From the Laboratory of Molecular Parasitology,
Department of Pathobiology and Center for Zoonoses Research, University
of Illinois at Urbana-Champaign, Urbana, Illinois 61802 and the
§ Department of Chemistry, University of Illinois at
Urbana-Champaign, Urbana, Illinois 61801
Received for publication, October 11, 2002, and in revised form, February 19, 2003
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ABSTRACT |
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We report the cloning and sequencing
of a gene encoding the farnesyl pyrophosphate synthase (FPPS) of
Trypanosoma brucei. The protein (TbFPPS) is an attractive
target for drug development because the growth of T. brucei
has been shown to be inhibited by analogs of its substrates, the
nitrogen containing bisphosphonates currently in use in bone resorption
therapy. The protein predicted from the nucleotide sequence of the gene
has 367 amino acids and a molecular mass of 42 kDa. Several sequence
motifs found in other FPPSs are present in TbFPPS, including an 11-mer
peptide insertion present also in the Trypanosoma cruzi
FPPS. Heterologous expression of TbFPPS in
Escherichia coli produced a functional enzyme that was
inhibited by several nitrogen-containing bisphosphonates, such as
pamidronate and risedronate. Risedronate was active in vivo
against T. brucei infection in mice (giving a 60% survival rate), but pamidronate was not effective. The essential nature of
TbFPPS was studied using RNA interference (RNAi) to inhibit the expression of the gene. Expression of TbFPPS
double-stranded RNA in procyclic trypomastigotes caused specific
degradation of mRNA. After 4 days of RNAi, the parasite growth rate
declined and the cells subsequently died. Similar results were obtained with bloodstream form trypomastigotes, except that the RNAi system in
this case was leaky and mRNA levels and parasites recovered with
time. Molecular modeling and structure-activity investigations of
enzyme and in vitro growth inhibition data resulted in
similar pharmacophores, further validating TbFPPS as the target
for bisphosphonates. These results establish that FPPS is essential for
parasite viability and validate this enzyme as a target for drug development.
The Trypanosoma brucei group of parasites causes
African trypanosomiasis (sleeping sickness) in humans, and nagana in
animals and is responsible for heavy socioeconomic losses in most
countries of sub-Saharan Africa (1). Therapy against African sleeping sickness is unsatisfactory because of the toxicity of currently available drugs, together with the development of drug resistance (2).
A number of bisphosphonates have recently been shown to have
significant activity against the proliferation of T. brucei
and other parasites in vitro (3, 4) and also have curative
effects in in vivo models of visceral (5) and cutaneous (6)
leishmaniasis. Bisphosphonates are pyrophosphate analogs in which the
oxygen bridge between the two phosphorus atoms has been replaced by a carbon substituted with various side chains. Several bisphosphonates are potent inhibitors of bone resorption and are in clinical use for
the treatment and prevention of osteoporosis, Paget's disease, hypercalcemia caused by malignancy, and tumor metastases in bone (7-10). Many bisphosphonates, such as pamidronate, alendronate, and
risedronate, are known to inhibit farnesyl pyrophosphate synthase (FPPS)1 (11-16), and in so
doing, they inhibit the formation of farnesyl pyrophosphate, a compound
used in protein prenylation and in the synthesis of dolichols,
ubiquinones, heme a, and sterols. Although a recombinant
FPPS from Trypanosoma cruzi has recently been shown to be
potently inhibited by bisphosphonates (17), the question as to whether
or not this enzyme is essential for trypanosomatid viability has
remained unresolved. Here, we report the results of RNA interference
(RNAi) studies together with in vitro and in vivo
inhibition studies that demonstrate that FPPS is indeed an essential
cellular component in T. brucei.
Materials--
Fetal bovine serum, Dulbecco's
phosphate-buffered saline, protease inhibitor mixture, geranyl
pyrophosphate (GPP), and isopentenyl pyrophosphate (IPP) were purchased
from Sigma. Fetal bovine serum (normal and dialyzed) was from Atlanta
Biologicals (Norcross, GA) and Serum Plus was from JRH Biosciences
(Lenex, KS). Restriction enzymes, T4 DNA ligase, Taq
polymerase, the Klenow fragment of DNA polymerase, Trizol reagent, and
0.24-9.5-kb RNA ladder were from Invitrogen. pCR2.1-TOPO
cloning kit, and Superscript II RT were from Invitrogen. Hybond-N nylon
membrane, PD-10 desalting column, and [ Culture Methods--
Procyclic forms of T. brucei,
strain ILTar 1, were grown in SDM-79 supplemented with 10% fetal
bovine serum (FBS). Procyclic form cell line 29-13 and bloodstream form
(cell line BF, with a single marker) co-expressing T7 RNA polymerase
and Tet repressor were gifts from G. A. M. Cross (Rockefeller
University) (21). Procyclic forms were grown in SDM-79 supplemented
with 15% FBS or dialyzed FBS in the presence of G418 (15 µg/ml) and
hygromycin (50 µg/ml). Bloodstream forms were grown in HMI-9 medium
(22) supplemented with 10% FBS or dialyzed FBS with 10% Serum Plus, and G418 (2.5 µg/ml). Cell densities were determined using a Neubauer chamber. Procyclic forms were diluted to 1 × 106
cell/ml and bloodstream forms to 0.5 × 105 cell/ml
and cultured in appropriate media. Growth curves were plotted by using
the product of the cell density and the dilution factor.
Cloning of the T. brucei FPPS Gene and DNA Sequencing--
A
fragment of 428 bp of the TbFPPS gene was amplified using
the oligonucleotide primers TbFPPS1
(5'-GGCTATTTGTTAGTGAGGCT-3') and TbFPPS2
(5'-CTTCTGGCGCTTGTAAGTCT-3') derived from a T. brucei genomic survey sequence (GenBankTM accession number
AQ651702) that showed pronounced similarity to FPPSs of other
organisms. Based on this sequence, we designed specific primers to
obtain the entire coding sequence using the 5'-rapid amplification of
cDNA ends system, version 2.0 (Invitrogen). The primer
TbFPPS2 was used for cDNA synthesis and the primers Tb-5'-SL (5'-AACGCTATTATTAGAACAGTTTCTG-3'),
complementary to the T. brucei 5'-spliced leader sequence,
and TbFPPS3 (5'-CCCACAGAGTTTCTACGCTG-3'), complementary to
sequences just upstream of the primer TbFPPS2, were used to
amplify the 5' end of the TbFPPS. The PCR was performed with
35 cycles of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 2 min, using 2.5 units of Pfu DNA polymerase, and cDNA as template, 200 pmol of each of the two primers, 1× Pfu
buffer, and 0.2 mM dNTPs in a total volume of 50 µl. For
3'-rapid amplification of cDNA ends, cDNA was synthesized using
oligo(dT) primer, and primers TbFPPS1 and oligo(dT) were
used to amplify the 3' end of the gene. Two oligonucleotide primers
based on the 5'- and 3'-untranslated regions of the TbFPPS
gene were then used to amplify the entire coding sequence, using
Pfu DNA polymerase and T. brucei genomic DNA as a
template. PCR products were cloned into the pCR2.1-TOPO and sequenced.
The predicted amino acid sequence of TbFPPS was aligned with the
sequences of other FPPSs by using the Biology Workbench 3.2 utility.2 The sequences
alignment and phylogenetic tree were done using CLUSTAL W (23) and
PHYLIP phylogeny packages (version 3.5c) (24) following the method of
Saitou and Nei (25).
Plasmid Constructs, Transfections, and Induction of RNAi--
A
fragment corresponding to the FPPS from T. brucei
(nucleotides 130-810) was amplified using the 5' primer
(CCGCTCGAGTTGGGCGGCAAATACAAC) and the 3' primer
(CCCAAGCTTGACTTTTCCTAGCCGCTC) having flanking XhoI and
HindIII sites, respectively. The resulting 680-bp fragment was cloned into the pCR2.1-TOPO, verified by sequencing, and ligated into the corresponding restriction sites of the pZJM vector (26). For
stable transfection of the procyclic form (cell line 29-13) and the
bloodstream form (cell line BF) via integration into a rDNA spacer
region, the vector was linearized by NotI digestion. Transfectants were selected using phleomycin (2.5 µg/ml) (18, 26).
The procyclic forms were cloned 1 day after transfection by limiting
dilution while the bloodstream forms were cloned immediately after
electroporation, by splitting cells into 24 wells of a microtiter plate. For induction of RNAi, cells were cultured in the presence of
tetracycline (1 µg/ml).
Southern and Northern Blot Analyses, and Reverse
Transcriptase-PCR--
Total genomic DNA from procyclic T. brucei was isolated by phenol extraction (27), digested with
different restriction enzymes, separated on a 1% agarose gel, and
transferred to nylon membranes. The blot was probed with a
[ Expression and Purification of TbFPPS from E. coli--
For
expression in E. coli, the entire coding sequence of the
TbFPPS gene was amplified by PCR. Oligonucleotide primers
for amplification of the FPPS coding region, ATG-TbFPPS
(5'-CTAGCTAGCATGCCAATGCAAATG-3') and TGA-TbFPPS
(5'-CCCAAGCTTCACTTCTGGCGCTTGTA-3'), were designed so that
NheI and HindIII restriction sites were
introduced at the 5' and 3' ends for convenient cloning in the
expression vector pET-28a+ to give pETbFPPS, which was
cloned and propagated in Escherichia coli DH5 Western Blotting and Generation of Antibodies against T. cruzi
FPPS--
Total trypanosome proteins (30 µg of protein/lane) were
subjected to SDS-polyacrylamide gel electrophoresis (10%).
Electrophoresed proteins were transferred to nitrocellulose using
a Bio-Rad Transblot apparatus. Membranes were probed with a 1:1,000
dilution of a rabbit anti-FPPS polyclonal antiserum prepared against
recombinant T. cruzi FPPS (17). Bound antibodies were
revealed by using goat anti-rabbit IgG (1:20,000) and the
ECLTM chemiluminescent detection kit (Amersham
Biosciences). To purify the T. cruzi FPPS antibodies, an
affinity matrix was prepared by coupling 5 mg of T. cruzi
FPPS to 1 ml of cyanogen bromide-activated Sepharose (Sigma). Immune
serum (3 ml) was diluted 2-fold with phosphate-buffered saline and
incubated with the affinity matrix overnight at 4 °C. Antibodies
were eluted with 100 mM glycine, pH 2.5, and the eluate was
neutralized with 0.1 volume of 2 M Tris-HCl, pH 8.0.
FPPS Assay and Product Analysis--
The activity of the enzyme
was determined by using the radiometric assay described previously
(17). One unit of enzyme activity is defined as the activity required
to incorporate 1 nmol of 4-[14C]IPP into
4-[14C]FPP in 1 min.
Treatment of T. brucei-infected Mice--
BALB/c mice (8 weeks
old) were infected intraperitoneally with 10,000 T. brucei
bloodstream trypomastigotes (monomorphic strain 427 from clone
MITat 1.4, otherwise known as variant 117). Starting the next day, mice
were then treated intraperitoneally with pentamidine or bisphosphonates
in 200 µl of 0.9% sodium chloride solution for the times and doses
indicated under "Results." Control mice received only the vehicle.
Molecular Modeling--
Using an avian FPPS x-ray structure as a
template (Protein Data Bank number 1UBW (29)), a homology model of
TbFPPS was built using default settings in the Modeler module of
Insight II (30). The majority of models constructed in this way were immediately discounted because of energy considerations and restraint violations, leaving a small group of structures that was further evaluated using the Procheck program (31). In the reported model, the
number of residues in the most favored region of the Ramachandran plot
and the overall G-factor were significantly better than typical for
2.6-Å resolution structures (91% and 0.0, respectively). This, together with the fact that the root mean square deviation of the
homology model from the template for non-loop atoms was only 0.41 Å,
lends confidence to the quality of the reported structure.
To model GPP in the TbFPPS active site, we transferred the GPP
coordinates from the template model into the TbFPPS structure. This
resulted in steric clashes between GPP and Tyr99, because
the template residue for Tyr99 in the avian crystal
structure is a F113S mutant and so provides little information about
the side chain geometry of Tyr99. To correct this, the
geometries of His98 and Tyr99 were therefore
optimized, because the template for His98 is also a mutant
(F112A) and hence also lacks important side chain information. Rotamer
searches for His98 and Tyr99 were performed to
minimize the GPP-Tyr99 and
His98-Tyr99 interactions. Hydrogen atoms were
added within Insight II to simulate a pH of 7.0, and GPP was taken to
be deprotonated. CHARMM27 potentials and partial charges were applied
to all atoms, except for the vinylic hydrogens in GPP, which were set
manually. Charges for GPP were obtained using the Gaussian 98 program
(32), using the x-ray coordinates of GPP to calculate
Merz-Singh-Kollman charges at the HF/6-31++G** level. The coordinates
of His98 and Tyr99 were then optimized by using
1000 steps of steepest descents minimization, then further optimized by
using the conjugate gradient algorithm to a gradient tolerance of
0.0001 kcal mol
To further understand the activities of the bisphosphonates
investigated (Fig. 1), we applied pharmacophore modeling techniques to
the TbFPPS inhibition results discussed later. The HipHop module in
Catalyst 4.6 (34) was used to identify the spatial relationships of the
chemical features common to the most active models. Compounds 1, 2, 3, 4, and
5 (Fig. 1) were used in pharmacophore generation, because
only these compounds were identified as "active" by Catalyst
default settings. Up to 255 best quality conformations of each compound
were built using the Confirm module in Catalyst 4.6. We allowed models
to contain two negative ionizable features, one positive charge
feature, and one custom endocyclic carbon atom feature, as input for
pharmacophore generation. Although HipHop did not identify any
pharmacophore that could map all four features in each compound, a
suitable hypothesis that did identify these features was found by
merging two HipHop models.
For comparison purposes, T. brucei cell growth inhibition
data (4) was used to build a quantitative pharmacophore model using the
Hypogen module in Catalyst 4.6 (34). For this model, we used the same
features as in the TbFPPS analysis, but pharmacophores found in this
way did not adequately describe the activities of the aliphatic
bisphosphonates. To account for these compounds, a hydrophobic
aliphatic feature was added to the Hypogen pharmacophore to describe
long chain compounds. The endocyclic carbon feature was modified to
also identify terminal methyl carbon atoms in short side chains (less
than 6 heavy atoms long), thus accounting for the increased potency of
olpadronate with respect to pamidronate. This pharmacophore gave
good correlation between predicted and experimental IC50
values, with R2 = 0.68 (data not shown).
Cloning of the TbFPPS Gene--
To screen for genes encoding FPPS
in T. brucei, the amino acid sequence of T. cruzi
FPPS (AF312690) was used to search the T. brucei data base
of The Institute for Genome Research (TIGR) using tBLASTn. This search
yielded six T. brucei genomic survey sequence clones. The
predicted amino acid sequence encoded by clone 6A5 (accession number
AQ651702) contained the C terminus of this protein. The fragment of the
T. brucei FPPS gene was amplified by PCR using
oligonucleotide primers complementary to the genomic survey sequence
with genomic T. brucei DNA acting as a template. The product
of the amplification (428 bp) was ligated into vector pCR2.1TOPO for
sequence analysis. This sequence enabled appropriate gene-specific
primers to be designed for the generation of 5'-end and 3'-end DNA
fragments using the 5'-rapid amplification of cDNA ends method (35)
and reconstruction of a full-length cDNA. The nucleotide sequence
of 2070 bp revealed an open reading frame of 1101 bp, which encodes a
367-amino acid protein with a predicted molecular mass of 42 kDa. Two
oligonucleotide primers based in the 5'- and 3'-untranslated regions of
this gene were then used to amplify the open reading frame from genomic
T. brucei DNA, to obtain a genomic clone. The open reading
frame was identical with the full-length cDNA. A BLAST search of
the protein data base showed that the amino acid sequence from T. brucei has 32-68% identity and 50-81% similarity with other
representative FPPS. The amino acid sequence of the T. brucei enzyme was aligned with the sequences of T. cruzi, human, and Saccharomyces cerevisiae FPPSs as
shown in Fig. 2A. All residues
involved in catalysis or binding (regions I-VII) identified in other
FPPSs (36) are present in the T. brucei enzyme.
A CLUSTALW alignment of FPPS peptide sequences from avian, some
mammalian, fungal, plant, and bacterial FPPSs was then used as the
basis for the generation of a phylogentic tree (Fig. 2B). The results suggested that T. brucei and T. cruzi
enzymes shared a most recent common ancestor. Plasmodium
falciparum and fungal and plant FPPSs were the closest outgroups
to the trypanosomatid sequences.
Southern blot analysis was performed with a PCR fragment that
encompasses the entire coding region of TbFPPS (Fig.
3A). Digestions with enzymes
that cut at sites not contained within the coding region of the gene
gave single bands (EcoRI, HindIII,
XhoI), whereas two bands were obtained with enzymes with
unique restriction sites in the open reading frame (KpnI,
SpeI, AvaI, HincII, NsiI),
suggesting that the gene was single copy per haploid genome.
Purification and Reaction Requirements of Recombinant
Protein--
TbFPPS was expressed in E. coli
BL21(DE3) as a fusion protein with an N-terminal polyhistidine tag.
Affinity chromatography on nickel-chelated agarose permitted a simple,
one-step purification. Enzyme purity was judged by using SDS-PAGE with
Coomassie Blue staining (Fig. 3B). The final protein
preparation catalyzed the incorporation of 4-[14C]IPP
into a hexane-extractable material when the allylic substrates, dimethylallyl pyrophosphate and GPP, were used (data not shown) (17).
4-[14C]IPP incorporation into the organic solvent
extractable material was linear with time for at least 60 min. The
radioactive assay was performed in the presence of different
concentrations of Mg2+ and Mn2+, to determine
their effect on the T. brucei FPPS. Mg2+ and
Mn2+ were added to the reaction mixture at concentrations
between 0.5 and 20 mM. As shown in Table
I, optimal levels of activity were
obtained by the addition of 5-10 mM Mg2+.
Enzyme activity was very low when the divalent cation was
Mn2+. The addition of 10 mM EDTA abolished FPPS
activity. Enzyme activity was also assayed between pH 6 and 10.5 using
a Tris-HCl (10 mM)/Tris glycine (10 mM) buffer.
Optimum activity was observed between pH 7.4 and 8.5 (Fig.
4A).
Kinetic Analysis--
Standard procedures were used to determine
kinetic parameters. Km and
Vmax values were obtained by a non-linear
regression fit of the data to the Michaelis-Menten equation (SigmaPlot
2000 for Windows). When the rate of FPP synthesis by the recombinant enzyme (10 ng) was measured in the presence of saturating IPP (47 µM) and varying GPP concentration between 0.15 and 200 µM, a Km value of 17.2 ± 3.9 µM and a Vmax value of 857.0 ± 8.7 units/mg, were calculated (Fig. 4B). When the
concentration of GPP was kept at 200 µM and the IPP
concentration was varied between 0.5 and 47 µM, the
Km value was 6.2 ± 0.6 µM and
the Vmax was 1,399.7 ± 1.3 units/mg (Fig.
4C).
Inhibition by Bisphosphonates--
Nine bisphosphonates
(1-9) together with the bromohydrin of
isopentenylpyrophosphate (Phosphostim, 10), shown in Fig. 1,
were tested for their ability to inhibit the T. brucei
enzyme. Ki values were calculated by using the Dixon
equation (37) and the IC50 values were obtained as described previously (17) (Table II). BPs
are known competitive inhibitors with respect to GPP of FPPSs of
different origins (15).
RNA Interference--
The essentiality of TbFPPS was
investigated by using RNAi. The inactivating fragment for RNAi was
inserted between the two opposing T7 promoters of the pZJM vector that
are both regulated by tetracycline repressors (18). Transfection of the
procyclic and bloodstream forms of T. brucei that express
the tetracycline repressor and the bacterial T7 RNA polymerase with
RNAi constructs directed against TbFPPS resulted in cells
that survived phleomycin selection. When production of double-stranded
RNA was induced by tetracycline in procyclic forms grown in medium
supplemented with 15% FBS, cell growth decreased as compared with
non-induced parasites after day two (Fig.
5A). When the induction of
RNAi was performed in a medium supplemented with 15% dialyzed FBS, instead of normal FBS, the parasites' growth rate declined after 2 days of tetracycline induction, and the cells subsequently died (Fig.
5B). Northern blot analysis of TbFPPS RNA from
cells grown with 15% dialyzed FBS in the absence or presence of
tetracycline for 24 h showed reduction of intact TbFPPS
RNA and a broad smear that probably represents degraded RNA (Fig.
5D). No mRNA could be found in these parasites as
analyzed by RT-PCR (Fig. 5E). Immunoblot analysis using an
affinity-purified antiserum raised against T. cruzi FPPS
(17) detected a band of 43.9 kDa in procyclic form lysates, which is
very close to the size predicted by its amino acid sequence (42 kDa).
This band was not detected in the RNAi-induced cells by day 4 (Fig.
5F, lane 3). Induction of TbFPPS
double stranded RNA in bloodstream forms caused an inhibition of cell
proliferation, but they still exhibited exponential growth even when
cultured in the presence of dialyzed serum (Fig. 5C,
circles). When mRNA levels were analyzed by Northern
blot (Fig. 5D) or RT-PCR (Fig. 5E), the target
mRNA was still detectable after addition of the antibiotic. Western
blot analysis also detected the presence of the TbFPPS protein (data
not shown).
Treatment of T. brucei-infected Mice with Bisphosphonates--
We
tested six bisphosphonates (1, 2, 3,
6, 7, and 8) for FPPS inhibition
activity on mice infected with T. brucei strain 427, variant
117. This strain gives an acute lethal infection in mice 5-6 days
after infection with 10,000 trypomastigotes. Risedronate was first
tested at doses of 5 mg/kg for 5 days and 10 mg/kg for 5 days, giving one dose per day. There was no significant protection against death
except for some delay in the death of the animals at a 10 mg/kg for 5 days (one mouse died at day 7, one at day 8, one at day 9, one at day
12, and one at day 17; all controls died at day 6) (Fig.
6). Increasing the dose to 10 mg/kg for 7 days or 15 mg/kg for 5 days resulted in toxicity, as indicated by
excessive panting and horripilation, and the animals died at the same
time as the controls. However, using a split dose regime (2× 5 mg/kg per day) for 5 days there was a 60% survival (one mouse died at day 7, another at day 14, whereas 3 mice survived more that 3 months without
parasitemia). All mice treated with the standard drug pentamidine (4 mg/kg for 4 days) survived more than 3 months without parasitemia.
Compounds 1, 2, 7, and 8 were also investigated at a dose of 10 mg/kg for 5 days (single or
split dose regiment) but there was toxicity, and no protection against
death was observed. Pamidronate (10 mg/kg intraperitoneal × 5 days) was also ineffective.
Homology Modeling--
We used the sequence information of TbFPPS
together with the known three-dimensional x-ray crystallographic
structure of the avian FPPS enzyme (29) to obtain the homology model of
the TbFPPS protein shown in Fig.
7A. In both the T. brucei and T. cruzi proteins, there is an 11-mer
insertion sequence (residues 184-194 in the T. brucei
sequence), which is shown highlighted in yellow on the left of Fig. 7A. In addition, the T. brucei sequence contains a second insertion, shown in
yellow on the right of Fig. 7A. The functions of these loops are not known. However, it is of interest to
note that of some 20 FPPS investigated, only the trypanosomatid parasites and, apparently, Plasmodium falciparum (38)
contain such insertions, and it is possible that these loops may play a
role in FPPS function in these organisms. As can be seen in Fig. 7,
this sequence and homology modeling information can also be used to
construct a three-dimensional picture of the conserved nature of each
residue in TbFPPS (Fig. 7B). Here, we show a color-coded "consensus" picture of different FPPSs: the most highly conserved residues are coded mauve, followed by decreasing
conservation: dark pink > light pink > blue > cyan > white.
The GPP substrate is docked into the hybrophobic cleft or active site
region, which for the most part is highly conserved across species
(mauve color). There are, however, some interesting
differences between the trypanosomatid and other FPPS sequences
investigated, in particular, the FF or YF residues located toward the
distal end of the hydrophobic pocket of most FPPSs (at positions 98 and
99) are changed to HY in both the T. cruzi and T. brucei proteins (and to SF in P. falciparum), offering
some potential for the development of novel inhibitors that might
hydrogen bond to these residues.
Structure-Activity Investigation--
To begin to carry out a
three-dimensional structure-activity relationship investigation of
TbFPPS inhibition by the compounds investigated, we utilized the
CatalystTM technique (34). The CatalystTM
program basically explores a wide range of conformations (up to 255)
for a given molecule and correlates particular spatial features with
observed experimental activity. In this way, a pharmacophore can be
generated that contains specific chemical features (such as aromatic
groups, positive charges, hydrogen bond donors/acceptors, and negative
ionizable groups) in specific locations, which correlate with activity.
When large amounts of Ki information are available,
a quantitative structure-activity relationship model can be developed,
whereas for a smaller data set, such as that we have obtained, the
pharmacophore contains the major features of importance in enzyme
inhibition, but is less suitable for quantitative predictions. Using
this approach (the HipHop feature in CatalystTM) we
obtained the pharmacophore shown in Fig.
8, A-C. This
pharmacophore contains four features of key importance in drug
activity: two negative ionizable groups (the bisphosphonates, shown in
blue); an endocyclic carbon (green) and a
positive charge (red). These features are shown superimposed on the
stick diagram structures of two of the more active species:
3-picolylaminomethylene bisphosphonate 1 (Fig.
8A) and risedronate (3, Fig. 8B). The
pharmacophore is also shown docked into the active site of TbFPPS
(+GPP) in Fig. 8C.
Next, we used the CatalystTM approach to develop a
pharmacophore for in vitro T. brucei growth
inhibition, using data obtained previously (4), to compare with the
enzyme inhibition results. In this case, test results for some 26 compounds are available, which enables use of the more quantitative
Hypogen feature of CatalystTM. Using Hypogen, we obtained a
pharmacophore for T. brucei growth inhibition that is quite
similar to that obtained for TbFPPS enzyme inhibition (obtained using
HipHop). This similarity can clearly be seen in Fig. 8D in
which we show, superimposed, the pharmacophores for enzyme inhibition
and for T. brucei cell growth inhibition. The T. brucei growth inhibition model contains an additional custom hydrophobic feature to account for, in a quantitative manner, the
activity of the long chain n-alkyl bisphosphonates (4).
The sensitivity of trypanosomatid protozoa to isoprenoid
biosynthesis inhibitors (39) offers a unique opportunity for drug target identification and the subsequent development of new
anti-trypanosomatid agents. FPPS plays a central role in metabolism
through the enzymatic generation of FPP, which is used for protein
prenylation, and for the synthesis of sterols, dolichols, heme
a, and ubiquinone, and is potently inhibited by
bisphosphonates (11-16). Stringent genetic validation of putative drug
targets is desirable before the rational design of inhibitory compounds
intended for chemotherapeutic use is undertaken (40). This study
validates FPPS as a drug target through the use of RNAi studies. It
provides genetic evidence that FPPS plays an essential cellular role in
T. brucei and demonstrates that the enzyme is vital for
parasite survival in vitro and in vivo. The
finding that a similar pharmacophore can be obtained by
structure-activity investigations of in vitro growth and
enzyme inhibition data further validates TbFPPS as the target of bisphosphonates.
The T. brucei FPPS gene, TbFPPS, was shown to be
present in the T. brucei genome and encodes for a functional
enzyme. As shown in the alignment in Fig. 2A, there is
considerable sequence similarity between different FPPSs. Heterologous
expression of TbFPPS in E. coli resulted in the
production of a recombinant enzyme that was generally similar to other
FPPSs with respect to its Mg2+ requirement, optimum pH, and
sensitivity to bisphosphonates. As shown in Table I and Fig.
4A, the protein we have expressed has optimum activity in
the presence of 0.5-10 mM MgCl2 and in the pH
range 7.4-8.5, basically as found with the human enzyme (41). In
addition, our results show that TbFPPS is potently inhibited by a
number of bisphosphonates (Table II). The most potent inhibitor found
is the aminomethylene bisphosphonate 1, having a
Ki of 7 nM, followed closely by its
isomer 2 and risedronate (3), both of which have
Ki = 10 nM. The next most potent
inhibitors found were the drugs incadronate (4,
Ki = 11 nM), the isoquinoline species
(5, Ki = 58 nM), and
pamidronate (6, Ki = 400 nM).
The general pattern of reactivity found here is similar to that found
for bisphosphonate inhibition of a recombinant human FPPS, where the
activity profile is again risedronate > incadronate > pamidronate (42). Likewise, the picolyl aminomethylene bisphosphonate 2 has been found to be a potent (IC50 = 25 nM) inhibitor of a plant FPPS (11) and this species is also
a potent inhibitor of bone resorption (43), consistent with activity
versus human FPPS. The imidazo(1,2-H)pyridyl
species 9 is relatively inactive, consistent with its poor
performance as a bone anti-resorptive agent (19), presumably because of
unfavorable steric interactions in the TbFPPS active site.
In addition to these investigations of aromatic bisphosphonate
inhibitors, we also tested whether aliphatic, nitrogen-free bisphosphonates might act as inhibitors of TbFPPS, because these compounds have been found to have activity against the growth of both
T. cruzi and Entamoeba histolytica, in
vitro (44, 45). The bisphosphonates 7 and 8 contain simple n-alkyl side chains containing 10 and 11 carbon atoms, and were found to have Ki values of
0.57 and 1.3 µM, respectively. These Ki values are still quite small and approach those
found with second generation bone resorption drugs such as pamidronate (Ki = 0.40 µM, Table II). Indeed, in
very early work, Shinoda et al. (46) proposed that species
such as 7 and 8 might be used in treating bone
resorption diseases. Presumably, although these species do not contain
the ammonium or pyridinium-like features associated with the most
potent inhibitors, they do provide an enhanced hydrophobic interaction
(van der Waals dispersion) in the active site of the enzyme, resulting
in at least moderate activity. The importance of this effect is also
seen on transition from pamidronate to olpadronate to ibandronate (47),
where successive N-alkylations and chain elongations result
in a potent FPPS inhibitor, having an IC50 of 20 nM against a human recombinant FPPS (42). The slightly
increased Ki for the longer chain species is not
inconsistent with this idea, because it has recently been shown by the
Novartis group (48) that increasing the side chain length of
N-containing bisphosphonates beyond a certain threshold basically abolishes all activity, most likely because they clash with
the aromatic rings located at the distal end of the hydrophobic pocket,
thought to be responsible for termination of isoprene chain elongation
by FPPS (29). In any case, it is of interest that the simple alkyl
species 7 has essentially the same activity as pamidronate.
We also tested the activity of one pyrophosphate species, the
bromohydrin of isopentenylpyrophosphate, 10. This compound is currently being developed as an anti-cancer drug and is known to
stimulate the growth and cytokine expression of This leads us finally to consider the potential use of these and
related compounds in chemotherapeutic intervention. Here, our initial
results indicated that procyclic (insect form) trypomastigotes were
extremely sensitive to the effects of RNAi, which generated a lethal
phenotype. In the bloodstream forms, RNAi only slowed growth, and was
not lethal. However, this was because of the leakiness of the vector in
these forms, because the mRNA was not completely eliminated after
induction of cells with tetracycline. Consequently, we carried out a
series of in vivo tests of selected bisphosphonates (1, 2, 3, 6, 7,
and 8) versus T. brucei-infected mice.
The most effective compound tested was risedronate (3),
which afforded a 60% cure (survival of 3/5 mice) at a dose of 2 × 5 mg/kg per day for 5 days, intraperitoneally (Fig. 6). This clearly
demonstrates that bisphosphonates can effectively suppress parasite
proliferation in vivo as well as in vitro and that bisphosphonates may thus be useful lead compounds for the synthesis of new drugs effective against African sleeping sickness.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP (3000 Ci/mmol) were obtained from Amersham Biosciences. [4-14C]IPP (57.5 mCi/mmol) was from PerkinElmer Life
Sciences. The pET-28a+ expression system and
BenzonaseTM nuclease were from Novagen (Madison, WI).
Nickel-nitrilotriacetic acid-agarose was obtained from Qiagen
(Valencia, CA). Pfu DNA polymerase was from Stratagene (La
Jolla, CA). The pZJM vector was a gift from Paul Englund (Johns Hopkins
University, Baltimore, MD) (18). Oligonucleotides were synthesized at
Integrated DNA Technologies, Inc. (Coralville, IA). All other reagents
were analytical grade. Bisphosphonates and the bromohydrin
pyrophosphate (Fig. 1) were synthesized
using standard methods (3, 19, 20) and were characterized by C/H/N
microanalysis and 1H NMR spectroscopy.
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Fig. 1.
Structures of bisphosphonates (1-9) and
the bromohydrin of IPP (phosphostim, 10).
-32P]dCTP-labeled TbFPPS. After
hybridization, the blot was washed three times in 2× SSC, 0.1%
SDS at 65 °C (SSC is 0.15 M NaCl, 0.015 M
sodium citrate). For the Northern blot analysis, total RNA was isolated
from procyclic and bloodstream forms using Trizol reagent. RNA samples
were subjected to electrophoresis in 1% agarose gels containing 1×
Mops buffer (20 mM Mops, 0.08 M sodium acetate, pH 7.0, 1 mM EDTA) and 6.29% (v/v) formaldehyde, after
boiling for 10 min in 50% (v/v) formamide, 1× Mops buffer, and 5.9%
(v/v) formaldehyde. The gels were transferred to a Hybond-N filter and hybridized with a probe containing the entire coding sequence of the
TbFPPS gene obtained by PCR. All Southern and Northern blots
were visualized by autoradiography. A fragment of the
-tubulin gene
was used as a control in Northern blots (26). Densitometric analyses of
Northern blots were performed by using a Kodak Digital Science Image
Station 440 CF. Comparison of levels of TbFPPS transcription between non-induced and induced cells was performed by taking as a
reference the densitometric values obtained with the TbFPPS transcripts from non-induced parasites. To analyze the level of TbFPPS mRNA in cells grown in the presence or absence of
tetracycline, total RNA was also isolated from equal cell numbers and
used for reverse transcriptase (RT)-PCR analysis using SuperScript II
RT and the same set of primers that were used to amplify the fragment (680 bp) that was cloned into the pZJM vector.
.
Double-stranded DNA sequencing was performed to confirm that the
correct reading frame was used, with the polyhistidine tag placed in
the N-terminal position. Subsequently, pETbFPPS was used to transform
E. coli BL21(DE3). Bacterial clones were grown in LB medium
containing 50 µg/ml kanamycin. When induction was performed,
bacterial cells transformed with pETbFPPS were first grown
to an A600 of 0.6 at 37 °C and then 1 mM isopropyl-
-D-thiogalactoside was added.
After 5 h of growth at 37 °C, cells were pelleted by centrifugation and resuspended in lysis buffer (5 mM
imidazole, 300 mM NaCl, 250 mM sucrose, 50 mM Tris-HCl, pH 7.2), incubated with 10 mg/ml lysozyme
for 15 min on ice, then sonicated. The lysate was incubated with
BenzonaseTM nuclease for 15 min on ice, then
centrifuged at 16,000 × g for 15 min. The
supernatant was mixed with nickel-nitrilotriacetic acid-agarose for
1 h at 4 °C, then loaded onto a column and washed with lysis
buffer in which the concentration of imidazole increased from 15 and 25 mM. Protein was eluted from the column with the same buffer
but containing 500 mM imidazole. The eluted fraction was
desalted with a PD-10 desalting column. Proteins were determined by the
method of Bradford (28) with bovine serum albumin as a standard and
analyzed for purity by SDS-polyacrylamide gel electrophoresis.
1 Å
1. This structure was
then uploaded to the Consurf
server3 and Rasmol (33) was
used to visualize the three-dimensional locations of highly conserved
residues in the TbFPPS model.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Sequence analysis of the FPPS from
T. brucei. A, comparison of the
deduced amino acid sequence of T. brucei with other FPPSs.
The deduced amino acid sequence of T. brucei FPPS
(GenBankTM accession number AY158342) is compared with the
sequences of T. cruzi (AF312690), human (P14324), and
S. cerevisiae (J05091) synthases. Similar residues are
shaded. The seven conserved regions I to VII are
underlined. B, unrooted tree based upon FPPS
amino acid sequences. The bar indicates a branch length
corresponding to 0.2 substitutions per site. Distances were calculated
using the Neighbor Joining (NJ) method of Saitou and Nei (25).
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Fig. 3.
Southern blot analysis and purification of
FPPS from E. coli. A, total genomic DNA was
digested with different endonucleases. The DNA fragments were separated
in 1% (w/v) agarose, transferred to a nylon membrane, and
hybridized with a 32P-labeled probe corresponding to the
FPPS coding sequence. B, a SDS-polyacrylamide gel was
stained with Coomassie Brilliant Blue. Lane 1, crude extract
from pET-28a+-transformed cell; lane 2, crude
extract of E. coli BL21(DE3)/pETbFPPS; lane 3,
soluble fraction from extract of E. coli BL21(DE3)/pETbFPPS;
lane 4, nickel column purified fraction.
Effect of divalent cations on FPPS from T. brucei
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Fig. 4.
Effect of pH and substrate concentration on
FPPS activity. FPPS activity was measured as described under
"Experimental Procedures" over a range of pH between 6 and 10.5 using Tris-HCl and Tris glycine buffers (A), and in the
presence of different concentrations of GPP (B) or IPP
(C). Insets in B and C
represent the linear transformation, by double reciprocal plot, of each
curve.
The effects of bisphosphonates on FPPS activity
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Fig. 5.
Effects of inhibition of TbFPPS expression by
tetracycline-inducible double-stranded RNA on cell growth.
Procyclic form RNAi constructs grown in medium supplemented with 15%
FBS (A) or 15% dialyzed FBS (B). Cells were
grown in the absence (diamonds) or presence of tetracycline
(1 µg/ml) (circles). C, bloodstream forms grown
in medium supplemented with 10% FBS or 5% dialyzed FBS and 10% Serum
Plus in the absence (triangles and diamonds,
respectively) or presence of tetracycline (1 µg/ml)
(circles). D, Northern blot analysis of
TbFPPS RNA from procyclic and bloodstream forms
(BSF) grown in the absence ( ) or presence (+) of
tetracycline (Tet). Total RNA was subjected to gel
electrophoresis before transfer to a nylon membrane, then hybridized
with the 32P-labeled probe corresponding to the FPPS coding
sequence. As a control, these blots were also reprobed with
-tubulin. E, RT-PCR analysis of total RNA from procyclic
(Pro) and bloodstream forms using primers specific for the
TbFPPS. The cells were grown in the absence (
) or presence (+) of
tetracycline and dialyzed FBS for 24 h. F, immunoblot
analysis of T. brucei procyclic form lysates (30 µg of
protein/lane) from uninduced parasites on days 0 (lane 1)
and 4 (lane 2) of the culture, and tetracycline-induced
cells for 4 days (lane 3).
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Fig. 6.
Survival data for T. brucei
brucei-infected mice. Squares, control group
(infected, untreated); triangles, treated group, 10 mg/kg
intraperitoneal, ×5; circles, treated group, 2× 5 mg/kg
per day intraperitoneal, for 5 days.
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Fig. 7.
Molecular models of TbFPPS.
A, TbFPPS homology model based on template avian x-ray
structure of FPPS + GPP (Protein Data Bank number 1UBW). The leftmost
insertion (loop) shown in yellow has only been found in
protozoan parasites. B, Consurf model illustrates sequence
variability. The color coding illustrates conservation in the following
order: white (highly variable) < cyan < blue < light pink < dark
pink < mauve (highly conserved).
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Fig. 8.
Pharmacophore models for TbFPPS inhibition
(A-C) and T. brucei
cell growth inhibition (D). In
A, the HipHop pharmacophore is shown superimposed on
1, in B on risedronate (3), in
C it is docked onto GPP in the TbFPPS active site.
D shows the HipHop pharmacophore superimposed on the Hypogen
pharmacophore for T. brucei growth inhibition (data from
Ref. 34). Graphics were generated with the Accelrys ViewerLite program
(www.accelrys.com).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
T cells (49),
but its precise molecular mechanism of action is unclear. Because FPPS,
in addition to its direct role in FPP synthesis, is also known to be
involved in binding to the tyrosine kinase domain of the fibroblast
growth factor receptor (50) and affects cell growth, it seemed possible
that this alternative role of FPPS might also occur in
T cells,
mediated by pyrophosphate (and indeed, bisphosphonate (51, 52))
binding. We therefore determined the Ki for the
bromohydrin pyrophosphate, 10. We obtained a
Ki = 103 µM, which would tend not to
support this possibility.
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ACKNOWLEDGEMENTS |
---|
We thank G. A. M. Cross (Rockefeller University) for generously providing trypanosome strains 29-13 and BF, P. Englund (Johns Hopkins University) for the RNAi vector pZJM, and Linda Brown for technical assistance. Preliminary sequence data for the T. brucei genomic survey sequence (GenBankTM accession number AQ651702) was obtained from The Institute for Genomic Research website.
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FOOTNOTES |
---|
* This work was supported in part by grants from the World Health Organization Special Programme for Research and Training in Tropical Diseases (to R. D. and to E. O.), the American Heart Association, National Center (to R. D.), and United States Public Health Service National Institutes of Health Grant GM-65307 (to E. O. and R. D.). The sequencing work was supported by an award from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Laboratory of Molecular Parasitology, Dept. of Pathobiology and Center for Zoonoses Research, College of Veterinary Medicine, University of Illinois, 2001 S. Lincoln Av., Urbana, IL. Tel: 217-333-3845; Fax: 217-244-7421; E-mail: rodoc@uiuc.edu.
Published, JBC Papers in Press, March 4, 2003, DOI 10.1074/jbc.M210467200
2 Workbench.sdsc.edu.
3 F. Glaser, T. Pupko, I. Paz, R. E. Bell, D. Bechor, E. Martz, and N. Ben-Tal, www.consurf.tau.ac.il.
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ABBREVIATIONS |
---|
The abbreviations used are: FPPS, farnesyl pyrophosphate synthase; FPP, farnesyl pyrophosphate; GPP, geranyl pyrophosphate; IPP, isopentenyl pyrophosphate; TbFPPS, Trypanosoma brucei farnesyl pyrophosphate synthase; FBS, fetal bovine serum; RT, reverse transcripase; RNAi, RNA interference; Mops, 4-morpholinepropanesulfonic acid.
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