Chemo-immunotherapeutic antimalarials targeting isoprenoid biosynthesis

Article abstract of DOI:10.1021/ml4000436

We synthesized 30 lipophilic bisphosphonates and tested them in malaria parasite killing (targeting parasite geranylgeranyl diphosphate synthase, GGPPS) and human γδ T cell activation (targeting human farnesyl diphosphate synthase, FPPS). Similar patterns of activity were seen in inhibiting human FPPS and Plasmodium GGPPS, with short to medium chain-length species having most activity. In cells, shorter chain-length species had low activity, due to poor membrane permeability, and longer chain length species were poor enzyme inhibitors. Optimal activity was thus seen with ～C ₁₀ side-chains, which have the best combination of enzyme inhibition and cell penetration. We also solved the crystal structure of one potent inhibitor, bound to FPPS. The results are of interest since they suggest the possibility of a combined chemo/immuno-therapeutic approach to antimalarial development in which both direct parasite killing and γδ T cell activation can be achieved with a single compound.

Full text of DOI:10.1021/ml4000436

Letter
pubs.acs.org/acsmedchemlett
Chemo-Immunotherapeutic Antimalarials Targeting Isoprenoid
Biosynthesis
,†,‡,¶
Wei Zhu, ‡ Yi-Liang Liu, Hong Wang,⊥,¶ Ke Wang, Kai Li, Joo Hwan No,
§,¶
§,¶
‡
‡
#
Yonghui Zhang,*
#
∥
#
⊥
Lawrence Ayong, Anmol Gulati, Ran Pang, Lucio Freitas-Junior, Craig T. Morita,
,
‡,§
and Eric Oldfield*
†
PrenylX Research Institute, Zhangjiagang 215600, People’s Republic of China
‡
§
∥
Department of Chemistry, Center for Biophysics & Computational Biology, School of Molecular and Cellular Biology, University
of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States
⊥
Division of Immunology, Department of Internal Medicine, the Interdisciplinary Graduate Program in Immunology, University of
Iowa Carver College of Medicine, Veterans Affairs Health Care System, Iowa City, Iowa 52242, United States
#
Center for Neglected Diseases Drug Discovery, Institute Pasteur Korea, Seongnam-si, Gyeonggi-do 463-400, South Korea
S Supporting Information
*
ABSTRACT: We synthesized 30 lipophilic bisphosphonates and
tested them in malaria parasite killing (targeting parasite
geranylgeranyl diphosphate synthase, GGPPS) and human γδ T
cell activation (targeting human farnesyl diphosphate synthase,
FPPS). Similar patterns of activity were seen in inhibiting human
FPPS and Plasmodium GGPPS, with short to medium chain-
length species having most activity. In cells, shorter chain-length species had low activity, due to poor membrane permeability,
and longer chain length species were poor enzyme inhibitors. Optimal activity was thus seen with ∼C side-chains, which have
10
the best combination of enzyme inhibition and cell penetration. We also solved the crystal structure of one potent inhibitor,
bound to FPPS. The results are of interest since they suggest the possibility of a combined chemo/immuno-therapeutic approach
to antimalarial development in which both direct parasite killing and γδ T cell activation can be achieved with a single compound.
KEYWORDS: Bisphosphonates, immunology, inhibitors, malaria
alaria is a major cause of mortality and morbidity from
(FPPS). This results in accumulation of the FPPS substrates,
isopentenyl diphosphate (IPP), and dimethylallyl diphosphate
(DMAPP), both of which are phosphoantigens that activate γδ
1
M
parasitic protozoan diseases worldwide and drug
2
,3
resistance is of concern. There is thus interest in new
drugs and new drug targets, as well as unconventional
1
2,15
T cells.
However, zoledronate has essentially no effect on
4
approaches involving host innate immunity. Activated γδ T
the intraerythrocytic form of the malaria parasites since it is
poorly membrane permeable. In contrast, lipophilic bi-
sphosphonates (containing N-alkyl side-chains) do kill the
cells are of interest in this context since they can not only kill
5
6
7
tumor cells, bacteria and influenza virus-infected cells, γδ T
cells can also produce TNF-α on activation, and TNF-α is
known to prevent the development of pre-erythrocytic stage
14
parasites. Here, the target is the Plasmodium geranylgeranyl
diphosphate synthase (GGPPS). This enzyme is unusual in that
it is structurally more similar to human FPPS than human
GGPPS and, unlike human GGPPS, is potently inhibited by
bisphosphonates. Inhibiting GGPPS in the parasite blocks
8
parasites. One class of drug molecules called bisphosphonates
are known to activate γδ T cells (containing the Vγ2 Vδ2 T cell
receptor), so these molecules might be used as immunomo-
9
dulators. Most bisphosphonates are, however, poorly taken up
16
17
10
formation of protein prenylation as well as carotenoid,
into cells and bind tightly to bone mineral. Recently, we
18
19
11
menaquinone, and vitamin E formation, Figure 1, and
showed that lipophilic bisphosphonates did not bind to bone
1
0
results in direct parasite killing. Plus, this killing effect is
mineral and, in addition, were more active in γδ T cell
14
1
2
blocked by the addition of geranylgeraniol, confirming a
GGPPS target.
activation than were the current bisphosphonate drugs used
to treat bone resorption diseases and cancer. We also
discovered that lipophilic bisphosphonates were active in
Here, we sought to find a lipophilic bisphosphonate that
would kill malaria parasites as well as activate γδ T cells, a
possible new route to malaria chemo-immunotherapy.
13
killing liver stage malaria parasites and that a lipophilic
analogue of the bisphosphate zoledronate was a potent
inhibitor of intraerythrocytic Plasmodium both in vitro and in
1
4
vivo in mice.
Received: January 31, 2013
Accepted: March 4, 2013
Bisphosphonates such as zoledronate (1) activate γδ T cells
by inhibiting the enzyme farnesyl diphosphate synthase
©
XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
Table 1. Enzyme Inhibition Together with γδ T Cell
Activation and P. falciparum Cell Growth Inhibition
side-chain
length
(n, OH/H)
TNF-α
IC50
(μM)
HsFPPS
IC50 (μM)
PvGGPPS P. falciparum
ID
IC50 (μM)
IC50 (μM)
1
0, OH
1, OH
2, OH
3, OH
4, OH
5, OH
6, OH
7, OH
8, OH
9, OH
10, OH
11, OH
12, OH
13, OH
14, OH
15, OH
0, H
0.10
0.080
0.049
0.034
0.030
0.049
0.044
0.040
0.036
0.080
0.23
0.21
0.49
4.2
170
210
160
190
250
170
81
0.13
0.15
0.12
0.16
0.14
0.12
0.12
0.11
0.10
0.12
0.12
0.32
0.40
2.7
>400
>400
>400
>400
390
97
2
3
4
5
6
7
25
8
47
14
9
23
9.6
Figure 1. Schematic illustration of pathways involved in zoledronate−
analogue activity in γδ T cells and in malaria parasites. Green = human
cell; cyan = malaria parasite. HMG-CoA = hydroxymethylglutaryl
coenzyme A; IPP = isopentenyl diphosphate; DMAPP = dimethylallyl
diphosphate; FPP = farnseyl diphosphate; GAP = glyceraldehyl-3-
phosphate; HMBPP = 4-hydroxyl-3-methyl-but-2-enyl diphosphate;
GGPP = geranylgeranyl diphosphate; TNF-α = tumor necrosis factor
α.
10
11
12
11
5.9
7.6
8.0
8.8
110
160
280
140
210
180
200
280
160
56
3.6
4.1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
5.6
44
42
3.2
>400
>400
>400
>400
>400
>400
51
60
7.1
0.14
0.19
0.08
0.066
0.079
0.11
0.058
0.044
0.040
0.043
0.10
0.28
0.37
0.35
3.0
0.16
0.16
0.17
0.14
0.13
0.20
0.21
0.17
0.16
0.10
0.11
0.14
0.31
0.38
1.1
1, H
2, H
3, H
4, H
5, H
22
6, H
8.6
7, H
36
7.2
8, H
17
4.1
9, H
12
3.3
10, H
11, H
12, H
13, H
14, H
15, H
11
1.3
10
2.5
13
2.7
51
8.8
94
120
150
8.3
210
4.5
We synthesized the 16 pairs of zoledronate (1) species (1−
2) shown in Figure 2a in which we varied the length of the
3
alkyl chain (n = 0 through n = 15 carbons) and the presence or
absence of the 1-OH group that is involved in bone-binding
and that has been proposed (with zoledronate, 1) to be
1
0,20
important in γδ T cell activation.
Synthesis and character-
ization details are provided in the Supporting Information. We
then tested all 32 compounds for human FPPS inhibition
activity. The most potent FPPS inhibitors were those with
medium length side-chains, and these were ∼3−10× more
potent than zoledronate itself, Figure 2b and Table 1. As the N-
alkyl chain length increases beyond C , FPPS inhibition
10
decreases, due presumably to the onset of steric repulsion with
the highly conserved Phe 98 and 99 residues in the FPPS active
21
site that limit chain elongation.
We next investigated the effects of chain-length and the
presence/absence of the 1-OH group on γδ T cell activation, as
22
determined in a TNF-α release assay. As can be seen in Table
1 and Figure 2c, there is a monotonic increase in activity
Figure 2. Chain length dependence of enzyme and cell growth
inhibition/activation and effects of the 1-OH group. (a) Structures of
compounds investigated; (b) HsFPPS; (c) γδ T cell activation/TNF-α
release; (d) PvGGPPS inhibition; (e) intraerythrocytic P. falciparum
cell growth inhibition.
beyond C with both series of compounds with increasing chain
4
length up to n ≈ 11, then activity rapidly decreases with n > 12.
The decrease in activity with the longer chain species occurs at
a longer chain length in cells than in FPPS inhibition due, we
believe, to the importance of hydrophobicity with the more
lipophilic species, which facilitates cell entry. These results also
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Figure 3. Structural results in stereo representation. (a) X-ray structure of HsFPPS/5 complex (cyan, PDB ID code 4GA3) superimposed on
PvGGPPS structure (purple, PDB ID code 3RBM). The Cα rmsd over 331 residues in 1.44 Å. (b) Comparison between the X-ray structures of 5
bound to HsFPPS, GPP (yellow), and FPP (green) bound to avian FPPS (PDB ID codes, 1UBX and 1UBW). The bisphosphonate 5 binds to the
allylic (GPP) site. Chain elogation in FPP is blocked by F98 and F99, corresponding to decreased HsFPPS inhibition by bisphosphonate inhibitors
with N-alkyl chains longer than ∼C . (c) Structures of HsPPPS/5 (cyan) overlaid on 29 (BPH-703; pink) bound to PvGGPPS (PDB ID code
10
3
RBM). The bisphosphonate, imidazolium, and N-alkyl side-chain structures are quite similar. Optimum activity in PvGGPPS is at ∼C , then steric
1
1
repulsion ensures.
25
clearly show, at least with the γδ T cell lines we have used, that
there is no major difference in activity due to the presence or
absence of the 1-OH group.
chemical investigation show that the imidazole nitrogen in
zoledronate also carries a +1 charge (due there to protonation),
when bound to FPPS.
To see how these lipophilic zoledronate derivatives bound to
FPPS, we obtained the X-ray crystallographic structure of 5
We next investigated the inhibition of Plasmodium vivax
GGPPS (PvGGPPS) and the direct killing of intraerythrocytic
parasites by 1−32. As can be seen in Figure 2d,e and Table 1,
several of the compounds most effective in inhibiting
PvGGPPS were also very effective in inhibiting P. falciparum
growth. The correlation between PvGGPPS and cell growth
inhibition was poor (R = 0.26) but improved to R = 0.80 on
addition of the log P and solvation energy descriptors reported
(
IC ≈ 30 nM) bound to human FPPS (HsFPPS), as shown in
50
Figure 3a (in cyan; PDB ID code 4GA3). Full crystallographic
data acquisition and structure refinement details are given in
Supporting Information Table S1. Compound 5 binds into the
2
3,24
same site as does zoledronate
with its two phosphonate
2
+
groups bound to the [Mg ] cluster, and there is a 0.7 Å rmsd
between the [Mg ] , bisphosphonate, and imidazole rings in
3
2
+
26
previously. The ability to inhibit FPPS and GGPPS with the
3
the two structures. The alkyl chain extends into the GPP/FPP
side-chain site, Figure 3b (FPPS structures, PDB ID code
same chain length compounds is likely due to their mimicking
the FPP product (in human FPPS) or the FPP substrate (in
Plasmodium GGPPS), together with the presence of the third
Asp in PvGGPPS that is essential for bisphosphonate binding
1UBX and 1UBW). The origin of the more potent FPPS
inhibition by the N-alkyl bisphosphonates over that seen with
the unsubstituted species is likely due to an enhanced
hydrophobic interaction as opposed to a purely Coulombic
interaction since the results of a solid-state NMR and quantum
2+
to [Mg ] with, and as can be seen in Figure 3c, the structures
3
of 5 bound to HsFPPS and 29 bound to PvGGPPS being very
similar (rmsd = 0.9 Å).
C
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ACS Medicinal Chemistry Letters
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In conclusion, the results we have presented here are of
interest for a number of reasons. First, we constructed a library
of 31 N-alkyl analogues of the bisphosphonate drug,
zoledronate, with and without 1-OH groups, and tested them
ABBREVIATIONS
■
GGPPS, geranylgeranyl diphosphate synthase; FPPS, farnesyl
diphosphate synthase; TNF-α, tumor necrosis factor α; IPP,
isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate;
GPP, geranyl diphosphate; FPP, farnesyl diphosphate
(
and zoledronate) for activity in inhibiting human FPPS. The
results show that medium chain length species inhibit human
FPPS most potently, while longer chain species are inactive,
due, we propose, to a steric clash with the FPPS chain-length-
determining residues Phe 98 and 99. Second, we investigated
the activity of all 32 compounds in γδ T cell activation: the
most active species had 10 ± 1 carbons in the N-alkyl side-
chain. We propose that the increased activity of these lipophilic
zoledronate−analogue bisphosphonates in cells compared with
zoledronate itself is due to the improved cell uptake of the
more lipophilic compounds. Third, we determined the X-ray
crystallographic structure of one potent inhibitor of human
FPPS bound to the enzyme, finding that the bisphosphonate
and imidazole groups occupied the same position as in
zoledronate bound to FPPS, as well as 29 bound to GGPPS.
Fourth, we find that the most potent Vγ2 Vδ2 T cell activators
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■
Thermodynamics of bisphosphonates binding to human bone: a two-
Corresponding Author
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*
E-mail: yhzhang30@yahoo.com (Y.Z.); eo@chad.scs.uiuc.edu
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Author Contributions
¶
These authors contributed equally.
Funding
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