1, KmCTP = 9.3 ± 2.5 µM, kcatCTP = 11.7 ± 1.2 sꢀ1)
using PhosphoWorks™ Fluorimetric Pyrophosphate Assay Kit (AAT Bioquest®, Inc.) which
directly measures pyrophosphate (PPi) released from the IspDꢀcatalyzed reaction (MEP + CTP
→ CDPꢀME + PPi,) (see Supporting Information, Figure S2). Previously reported kinetic
parameters of the substrate CTP for this construct were KmCTP = 59 µM and kcatCTP = 0.43 sꢀ1.14 In
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addition, Wu and colleagues reported kinetic parameters of the substrate MEP KmMEP = 61 µM,
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= 0.16 sꢀ1 using a histidineꢀ and maltose binding proteinꢀtagged PfIspD protein.12
MEP
kcat
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However, in both cases different detection assays (EnzChek Phosphate and Pyrophosphate
Assay Kits) were used to indirectly measure release of pyrophosphate through its subsequent
conversion to phosphate. After the kinetic parameters were determined, we first studied the
inhibitory properties of 1a, its three diastereomers 5a, entꢀ1a, entꢀ5a, and the antimalarial FOS
(Table 1). As was first reported by Wu et al,12 we confirmed that among the 4 stereoisomers of
MMV008138, (1R,3S)ꢀconfigured 1a is the most potent inhibitor of PfIspD (IC50 = 44 ± 15 nM).
Thus, the stereochemical dependence of P. falciparum growth inhibition potency correlates very
well with PfIspD target engagement. Furthermore, as expected, the IspC (DXR)ꢀtargeting
antimalarial FOS is a poor inhibitor of PfIspD (~4% inhibition at 10 ꢁM). We would also note that
the toxicological selectivity of 1a is much higher than that of FOS: whereas 1a has no effect on
E. coli growth at 2,000ꢀfold of its P. falciparum growth inhibition IC50 value, FOS shows an E.
coli MIC only 14ꢀfold higher than its P. falciparum growth inhibition IC50 value. This toxicological
selectivity is expected, since 1a does not engage EcIspD.12, 14 Interestingly, while (1S,3R)ꢀ
configured ent-1a and (1S,3S)ꢀconfigured 5a have no effect on E. coli growth at 250 µM and
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125 µM, (1R,3R)ꢀconfigured ent-5a inhibited 18 ± 6 % of E. coli growth at 250 µM.
As alluded to above, previously we observed a very tight Dꢀring SAR for analogs of 1a;
only analogs bearing at least one sterically small, electronꢀwithdrawing substituent at 2’ and/or
4’ were potent P. falciparum growth inhibitors (e.g. 1e-g).13 To further test this hypothesis, 17
new acid analogs of 1a were prepared and compared to previously synthesized analogs (Table
2). As can be seen, P. falciparum growth inhibition potency is retained in compounds 1hꢀl that
feature single replacement of the Cl atoms of 1a with F (1h, i), Br (1j, k), or both Cl atoms of 1a
with Br (1l). Within experimental error, compounds 1j and 1k are equipotent to 1a, but
compounds featuring 2’ꢀI,4’ꢀF, 2’ꢀF,4’ꢀI, or 2’ꢀBr,4’ꢀI substitution (1m-o) were less potent growth
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inhibitors. Nevertheless, compounds 1eꢀn all showed full rescue upon addition of 200 ꢁM IPP,
confirming their action as MEP pathway inhibitors. However, replacement of one of the Cl atoms
of 1a with OMe, OH or CO2H (1pꢀs) significantly reduces growth inhibition potency. Compounds
featuring very large electronꢀwithdrawing groups (1t) or electronꢀreleasing substituents at 2’,4’
(1u, 1v) are also poor P. falciparum growth inhibitors. A few compounds featuring 3’,4’ꢀ
disubstitution (1w, 1x), triꢀ and tetraꢀsubstitution (1y, 1z, 1aa), and heterocyclic modification
(1ab, 1ac,) were also explored, and found to be poor P. falciparum growth inhibitors. Two
modifications of the CꢀD ring junction of 1a were explored. Compound 1ad features the insertion
of a CH2 unit between the C and D rings of 1a, and this modification abrogated growth inhibition.
Secondly, preparation of a derivative of 1a that featured replacement of the C1ꢀH with methyl
was attempted via a ketone PictetꢀSpengler reaction with 2,4ꢀdichlorophenyl methyl ketone
(vide infra). Although the expected product of this reaction could not be isolated, compounds
1ae and 1af were successfully prepared by ketone PictetꢀSpengler reactions with 4ꢀ
chlorophenyl methyl ketone and acetophenone. However, these compounds also did not inhibit
growth of the parasite below 5 ꢁM. Thus, the new compounds synthesized reinforce our earlier
conclusion that potent growth inhibition by analogs of 1a requires 2’,4’ꢀdisubstitution of the Dꢀ
ring, featuring at least one electronꢀwithdrawing substituent.
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The PfIspD inhibition potencies of these compounds largely follow the same trend
(Tables 1, 2). Unsubstituted or monosubstituted Dꢀring derivatives (1bꢀ1d, IC50 = 510 to >5,000
nM) are weaker inhibitors than 1a (IC50 = 44 ± 15 nM). Close analogs of 1a featuring 2’,4’ꢀ
disubstitution with at least one small electronꢀwithdrawing group (1eꢀ1l) feature PfIspD IC50
values ranging from 31 – 260 nM, consistent with their potent growth inhibition (IC50 = 320 – 860
nM). Note that several potent compounds (1a, 1e, 1iꢀ1l) have PfIspD IC50 values close to the
nominal enzyme concentration in the assay (60 nM), and thus these values represent our
current best estimates. This nominal enzyme concentration represents a practical current lower
limit, based on the sensitivity of the assay (0.1 µM of PPi); if assay at lower nominal enzyme
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concentration could be performed, it is possible that the measured IC50 values of these
compounds would be lower. Therefore, differences in the measured PfIspD IC50 values of 1a,
1e, and 1iꢀ1l (31 – 100 nM) may not be reflective of actual differences in targetꢀsite affinity of
these compounds. Finally, as was seen for growth inhibition, replacement of the C2’ and C4’ Cl
groups of 1a with CF3, Me, or OMe (1tꢀ1v), or 3’,4’ꢀCl2 substitution (1w) significantly reduced
PfIspD inhibition. Thus, the antimalarial activity of Dꢀring analogs of 1a appears very well
correlated to the extent of their engagement with PfIspD. This tight SAR suggests that the Dꢀ
ring projects into a very wellꢀdefined cavity of PfIspD, and is not solventꢀexposed. The
requirement that at least one of the substituents at C2’ or C4’ must be a halogen (cf. 1a vs 1e/1f
vs 1u) is curious, and could be a consequence of a number of phenomena. It is possible that
halogenꢀbonding21 to one or more PfIspD backbone carbonyl groups contributes to affinity, as
was characterized crystallographically for the 1,3ꢀdihalobenzene moieties present in the
pseudillin inhibitors of A. thaliana IspD.22
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We then examined several of the carboxylic acid replacement analogs of 1a disclosed in
our earlier publication (3a, 6a, 7a, 7e, 11a), and six new ones (7iꢀ7k, 8a, 9a, 10a, Table 3). As
mentioned earlier methyl ester (3a) was a poor P. falciparum growth inhibitor, and as expected
this compound does not measurably inhibit PfIspD at 1,000 nM. The 1° amide 6a does inhibit
growth of P. falciparum in the 1 ꢁM range, and its potency to inhibit PfIspD is similar. Methyl
amides (7a, 7e, 7iꢀk) proved to be excellent replacements for the carboxylic acid group, both for
growth inhibition (IC50 = 190 – 506 nM) and for PfIspD inhibition (IC50 = 21 – 360 nM). With
respect to growth inhibition, 7j, 7k are equipotent to 1a (within error), and 7a, e, iꢀk all show
100% rescue upon supplementation with 200 ꢁM IPP. The methyl amides also show very weak
inhibition of E. coli growth at 250ꢀ500 ꢁM. Finally, the ethyl amide (8a), N,Nꢀdimethyl amide
(9a), 1° alcohol analog (10a) and racemic desꢀcarboxy derivative 11a showed poor growth and
PfIspD inhibition. Thus, within this limited series antimalarial activity is also wellꢀcorrelated to
PfIspD engagement. These data and those in Table 1 show that the carboxylate group of 1a
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plays a critical role in interacting with PfIspD; however, the nature of this interaction is not clear.
A purely electrostatic interaction of the carboxylate of 1a with PfIspD is ruled out by the potency
of methyl amide 7a. Yet the carboxylate cannot simply serve as a hydrogenꢀbond acceptor,
since the isosteric 1° amide 6a does not potently engage PfIspD. In any event, the carboxylate
and methylamide groups of 1a and 7a must project into a rather constricted pocket, since ethyl
amide 8a and N,Nꢀdimethyl amide 9a dramatically lose potency relative to methyl amide 7a.
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PfIspD inhibitor 1a is cytocidal in late trophozoite-early schizont stages but not in ring
stage. In our previous study we demonstrated that 1a (MMV008138) targets the apicoplast,
where it inhibits elongation and disturbs the mitochondrial membrane; these effects are
reversed upon IPP supplementation.11 However, the parasite’s development was arrested at the
early schizont stage, since DNA replication (assessed by Hoechst staining) was still observed
after 40 h of treatment with 2.5 µM of 1a.11 Therefore, to better assess if 1a is cytocidal and
parasite stageꢀspecific we determined the 50% lethal concentration (LC50) at 72 h using highly
synchronous cultures starting at ring stage and bolus incubation times ranging from 6 to 24 h. At
each indicated time, 1a was washed out and parasites were returned to culture to complete 72 h
growth, at which point growth was measured by SYBR Green assay (Figure 2). In parallel,
cultures were smeared and stained with Giemsa for stage assessment of 1aꢀtreated and control
parasites at the time that 1a was washed out. The SYBR Green assay at 72 h confirmed that
parasites were able to grow similarly to untreated controls following 6 and 12 h incubation with
1a, therefore, the LC50 values could not be determined. However, when incubation with 1a was
extended to the lateꢀtrophozoite/early schizont stage (18 h), the LC50 value was 1.20 ± 0.08 ꢁM.
When the drug incubation was extended to 24 h, the measured LC50 value further decreased to
0.46 ± 0.02 ꢁM, which was very close to the measured IC50 value at 72 h performed
simultaneously with these assays (0.35 ± 0.03 µM). The cytocidal action of 1a, therefore,
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manifests at 18ꢀ24 h, which is the time needed for parasites to progress from ring to late
trophozoite/early schizont stage. This transition marks the period of highest metabolic activity of
the MEP pathway, as discussed below (Figure 3). The absence of growth inhibition when
cultures were treated for only 6 or 12 h is interesting (Figure 2A), given that the MEP pathway is
known to be active through the entire asexual intraerythrocytic cycle.18, 23 Analysis of MEP
pathway intermediates at ring (12 h postꢀinvasion, hpi), trophozoite (19 hpi) and schizont (33 h
postꢀinvasion) showed that IPP concentrations increase markedly between 19 and 33 hpi
(Figure 3), which is in agreement with our cytocidal analysis showing that the measured LC50
value in cultures exposed for only 24 h was very close to the IC50 value measured at 72 h.
Cassera and coꢀworkers have previously shown that transcript abundance of genes involved in
the MEP pathway, and the isoprenoid products dolichol and ubiquinone all peak at the schizont
stage.23 Therefore, one potential explanation for the lack of growth inhibition observed at 72 h
when 1a was present for only 6 or 12 h is that isoprenoid products were present at a sufficient
level at the time that 1a treatment started to allow parasites to progress through ring and early
trophozoite stages until 1a was washed out.
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Methylamide derivative 7a inhibits CDP-ME formation in vivo better than the carboxylic
acid 1a. In 2015, we reported that the methylamide derivative 7a was equipotent to 1a for P.
falciparum growth inhibition,13 and in this study, we determined that their PfIspD inhibition
values are identical within experimental error (44 ± 15 nM and 57 ± 10 nM respectively, Table 1
and 3). As a further means to compare these compounds, we assessed in vivo IspD inhibition in
P. falciparum at schizont stage by quantitative liquid chromatographyꢀmass spectrometry (LCꢀ
MS/MS, Figure 4) in highly synchronous schizont stage parasites treated for 2 and 4 h with 1
µM 1a or 7a (~4 times the IC50 value). FOS was used as positive control at 10 µM (~10 times
the IC50 value). After 2 h of drug exposure, 7a caused 93 ± 5% decrease in the cellular levels of
the PfIspD product CDPꢀME, while 1a caused 67 ± 2% decrease. The 27% greater reduction
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caused by 7a was statistically significant by the BenjaminiꢀHochberg significance test. After 4 h
of treatments, 1a caused 84 ± 8% decrease in the levels of CDPꢀME while the reduction caused
by 7a remained the same. Interestingly, FOS treatment did not cause significant reduction of the
CDPꢀME levels after 2 h nor 4 h of drug exposure. As mentioned above, FOS does not inhibit
the enzymatic activity of recombinant PfIspD (Table 1). Previously, Zhang and colleagues
reported that parasites treated with 5 µM FOS for 10 h starting at ring stage presented
increased levels of MEP and an unknown metabolite, presumptively identified as 2ꢀCꢀ
methylerythrose 4ꢀphosphate, and reduced levels of CDPꢀME.24 Accumulation of MEP and the
potential 2ꢀCꢀmethylerythrose 4ꢀphosphate was unexpected and the authors hypothesized that
FOS may be indirectly inhibiting PfIspD through the intermediate 2ꢀCꢀmethylerythrose 4ꢀ
phosphate that accumulates following FOS treatment or through other control mechanisms that
remain to be identified. Because our experiments involved shorter treatments at a different P.
falciparum stage, the results cannot be directly compared; however, in both cases FOS induced
unexpected levels of metabolites downstream of its enzyme target DXR, suggesting that FOS is
triggering other control mechanisms that regulate the MEP pathway in the malaria parasite.
On the other hand, the reduction of the cellular levels of the methylerythritol
cyclodiphosphate (MEcPP) and the final MEP pathway product, IPP, did not show significant
differences among the three treatments after 2 h and 4 h of exposure to 1a, 7a and FOS as
assessed by the BenjaminiꢀHochberg significance test. However, the overall reduction of the
MEcPP and IPP levels was greater after 4 h of treatment with the three inhibitors where >90%
reduction of the IPP levels was achieved (Figure 4). Overall, these results are in agreement with
the results described above showing that 1a is cytocidal in late trophozoiteꢀearly schizont stages
where there is a higher demand of isoprenoid products such as dolichol and ubiquinone.23
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Human IspD is not targeted by 1a. Isoprenoid synthase domainꢀcontaining (IspDꢀlike) proteins
belong to a large family of glycosyltransferases conserved from bacteria to mammals and
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defective function of a IspDꢀlike protein disrupts glycosylation of αꢀdystroglycan causing
Walker–Warburg syndrome, a congenital muscular dystrophy accompanied by a variety of brain
and eye malformations.25ꢀ26 Recently, functional studies revealed that human IspD is a ribitolꢀ5ꢀ
phosphate cytidyltransferase.27ꢀ29 Because of the possibility that drugs directed against IspD
enzymes from pathogenic bacteria and parasites could also inhibit the human IspD enzyme
causing undesired side effects, the validity of IspD as drug target has been questioned.29ꢀ30 In
order to assess if 1a inhibits human IspD activity, its enzymatic activity was measured by LCꢀ
MS/MS to monitor CDPꢀribitol formation in the presence of 20 and 200 µM of 1a. The CDPꢀ
ribitol formation was not inhibited by the presence of 1a at either concentration (Figure 5). This
result was expected since 1a has been shown to be active against P. falciparum and P. vivax
but not bacterial or plant IspD homologues.12, 14 Therefore, our results support the validity of
PfIspD as drug target and the further development of this chemotype for malaria prophylaxis
and pharmacotherapy. As other PfIspD inhibitor chemotypes emerge, testing against hIspD will
help elucidate the structural basis of this important selectivity.
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METHODS
Synthesis of Analogs of 1a. Compound 1a and its Dꢀring variants 1bꢀae were prepared by
PictetꢀSpengler reaction of the requisite aldehydes with (S)ꢀTrpꢀOMe as described in Scheme 1.
The transꢀconfigured methyl ester intermediates 3aꢀac were separated from their cisꢀisomers by
column chromatography on silica gel (using dichloromethaneꢀethyl acetateꢀhexanes mixtures to
elute, as described in the Supporting Information). The relative configuration of each isomer was
determined by 13C NMR according to Cook’s method,31 and in every case examined thus far,
the cisꢀisomer eluted before the transꢀisomer. Compounds 1hꢀs, 1w, 1yꢀaf have not been
previously described. C1ꢀMethyl analogs 1ae and 1af were prepared by ketone PictetꢀSpengler
reaction32 with (S)ꢀTrpꢀOMe (Scheme 2). Note that application of this protocol to 2,4ꢀ
dichlorophenyl methyl ketone did not give the expected C1ꢀmethyl analog of 1a. Methyl ester
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3a, 1° amide 6a, methyl amides 7a, 7e, and (±)ꢀ11a were prepared as described previously.13
New methyl amides 7i-k were prepared by treating the corresponding methyl esters 3iꢀk with
methylamine in ethanol. Ethyl amide 8a and N,Nꢀdimethyl amide 9a were prepared by HATU
coupling of 1a with ethylamine and dimethylamine, respectively. The 1° alcohol analog 10a was
prepared by LiAlH4 reduction of 1a.
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P. falciparum Culture. Parasites Dd2 (MRAꢀ150) strain were maintained in O+ human
erythrocytes at 4% hematocrit in RPMI 1640 media supplemented with 2 g/L glucose (Sigmaꢀ
Aldrich), 2.3 g/L sodium bicarbonate (SigmaꢀAldrich), 50 mg/L hypoxanthine (SigmaꢀAldrich),
5.94 g/L HEPES, 20 mg/L gentamycin (GIBCO Life Technologies), and 5 g/L Albumax I (GIBCO
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Life Technologies). Parasites were kept at 37 C under reduced oxygen conditions (5% CO2,
5% O2, and 90% N2). Synchronous cultures in ring stage (>95%) were obtained by two cycles of
5% sorbitol treatment.
P. falciparum Growth Inhibition (IC50) and IPP Reversal of Growth Assays. The effects of
1a and analogs were evaluated against P. falciparum, Dd2 strain, by SYBR Green assay as
described previously in a 72 h assay since we previously reported that this class of apicoplastꢀ
targeting compounds do not present a delayed death phenotype.11 Studies were performed with
Dd2 strain since it is chloroquine resistant and we have previously showed that there is no
significant differences between resistant and sensitive P. falciparum strains.11 Briefly, ring stage
parasite cultures (100 µL per well, with 1% hematocrit and 1% parasitemia) were grown for 72 h
in the presence of increasing concentrations of the inhibitor under reduced oxygen conditions
(5% CO2, 5% O2, and 90% N2) at 37 oC. After 72 h in culture, parasite viability was determined
by DNA quantitation using SYBR Green I as described previously.33 The halfꢀmaximum
inhibitory concentration (IC50) values were calculated with GraphPad Prism (GraphPad
Software, Inc.) using nonlinear regression curve fitting. The reported values represent averages
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of at least three independent experiments performed in triplicate, using 10ꢀpoint serial dilutions,
with standard errors of the mean (S.E.M.). The range for serial dilutions was adjusted
accordingly for each analog after the first screening to set the IC50 value in the middle of the
concentration range.
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To establish reversal of growth inhibition by IPP, ring stage parasite cultures were grown
for 72 h in the presence of increasing concentrations of drug, and in the presence or absence of
200 µM IPP. The reported values represent averages of at least two independent experiments.
S.E.M. values are only indicated for mean values below 100%. The parasite’s growth inhibition
and recovery was assessed by SYBR Green as described previously.11
Cytocidal (LC50) Assay. In order to determine the concentration of a bolus dose of the reꢀ
synthesized 1R,3SꢀMMV008138 (1a) that kills 50% of parasites (LC50), P. falciparum (Dd2
strain) cultures were exposed to increasing concentrations of 1a and then the drug was washed
away at 6, 12, 18, and 24 h to also probe stage specificity for the activity of 1a as described
previously.34 Briefly, following the bolus dose incubation, plates were centrifuged at 700 x g for 3
min and 1aꢀcontaining medium was removed. Cell pellets were washed three times with 100 µl
of medium using the same centrifuge settings and then reꢀsuspended in 100 µl of media without
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the inhibitor. Washed plates were then incubated at 37 C to complete a total of 72 h after
setting the assays, and growth of surviving parasites was assessed by SYBR Green. The halfꢀ
maximum lethal concentration (LC50) values were calculated with GraphPad Prism software
(GraphPad Software, Inc.) using nonlinear regression curve fitting, and the reported values
represent averages and S.E.M. of at least three independent assays, with each assay
performed in triplicate. Parasites untreated or treated with 1a were smeared and stained with
Giemsa before washing the inhibitor in order to assess stage development at the time that 1a
was removed.
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E. coli Growth Inhibition Assays. In order to investigate the effect of selected compounds
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against E. coli, strain BL21(DE3), an overnight culture of E. coli cultivated at 37 C at 200 rpm
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agitation was diluted 100ꢀfold into LB broth medium and incubated to an OD600 of ∼0.6. The
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culture was then diluted 10,000ꢀfold into LB broth medium. Then, 750 ꢂL of this E. coli inoculum
was inoculated into a culture tube containing 750 ꢂL of the test compounds previously diluted in
LB broth medium at three final concentrations (500 ꢁM, 250 ꢁM, and 125 ꢁM). The final DMSO
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concentration was 5%. Cultures were incubated for 18 h at 37 C and 200 rpm agitation. The
following controls were performed: 100 ꢁM fosmidomycin (FOS) treatment which targets the
MEP pathway in E. coli, media without inoculum, 5% DMSO (vehicle), and control with inoculum
alone (untreated). After 18h incubation, bacteria growth was measured using a cell density
meter. The percentage of growth was normalized to that of untreated control bacteria and
potential inhibition of growth of 1a analogs was determined by comparison to the 5% DMSO
control, which does not affect E. coli growth, as we described previously.13 The data represents
the mean and S.E.M. of two independent experiments.
P. falciparum IspD Protein Purification and IspD enzymatic Assay Conditions. Plasmid
containing wildꢀtype PfIspD (pBG1869 PfIspD) as described in Imlay et al, was a gift from Dr.
Audrey Odom.14 pBG1869 PfIspD was transformed into Arctic Express (DE3) RIL E. coli cells
(Stratagene), followed by protein expression and purification as described previously (see
Supporting Information, Figure S1).14 The effect of 1a analogs on inhibiting PfIspD enzymatic
activity was measured using PhosphoWorks™ Fluorimetric Pyrophosphate Assay Kit (AAT
Bioquest®, Inc.) following manufacturer’s instructions.35ꢀ37 This fluorimetric assay directly
measures pyrophosphate (PPi) with a linear range of 0.3 to 30 µM (detection limit of 0.1 µM) as
described by the manufacturer. We measured PPi released in the enzymatic reaction catalyzed
by PfIspD (MEP + CTP → CDPꢀME + PPi). The reactions were performed in a solid black 96ꢀ
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well microplate in a final volume of 50 ꢁL (25 ꢁL test samples and 25 ꢁL assay solution)
according to the manufacturer’s protocol. Briefly, various concentrations of 1a analogs were
incubated for 10 minutes with 60 ꢁM CTP, 60 ꢁM MEP, 100 mM TrisꢀHCl (pH 7.4) and 1.6 mM
MgCl2. Reactions were initiated by adding 60 nM PfIspD. In assays with variable MEP, 100 µM
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CTP was used and in assays with variable CTP, 100 µM MEP was used. Fluorescence signal
was continuously monitored at 316 nm (Ex) and 456 nm (Em) using a Cytationꢀ5 multiꢀmode
plate reader (BioTek). The halfꢀmaximum inhibitory concentration (IC50) values were calculated
using GraphPad Prism software (GraphPad Software, Inc.) from a 7ꢀdata points curve from at
least three independent experimental replicates. The data represents the mean and S.E.M. A
reaction mix without PfIspD was performed in all assays and used for background subtraction.
The range for serial dilutions was adjusted accordingly for each analog after the first screening
to set the IC50 value in the middle of the concentration range.
Human IspD Protein Enzymatic Assay. Human IspD (hIspD) recombinant protein and its
substrate ribitolꢀ5ꢀphosphate were a generous gift from Dr. Lance Wells and Dr. Osman Sheikh
(University of Georgia).28ꢀ29 The effect of 1a on inhibiting hIspD enzymatic activity was measured
by LCꢀMS/MS to monitor CDPꢀribitol formation as described below for the detection of the MEP
intermediates.10 Briefly, the enzymatic assay was performed using 2 ꢁM hIspD, 50 mM TrisꢀHCl
(pH 7.2), 2 mM MgCl2, 2 mM DTT, 1 mM CTP and 1 mM ribitolꢀ5ꢀphosphate in the absence or
presence of 1a at 20 and 200 µM. Reactions were carried out at 37 ºC for 16 h. The reported
mean and S.E.M values were calculated from two independent assays.
MEP Pathway Metabolite Profiling. Mycoplasmaꢀfree parasite cultures were tightly
synchronized and grown to recover ring, trophozoite and schizont stages for MEP pathway
intermediates level assessment or early schizont stage (~27 h postꢀinvasion) for drug
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treatments and MEP pathway intermediates level assessment. Cultures in early schizont stage
were treated for 2 and 4 h with 0.1% DMSO (control), 1 µM 1a, 1 µM 7a or 10 µM FOS. In all
cases, parasites from infected erythrocytes were released from the host cell by lysis with 0.03%
saponin in cold PBS containing 2 g/L glucose and washed three times with PBS/glucose by
centrifuging 7 min at 10,000 × g at 4 oC. Metabolite extraction was performed by adding 250 ꢂL
per sample of iceꢀcold extraction solvent consisting of chloroform, methanol, acetonitrile (2:1:1,
v/v/v) containing 1 ꢂM isopentenyl Sꢀthiolodiphosphate (ISPP) as internal standard. Samples
were sonicated for 10 min in a water bath sonicator with ice. Then, 500 ꢂL of iceꢀcold MS water
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was added, mixed by vortex and centrifuged at 12,000 x g at 4 C for 4 min. The polar upper
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phase was transferred to a 10 kDa centricom tube and centrifuged at 12,000 x g at 4 C for 10
min. The flow through was lyophilized, resuspend in 120 ꢂL of iceꢀcold MS water and
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centrifuged at 12,000 x g at 4 C for 10 min. Samples were transferred to a total recovery vial
(Waters) and two injections of 40 ꢁL of each sample underwent LCꢀMS/MS analysis as
previously described using ionꢀpair reversed phase ultraꢀperformance liquid chromatography in
tandem with mass spectrometry (IPꢀRPꢀUPLCꢀMS/MS).38 Data were acquired using MassLynx
Software, v. 4.1, and processed using TargetLynx™ Application Manager (Waters). Relative
quantification of the level of each metabolite was performed by determining the analyteꢀtoꢀ
internal standard ratio (response) calculated by dividing the area of the analyte peak by the area
of the internal standard (ISPP) peak. The response of each detected metabolite is expressed as
the mean and S.E.M. of two biological replicates.
ACKNOWLEDGMENTS
We thank the National Institutes of Health (AI128362 (PRC), AI082581 (PRC), and AI108819
(MBC)), the Fralin Life Science Institute at Virginia Tech, and the Virginia Tech Center for Drug
Discovery, for financial support. The following reagent was obtained through the MR4 as part of
the BEI Resources Repository, NIAID, NIH: Plasmodium falciparum Dd2 strain, MRAꢀ150,
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deposited by D Walliker. We thank Dr. Audrey Odom (Washington University) for the gift of the
plasmid containing PfIspD for protein expression. We thank Dr. Lance Wells and Dr. M. Osman
Sheikh for the gift if the human IspD protein and ribitolꢀ5ꢀphosphate.
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ASSOCIATED CONTENT
Supporting Information
The following file (45 pages) is available free of charge on the ACS Publications website
doi:xxxx. Synthetic procedures and analytical characterization data for all the new compounds
described in the paper, SDSꢀPAGE analysis of the PfIspD used for enzyme inhibition studies
(Figure S1), and determination of kinetic parameters for PfIspD (Figure S2).
Abbreviations
MEP, methylerythritol phosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl
pyrophosphate; CTP, cytidine diphosphate; CDPꢀME, cytidine diphosphate methylerythritol;
MEcPP, methylerythritol cyclodiphosphate; FOS, fosmidomycin; SAR, structureꢀactivity
relationships; hpi, hours postꢀinvasion; IC50, halfꢀmaximum inhibitory concentration; LC50, halfꢀ
maximum lethal concentration.
Author Contributions
The study and experiments were designed by M.B.C, P.R.C., D.J.S., M.M.T., M.G., E.F.M., Zꢀ
K.Y., R.E., M.E.S., M.L.FꢀM., J.H.B., M.A.C., P.M.K. Experiments were conducted by M.G.,
E.F.M., ZꢀK.Y., R.E., M.E.S., M.L.FꢀM., J.H.B., M.A.C., P.M.K. All authors analyzed the data.
M.B.C., P.R.C., M.G., M.M.T., and D.J.S. wrote the manuscript, and all authors have read and
given approval to the final version.
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Cowan, N.; D'Alessandro, S.; Le Dang, N.; Delves, M.; DeRisi, J. L.; Du, A. Y.; Duffy, S.; Abd Elꢀ
Salam ElꢀSayed, S.; Ferdig, M. T.; Fernandez Robledo, J. A.; Fidock, D. A.; Florent, I.; Fokou,
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Figure 1. MMV008138 as originally disclosed, its active stereoisomer 1a ((1R,3S)ꢀ
MMV008138), and selected analogs.
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7
O
O
Oꢀ
Oꢀ
8
9
3S
+
+
NH2
NH2
C
Cl
Cl
B
A
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1R
2'
N
N
H
H
D
4'
4'
Cl
Cl
MMV008138
1a
(1R,3S)ꢀMMV008138
O
Oꢀ
R
3S
3S
+
NH
NH2
Cl
X
1R
1R
2'
2'
N
N
H
H
4'
4'
Cl
Y
R
Y
H
H
Me
Me
X
H
Cl
Cl
Me
1b
1c
1e
1u
CO2Me
CONH2
CONHMe
H
3a
6a
7a
(±)ꢀ11a
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Scheme 1. Synthesis of 1a and its Dꢀring analogs.
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Scheme 2. Synthesis of C1ꢀmethyl analogs of 1a.
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Table 1. PfIspD inhibition potency of MMV008138 stereoisomers and FOS.
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7
8
O
O
Oꢀ
Oꢀ
O
3S
3S
HO
+
+
C
NH2
NH2
P
N
H
HO
Cl
Cl
B
A
O
OH
1R
1S
9
2'
N
N
H
D
H
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Fosmidomycin (FOS)
1a
4'
5a
Cl
Cl
Compound
configuration
P. falciparum
growth
% Recovery (200
PfIspD IC50 (nM)d
% E. coli growth
inhibition (18 h)e
µM IPP)c
inhibition IC50
(nM)a
1a
5a
entꢀ1a
entꢀ5a
FOS
(1R,3S)
(1S,3S)
(1S,3R)
(1R,3R)
NA
250 ± 70b
>10,000b
>10,000b
3,000 ± 200b
880 ± 70b
100 @ 2.5 ꢁMb
44 ± 15
NI @ 500 ꢁMb
NI @ 125 ꢂM
NI @ 250 ꢁM
18 ± 6 @ 250 ꢂM
11.99 ± 0.02 ꢁM (MIC)
NDb
0% inh. @ 1 ꢁM
0% inh. @ 1 ꢁM
27 ± 5 % inh. @ 1 ꢁM
4 ± 2 % inh. @ 10 ꢁM
NDb
60 ± 5 @ 10 ꢁMb
100b
13
aMRAꢀ150, chloroquineꢀresistant (intermediate), pyrimethamineꢀresistant, mefloquineꢀresistant. Data reported previously. cDrug
at indicated concentration; ND signifies ‘not determined.’ Recombinant P. falciparum IspD IC50 values measured at [PfIspD] of 60
nM. Percent growth inhibition at the indicated concentration (NI signifies ‘no inhibition’), or the minimum inhibitory concentration
(MIC). Values represent averages of at least two independent assays ± S.E.M.
b
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e
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Table 2. Growth inhibition (P. falciparum, E. coli) and PfIspD inhibition of Dꢀring variants of 1a.
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Compounda
X
P. falciparum growth
inhibition IC50 (nM)c
>10,000d
% Recovery
(200 µM IPP)e
ND
PfIspD IC50 (nM)f
% E. coli growth
inhibition (18 h)h
NI @ 250 ꢁM
NI @ 250 ꢁM
NI @ 250 ꢁM
NI @ 500 ꢁMc
NI @ 500 ꢁMc
NI @ 500 ꢁMc
NI @ 250 ꢁM
NI @ 250 ꢂM
NI @ 250 ꢁM
NI @ 125 ꢁM
NI @ 125 ꢂM
NI @ 250 ꢁM
NI @ 125 ꢂM
ND
1b
1c
1d
1e
1f
1g
1h
1i
H
2’ꢀCl
4’ꢀCl
2’ꢀCl,4’ꢀMe
2’ꢀMe, 4’ꢀCl
2’,4’ꢀF2
>5,000g
~1,000
510 ± 90
82 ± 10
260 ± 50
230 ± 10
140 ± 30
100 ± 10
34 ± 11
31 ± 4
84 ± 14
140 ± 70
130 ± 20
ND
3,280 ± 990d
1,170 ± 60d
410 ± 40d
700 ± 90d
780 ± 175d
860 ± 80
60 ± 5 @ 10 µM
50 ± 7 @ 10 µM
100 @ 2.5 ꢁM
100 @ 2.5 ꢁM
100 @ 5 ꢁM
100 @ 5 ꢁM
100 @ 10 ꢁM
100 @ 5 ꢁM
100 @ 5 ꢁM
100 @ 10 ꢁM
100 @ 10 ꢁM
100 @ 10 ꢁM
80 ± 5 @ 10 ꢁM
ND
ND
ND
ND
ND
ND
ND
ND
ND
2’ꢀF, 4’ꢀCl
2’ꢀCl,4’ꢀF
2’ꢀCl, 4’ꢀBr
2’ꢀBr,4’ꢀCl
2’,4’ꢀBr2
2’ꢀI, 4’ꢀF
2’ꢀF, 4’ꢀI
2’ꢀBr, 4’ꢀI
2’ꢀCl, 4’ꢀOMe
2’ꢀOMe, 4’ꢀCl
2’ꢀCl, 4’ꢀOH
2’ꢀCl, 4’ꢀCO2H
2’,4’ꢀ(CF3)2
2’,4’ꢀMe2
2’,4’ꢀ(OMe)2
3’,4’ꢀCl2
3’,4’ꢀ(OMe)2
2’,6’ꢀF2,4’ꢀCl
2’,3’,4’ꢀF3
2’ꢀBr, 4’ꢀF, 5’ꢀOMe
na
na
na
na
na
433 ± 55
320 ± 60
360 ± 40
590 ± 20
970 ± 180
3343 ± 496
1,500 ± 200
>5,000
2,500 ± 600
>5,000
1j
1k
1l
1m
1n
1o
1p
1q
1rb
1s
1t
1u
1v
1w
1x
1y
1z
1aa
1ab
1ac
1ad
1ae
1af
ND
ND
ND
ND
ND
ND
ND
ND
0% inh. @ 10,000
>10,000d
>5,000g
~1,000
>5,000g
0% inh. @ 500 nM
ND
NI @ 250 ꢂM
NI @ 250 ꢂM
NI @ 250 ꢂM
NI @ 250 ꢁM
ND
ND
ND
ND
ND
ND
ND
70% inh. @ 10,000d
>20,000d
>10,000
>20,000d
1,800 ± 150
~ 20,000
>10,000
>10,000
> 5,000
>10,000
>10,000
>5,000
ND
ND
ND
ND
ND
ND
ND
ND
80 ± 8 @ 10 ꢁM
ND
ND
ND
ND
ND
ND
ND
ND
ND
aAll compounds are transꢀconfigured and derived from (S)ꢀTrpꢀOMe; all are (1R,3S) except for 1ac, which is (1S,3S) due to a Cahnꢀ
IngoldꢀPrelog priority switch. bThis compound was tested as an approximate 1:1 mixture of the (1R,3S) and (1S,3S)ꢀstereoisomers.
cMRAꢀ150, chloroquineꢀresistant (intermediate), pyrimethamineꢀresistant, mefloquineꢀresistant. Data reported previously. eDrug
13
d
f
at indicated concentration; ND signifies ‘not determined.’ Recombinant P. falciparum IspD IC50 values measured at [PfIspD] of 60
g
nM. Approximately 10% inhibition at 1,000 nM. hPercent growth inhibition at the indicated concentration; NI signifies ‘no inhibition.’
Values represent averages of at least two independent assays ± S.E.M.
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1
2
3
4
5
Table 3. Growth inhibition (P. falciparum, E. coli) and PfIspD inhibition of C3ꢀcarboxy
replacement variants of 1a.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compounda
X
P. falciparum
growth inhibition
IC50 (nM)b
6,800 ± 1400c
1,200 ± 100c
190 ± 30c
% Recovery (200
PfIspD IC50 (nM)e
% E. coli growth
inhibition (18 h)f
µM IPP)d
3a
6a
7a
7e
7i
2’,4’ꢀCl2
2’,4’ꢀCl2
2’,4’ꢀCl2
2’ꢀCl, 4’ꢀMe
2’ꢀCl, 4’ꢀF
2’ꢀCl,4’ꢀBr
2’ꢀBr, 4’ꢀCl
2’,4’ꢀCl2
0% inh. @ 1,000
~1,000
57 ± 10
360 ± 40
278 ± 27
21 ± 6
31 ± 4
~1,000
~5% inh. @ 1,000
ND
NI @ 250 ꢂM
NI @ 250 ꢂM
NI @ 500 ꢁMc
NI @ 500 ꢁMc
21 ± 3 @ 250 ꢂM
NI @ 250 ꢁM
NI @ 250 ꢁM
NI @ 250 ꢂM
NI @ 250 ꢂM
ND
20 ± 10 @ 20 µM
50 ± 7 @ 10 µM
100 @ 2.5 ꢁM
100 @ 2.5 ꢁM
100 @ 2.5 ꢁM
100 @ 5 ꢁM
100 @ 5 ꢁM
ND
340 ± 50c
506 ± 45
300 ± 40
340 ± 60
~5,000
7j
7k
8a
9a
10a
11a
2’,4’ꢀCl2
2’,4’ꢀCl2
2’,4’ꢀCl2
> 20,000
ND
ND
0 @ 20 ꢁM
>10,000
10,000 ± 1,600c
7% inh. @ 1,000
45 ± 10 @ 250 ꢂM
aAll compounds are transꢀconfigured, derived from (S)ꢀTrpꢀOMe, and are (1R,3S). MRAꢀ150, chloroquineꢀresistant (intermediate),
pyrimethamineꢀresistant, mefloquineꢀresistant. cData reported previously.
determined.’ eRecombinant P. falciparum IspD IC50 values measured at [PfIspD] of 60 nM. fPercent growth inhibition at the indicated
b
13
dDrug at indicated concentration; ND signifies ‘not
concentration; NI signifies ‘no inhibition.’ Values represent averages of at least two independent assays ± S.E.M.
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2
3
4
5
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7
8
9
10
11
12
13
14
15
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17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
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59
60
Figure 2. LC50 value of 1a decreases with increasing drug exposure time. (A) Parasite growth
was measured at 72 h by SYBR Green assay in cultures where 1a was washed out at 6 or 12 h,
and parasites were returned to culture to complete 72 h. A representative growth inhibition
curve at 72 h from parasites continuously exposed to 1a is also shown. (B) 1a becomes
cytocidal at 18 h drug bolus time when parasites reach lateꢀtrophozoite/early schizont stages.
The reported values represent averages and S.E.M. of at least three independent assays.
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