Design, synthesis, calorimetry, and crystallographic analysis of 2-alkylaminoethyl-1,1-bisphosphonates as inhibitors of trypanosoma cruzi farnesyl diphosphate synthase

Article abstract of DOI:10.1021/jm300425y

Linear 2-alkylaminoethyl-1,1-bisphosphonates are effective agents against proliferation of Trypanosoma cruzi, the etiologic agent of American trypanosomiasis (Chagas disease), exhibiting IC₅₀ values in the nanomolar range against the parasites.

Full text of DOI:10.1021/jm300425y

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Article
Design, Synthesis, Calorimetry and Crystallographic analysis
of 2-Alkylaminoethyl-1,1-Bisphosphonates as inhibitors
of Trypanosoma cruzi Farnesyl Diphosphate Synthase
Srinivas Aripirala, Sergio Hernan Szajnman, Jean Jakoncic, Juan Bautista
Rodriguez, Roberto Docampo, Sandra B. Gabelli, and L. Mario Amzel
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm300425y • Publication Date (Web): 20 Jun 2012
Downloaded from http://pubs.acs.org on June 26, 2012
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Journal of Medicinal Chemistry
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Design, Synthesis, Calorimetry and Crystallographic
analysis of 2ꢀAlkylaminoethylꢀ1,1ꢀBisphosphonates as
inhibitors of Trypanosoma cruzi Farnesyl Diphosphate
Synthase
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Srinivas Aripiralaa, Sergio H. Szajnmanc, Jean Jakoncicb, Juan B. Rodriguezc, Roberto Docampod,
Sandra B. Gabellia*, and L. Mario Amzela*
aDepartment of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205, USA;
bBrookhaven National Laboratory, National Synchrotron Light Source, Upton, NY 11973
cDepartamento de Química Orgánica and UMYMFOR (CONICET–FCEyN), Facultad de Ciencias
Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, C1428EHA,
Buenos Aires, Argentina
dCenter for Tropical and Global Emerging Diseases and Department of Cellular Biology, University of
Georgia, Athens, Georgia 30606
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ABSTRACT: Linear 2ꢀalkylaminoethylꢀ1,1ꢀbisphosphonates are effective agents against proliferation
of Trypanosoma cruziꢀꢀthe etiologic agent of American trypanosomiasis (Chagas disease)ꢀꢀ exhibiting
IC50 values in the nanomolar range against the parasites. This activity is associated with inhibition at the
low nanomolar level of the T. cruzi farnesyl diphosphate synthase (TcFPPS). Xꢀray structures and
thermodynamic data of the complexes TcFPPS with five compounds of this family show that the
inhibitors bind to the allylic site of the enzyme with their alkyl chain occupying the cavity that binds the
isoprenoid chain of the substrate. The compounds bind to TcFPPS with unfavorable enthalpy
compensated by a favorable entropy that results from a delicate balance between two opposing effects:
the loss of conformational entropy due to freezing of single bond rotations, and the favorable burial of
the hydrophobic alkyl chains. The data suggest that introduction of strategically placed double bonds
and methyl branches should increase affinity substantially.
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KEYWORDS. Bisphosphonate, Trypanosoma cruzi, Chagas disease, farnesyl diphosphate synthase,
FPPS, IPP, DMAPP, ITC, farnesyl pyrophosphate synthase, mevalonate pathway.
INTRODUCTION
American trypanosomiasis (Chagas disease) is a major parasitic disease that affects millions of
individuals worldꢀwide1, 2. T. cruzi, the etiologic agent of American trypanosomiasis, has a complex life
cycle in which it passes from a bloodꢀsucking Reduviid insect vector to mammals.3 It multiplies in the
insect gut as an epimastigote form and is spread as a nonꢀdividing metacyclic trypomastigote from the
insect feces by contamination of intact mucosa or of wounds produced by the bloodꢀsucking activity of
the vector. In the mammalian host, the parasite proliferates intracellularly in the amastigote form and is
subsequently released into the blood stream as a nonꢀdividing trypomastigote.3 In humans, spread of
Chagas disease can also take place via the placenta or by blood transfusion.4, 5 The occurrence of
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American trypanosomiasis in countries where the disease is not endemic has been attributed to the
second mechanism.4, 5 Chemotherapy for this neglected disease, based on old and empirically discovered
drugs, is not very effective.6 Thus, it is critical that we develop new safe drugs based on knowledge of
the biochemistry and physiology of the microorganism. 2ꢀalkylaminoethylꢀ1,1ꢀbisphosphonates have
emerged as a new avenue for the development of compounds active against Chagas disease.
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Bisphosphonates of general formula 1 (Figure 1) are metabolically stable pyrophosphate (2)
analogues in which a methylene group replaces the oxygen atom bridge between the two phosphorus
atoms of the pyrophosphate moiety. Substitution at the carbon atom with different side chains has
generated a large family of compounds.7ꢀ10 Bisphosphonates became compounds of pharmacological
importance since calcification studies were done more than 40 years ago.11ꢀ13 Currently, several
bisphosphonates (Figure 1) such as pamidronate (3), alendronate (4), risedronate (5), and ibandronate (6)
are in clinical use for the treatment and prevention of osteoclastꢀmediated bone resorption associated
with osteoporosis, Paget's disease, hypercalcemia, tumor bone metastases, and other bone diseases.
Selective action on bone is based on binding of the bisphosphonate moiety to bone mineral.14 It has
been postulated that the parasite’s acidocalcisomes, organelles equivalent in composition to the bone
mineral, may accumulate bisphosphonates and facilitate their antiparasitic action.14 In the case of bone,
bisphosphonates act by a mechanism that leads to osteoclast apoptosis.15 The site of action of
aminobisphosphonates has been narrowed down to the isoprenoid pathway and, more specifically, to
inhibition of protein prenylation.16 Within the isoprenoid pathway, farnesyl pyrophosphate synthase
(FPPS; also called farnesyl diphosphate synthase) was identified as the main target of
bisphosphonates.17ꢀ22 FPPS catalyses two consecutive 1´ꢀ4 condensation reactions between an allylic
(DMAPP or GPP) and a homoallylic substrate (IPP) to give a final product FPP. These reactions
constitute the two committed steps in the biosynthesis of farnesyl pyrophosphate. In the first step it
catalyzes the 1´ꢀ4 condensation of one molecule of IPP (homoallylic substrate) and one molecule of
DMAPP (allylic substrate) to give GPP. In the second step it condenses one molecule of GPP and one
molecule of IPP. Inhibition of the enzymatic activity of FPPS blocks farnesyl pyrophosphate and
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geranylgeranyl pyrophosphate formation, compounds which are required for the postꢀtranslational
prenylation within osteoclasts of small GTPases such as Rab, Rho and Rac.23
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Figure 1. General formula and chemical structure of pyrophosphate and bisphosphonates. 1ꢀgeneral
bisphosphonate; 2ꢀpyrophosphate; 3ꢀ6ꢀrepresentative FDAꢀapproved bisphosphonates clinically
employed for different bone disorders: 3, palmidronate; 4, alendronate; 5, residronate; 6, ibandronate.
Besides their effectiveness in longꢀterm treatment of bone disorders, bisphosphonates exhibit a wide
range of biological activities that include, in addition to stimulation of γδ T cells of the immune
system,24 antibacterial,25 herbicidal,26 antitumor27ꢀ30 and antiparasitic activities.31ꢀ35
In vivo assays showed that risedronate can significantly increase survival of T. cruzi-infected mice.36
Besides being effective growth inhibitors of T. cruzi in in vitro and in vivo assays without toxicity to the
host cells14, bisphosphonates were found to be also effective against pathogenic trypanosomatids other
than T. cruzi. Those include T. brucei rhodesiense, Leishmania donovani, and L. mexicana as well as
apicomplexan parasites such as Toxoplasma gondii and Plasmodium falciparum.33, 34, 37ꢀ43 These results
point to bisphosphonates as potential candidates for chemotherapy of a range of neglected infectious
diseases. They have the advantage, among other favorable characteristics, that they are relatively
inexpensive and easy to synthesize. Furthermore, one may assume a low toxicity for bisphosphonateꢀ
containing drugs considering that many bisphosphonates are FDAꢀapproved drugs that have been widely
used for many years in the longꢀterm treatment of bone disorders.
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Figure 2. Bisphosphonate drugs used in this study40. [2ꢀ(nꢀpropylamino)ethaneꢀ1,1ꢀdiyl]bisphosphonic
acid (BR25 = 10); [2ꢀ(nꢀpentylamino)ethaneꢀ1,1ꢀdiyl]bisphosphonic acid (BR6 = 11); [2ꢀ(nꢀ
hexylamino)ethaneꢀ1,1ꢀdiyl]bisphosphonic acid (BR18 = 12); [2ꢀ(nꢀheptylamino)ethaneꢀ1,1ꢀ
diyl]bisphosphonic acid (BR11 = 1338, 40, 42); [2ꢀ(cyclohexylamino)ethaneꢀ1,1ꢀdiyl]bisphosphonic acid
(BR28 = 14).
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In this paper we report structural and thermodynamic studies of the interaction of five 2ꢀ
alkylaminoethylꢀ1,1ꢀbisphosphonates with T. cruzi FPPS (TcFPPS; Figure 2). The structures show that
the inhibitors bind to the allylic site of the enzyme with the phosphates of the bisphosphonates
coordinating three Mg2+ ions that bridge the compound to the enzyme in a manner similar to that
observed for the physiological substrates.44ꢀ46 The alkyl chains of the inhibitors bind within a long
cavity normally occupied by the isoprenoid chain of the allylic substrate (Figure 3). The inhibitors bind
to TcFPPS with high affinity despite having unfavorable enthalpy of binding. The favorable entropy
that results from burying the hydrophobic alkyl chain is the main binding driving force.
Although several bisphosphonate families have been shown to inhibit the trypanosomal FPPS, the lack
of pharmacokinetic studies on these compounds suggests that it is still important to expand the number
of compounds in the pipeline, especially with compounds of high affinity.
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Figure 3. Allylic and homoallylic sites of FPPS. The allylic site is the part of the active site occupied by
Mg and the bisphosphonate 10. The Homoallylic site is occupied by IPP. Magnesiums are shown in cpk
model while the ligands 10 and IPP are shown as a stick model. The surface shows positive potential as
blue and negative as red.
The structural and thermodynamic information presented here provides the basis for the design of novel,
more effective compounds for the treatment of Chagas disease. In particular, new inhibitors with
strategically placed double bonds and methylꢀgroup branches are predicted to have significantly
increased affinity.
RESULTS AND DISCUSSION
Structure of the inhibitor complexes
Like the FPPS from other species, including humans, 17, 47 the farnesyl diphosphate synthase of T. cruzi
(TcFPPS) is a physiological homodimer (monomers A and B). The structures of TcFPPS in complex
with five bisphosphonates 10ꢀ14 (resolutions between 2.01 Å and 3.0 Å; Table 1) each contained, in
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Table 1. Structures of TcFPPS complex: Data collection and Refinement Statistics
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Crystal
TcFPPS+IPP
11 + Mg2+
TcFPPS+
13 + Mg2+
TcFPPS+IPP
12 + Mg2+
P6122
TcFPPS+IPP
14 + Mg2+
TcFPPS+IPP
10 + Mg2+
Space group
8
9
a = 103.2
b = 103.2
c = 386.6
BNLꢀX6a
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Cell
dimensions(Å)
a = 57.9; b = 57.9; c = 392.4
BNLꢀX6a
50.0ꢀ2.1
BNLꢀX6a
50.0ꢀ2.01
(2.04ꢀ2.01)
BNLꢀX6a
50.0ꢀ2.65
(2.70ꢀ2.65)
BNLꢀX6a
50.0ꢀ3.05
(3.1ꢀ3.05)
Xꢀray Source
50.0ꢀ2.35
Res(Å)
(HighRes shell)
(2.14ꢀ2.1)
(2.39ꢀ2.35)
Measured
Reflect.
173,165
152,558
174,663
79,815
453,674
Unique
Reflections
22,705
27,252
12,287
7,407
49,874
23.6 (4.3)
93.2 (98.2)
11.3 (50.9)
34.3 (6.2)
97.8 (98.7)
8.0 (34.6)
38.0 (7.8)
95.7 (99.8)
10.5 (47.3)
46.9 (16.8)
87.8 (90.3)
6.9 (14.3)
26.6 (2.2)
96.0 (93.7)
9.0 (52.0)
I/σ
Completeness
(%)
Rmerge (%)
Refinement
22.1
28.4
1
20.3
26.7
1
23.6
30.7
1
23.1
29.6
1
22.0
28.5
3
Rcryst (%)
Rfree
Monomer in ASU
Total Atoms
Protein atoms
Water molecules
3,037
2,900
89
3,205
2,900
256
2,960
2902
23
2,916
2854
3
9,014
8701
161
R.m.s deviations
0.021
1.82
0.02
1.83
0.008
1.13
0.007
0.97
0.009
1.21
Bond length (Å)
Angle (°)
Bfactor(Å2)
31.14
36.23
58.19
35.01
28.33
27.25
59.30
35.59
51.53
40.18
51.17
34.12
46.81
47.54
49.13*
26.10
33.36
29.54
44.72
30.51
Protein
Allylic
Homoallylic
H20
*Occupancy : 0.6
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ꢀ2
addition, 3 divalent cations (Mg2+) and isopentenyl pyrophosphate (IPP) or SO4 (13ꢀTcFPPS complex
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has SO4 ). Crystals of the complexes belong to space group P6122; four of the complexes have an
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average cell dimension of 392 Å along cꢀaxis, 5 Å shorter than the equivalent dimension in the apo
structure48 (Table 1) indicating that the structures of the complexes pack more compactly than that of the
apo enzyme.
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The structures were determined by direct refinement or by molecular replacement (complex 10ꢀ
TcFPPS) using the structure of the Trypanosoma cruzi FPPS in complex with alendronate and IPP
(1YHM). After initial refinement, 2FoꢀFc maps showed excellent density for the bound inhibitors in the
region corresponding to the allylic site. In the homoallylic site, electron density for IPP is seen in four
structures – 10, 11, 12 and 14; in 13 a SO4ꢀ2 ion occupies the homoallylic site.
In all cases, three Mg2+ ions bridge the phosphates of the inhibitors to the protein. The conserved
aspartate residues of the twoꢀaspartate rich motifs DDXXD (first aspartate rich motif: FARM, residues
98ꢀ102; second aspartate rich motif: SARM, residues 250ꢀ254) bind three divalent cations (Mg2+) that
are in turn coordinated by the phosphate backbone of the bisphosphonates (Figure 4a; 11). The IPP is
bound to the enzyme by interacting directly with arginine residues (Arg51, Arg108, and Arg360 (Figure
4b). The conserved RRG sequence (residues 107ꢀ109) of the loop following the FARM region and
residues GK (263 and 264) in the loop following the SARM region are in the conformations usually
seen in the closed form of the enzyme with both the allylic and homoallylic sites occupied. In all five
structures the bisphosphonate, occupies the allylic site, and interacts with 3 divalent Mg2+ ions. Ligand
waters complete the octahedral coordination of the ions.
Structural alignment of the four complexes with nꢀalkyl chains (10ꢀ13) shows that the phosphate
backbones of the bisphosphonates interact with the same residues of the protein, located near the top of
the active site (Figure 4c; Figure 5a). However, deeper into the active site there are a small number of
significant differences (Figure 5b). In the 12 and 13 complexes, Tyr94 and Gln167 both move to
accommodate the bisphosphonates with the longer alkyl chains. In the complex with 13, the
bisphosphonate with the longest alkyl chain, the end of the inhibitor bound to monomer A is at a
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distance of 3.5 Å from Ile 129 from monomer B. This additional favorable van der Waals interaction
contributes to the tighter binding of 13 (Table 2) in comparison to the slightly shorter 12 (one fewer
carbon).
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It was proposed in earlier studies that His93 and Tyr94 (TcFPPS numbering) form the “floor” of the
allylic site and determine the maximum length of the allylic substrates that can bind to the enzyme49 and
by extension the length of the inhibitors. The complexes 13ꢀTcFPPS and 12ꢀTcFPPS show that Tyr94
adopts a different conformation to accommodate longer alkyl chains (Figure 5b). Earlier studies with
avian FPPS revealed that when Phe112 and Phe113 (equivalent to His93 and Tyr94 of TcFPPS) were
replaced by residues with smaller side chains (Ala and Ser respectively), the mutated enzyme produced
geranyl geranyl diphosphate (20 carbons)50. Also, in some species, geranyl geranyl diphosphate
synthases contained smaller amino acids such as serine or threonine in the position equivalent to
TcFPPS Tyr94 indicating that Tyr94 may be important in determining final product length51. These
observations point to His93, Tyr94 of chain A and Ile129 of the B chain as the residues that determine
maximum permissible alkyl chain length.
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Comparison of the 14ꢀTcFPPS and 10ꢀTcFPPS complexes
In the 14ꢀTcFPPS structure, the cyclohexyl moiety adopts a chair conformation. Structural alignment of
the 14ꢀTcFPPS and 10ꢀTcFPPS complexes shows that packing of 14 to the enzyme is not as tight as that
of 10. Also, residue Gln167, one of the conserved residues in αꢀhelix F, adopts a different conformation
in the two complexes (Figure 4d.). The same is true about another conserved residue, Tyr211: in 14 its
hydroxyl points away from bisphosphonate moiety. Interestingly, in the 14 complex Tyr211 points
towards the isoprenyl chain of the bound IPP and, as a result, it affects the IPP conformation.
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Figure 4. Binding of bisphosphonates in various complexes of TcFPPS. (a) TcFPPS in complex with
11, IPP and 3 divalent cations. Water molecules are shown as red spheres and Mg2+ in white. Protein
backbone and residues of the active site are shown in green color. 11 and IPP are shown in cream color.
Residues from the FARM and SARM are shown coordinating with Mg2+ ions. The basic amino acids
Arg360 and Arg51 are observed to be interacting with the diphosphate moiety of IPP. (b) Homoallylic
site of TcFPPS in complex with 11 (cream) and IPP (cream). IPP interacts with the basic amino acids
Arg51 and Arg360. Arg107 and Arg108, from the loop after first aspartate rich region, interact with the
inhibitor in the allylic site (11). (c) Structural overlap of the four BPs with n-alkyl chains (10ꢀ13) in
complex with TcFPPS + Mg2+ + IPP (green). (d) Structural overlap of TcFPPS in complex with 10
(green) and with 14 (magenta). Residues in the TcFPPSꢀ10 complex are shown in green color and those
of TcFPPSꢀ14 complex in magenta. Key differences in the conserved interactions of the ligands with
residues of the active site are shown.
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Inhibitor Affinities
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Bisphosphonates derived from fatty acids have become interesting potential antiparasitic agents,
especially 2ꢀalkylaminoethyl derivatives, which were shown to be potent growth inhibitors of the most
clinically relevant form of T. cruzi with IC50 values in the nanomolar range against the target enzyme38,
41. Compounds 10−14 are representative members of the 2ꢀalkylaminoethyl family of bisphosphonates,
which have proven to be far more efficient growth inhibitors of trypanosomatids than their parent drugs
1ꢀhydroxyꢀ, 1ꢀalkylꢀ, and 1ꢀaminoꢀbisphosphonates such as compounds 7, 8 and 9 (Figure 6).39, 41, 42
Compounds 10−14 inhibit the enzymatic activity of TcFPPS with IC50 values of 38 nM, 1.84 ꢀM, 0.49
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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41
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀM, 58 nM, 13 nM, respectively (Table 2).40
Thermodynamic Data
The interactions of TcFPPS with these 2ꢀalkylaminoethyl bisphosphonates were studied by isothermal
titration calorimetry at 28 °C. (ITC data for reversible binding of 14 to TcFPPS could not be obtained.)
The four compounds with nꢀalkyl chains, bind to the target enzyme with a positive, unfavorable enthalpy
change (Figure 7; Table 2), in agreement with previous studies with other bisphosphonates52. This
unfavorable enthalpy is compensated by large favorable entropy that is itself determined by the
difference between to opposite effects. As the inhibitor molecules bind, the single bond rotations around
the C—C bonds of the alkyl chain become frozen in the complex resulting in a loss of conformational
entropy that becomes larger as the number of carbons in the alkyl chain increases. At the same time, the
favorable entropy from the burial of the hydrophobic alkyl chain, also increases with chain length,
resulting in a very fine balance between these two effects. The values of the unfavorable binding
enthalpy also vary significantly among the inhibitors. 12 is the most unfavorable by 1.5ꢀ2.0 Kcal/mol.
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7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Figure 5. Allylic site binding of inhibitors in various TcFPPSꢀBPs complexes. (a) TcFPPS in complex
with 11 and 3 divalent cations. Water molecules are shown as red spheres, Mg2+ in white. The TcFPPS
protein backbone and some residues in the active site are shown in green color. 11 is shown as a stick
model in green color. Residues from the first and second aspartate rich regions are shown coordinating
the Mg2+ ions. (b) Structural overlap of TcFPPS in complex with four n-alkyl chain bisphosphonates 10ꢀ
13. Residues His93, Tyr94, Ile129 (monomer B) at the “bottom” of the allylic site are shown. Monomer
A of the TcFPPSꢀ11 complex is shown in green, the TcFPPSꢀ13 complex in pink, TcFPPSꢀ12 complex
in brown, TcFPPSꢀ10 complex in violet while monomer B is shown in red, cyan, yellow and blue
respectively. The distance between the terminal carbon of the longest bisphosphonate 13 and the Ile 129
is 3.5 Å.
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23
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60
The high affinity of 14 (IC50 13 nM) can be rationalized based on these same arguments. TcFPPS
binds 14 in a manner similar to 10, suggesting that it would have a similar enthalpy of binding (not
measured), although somewhat more unfavorable due to changes in the conformations of Gln167 and
Tyr211 described above. However, 14 buries a large hydrophobic surface without the loss of
conformational entropy of the nꢀalkyl chain experienced by the other inhibitors: the conformational
flexibility of the ring is highly restricted even in the unbound state.
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9
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40
41
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. Chemical structure of representative bisphosphonic acids derivatives.
Furthermore, binding in the more stable chair conformation not only reduces the loss of entropy but
also avoids the enthalpic penalty of binding the less favorable boat conformation.
The inhibition data (IC50) of bisphosphonates 10ꢀ13 against TcFPPS are in excellent agreement with
the Kd’s obtained in the ITC experiments (Table 2). These results and the structural data taken together
indicate that inhibition results from binding of these inhibitors to the allylic portion of the catalytic site
of the enzyme. It is likely that other closely related bisphosphonates that effectively inhibit the
enzymatic activity of TcFPPS also do so by binding to the allylic site.41 The data for 13 can also be fit
using two different sites per dimer (see figure 7 and Table 2 and their footnotes). This is similar to
previous data on other nitrogenꢀcontaining bisphosphonates52. One reason for this behavior of 13 may be
its size. Like some of the other large bisphosphonates, binding of 13 to one site of the dimer, modifies
the affinity of the other monomer.
Towards the design of new bisphosphonate TcFPPS inhibitors
As mentioned above, binding of these inhibitors is enthalpically unfavorable. The favorable entropy,
which dominates the favorable freeꢀenergy, results from a delicate balance two opposing effects: the
unfavorable loss of conformational entropy, due to freezing of single bond rotations of the inhibitor (and
binding site side chains), and the favorable increase of entropy associated with burial of the hydrophobic
alkyl chains. With the shortest compound, nꢀpropyl, the balance produces the tightest binding of the
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Table 2. Isothermal Titration Calorimetry Studies on the Binding of Bisphosphonates to TcFPPS
1
2
3
4
5
6
7
8
Carbons in
ꢁG
ꢁH
ꢁS
1/Ka
(nM)
IC*
50
Lig
10
nꢀalkyl
(kcal/ mol)
(kcal/mol)
(cal/mol
/K)
(nM)
38.0
1840
3
5
ꢀ10.43
ꢀ8.15
6.35 ± 0.07
5.22 ± 0.11
56.1
25.0 ± 6.3
1030 ±170
11
44.7
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
12
13
6
7
ꢀ8.72
ꢀ9.88
7.87 ± 0.11
5.65 ± 0.09
55.4
51.9
400 ± 63
58.8 ± 20.4
†(10.2 ± 7.3;
38.3 ± 11.2)
490
58.0
†( 6.39 ± 0.36;
4.62 ± 0.44)
*IC50 were calculated before40 †Values calculated using 2ꢀsite model.
series. Increasing the length of the alkyl chain to pentyl or hexyl reduces the affinity by over an order of
magnitude. This change seems to imply that by the addition of two or three methylenes, the increase in
the loss of conformational entropy is greater than the additional entropy gain due to burial of the longer
chain (more so for the pentyl than for the hexyl). This tendency is reversed when the nꢀalkyl chain is
seven carbons long (13 vs. 12, Table 2). This observation suggests that increasing the alkyl chain
further could generate compounds with higher affinity. However, analysis of the structure of the
complex of TcFPPS with the nꢀheptyl inhibitor shows that increasing the nꢀalkyl chain past seven
carbons would result in clashes with residues of the enzyme: in any of its possible positions the eighth
carbon would clash with either Tyr94, His93, or Ile129 of chain B.
Comparison of the structure of the 13ꢀTcFPPS complex with the previously determined structure of
the chicken FPPS (GgFPPS) in complex with GPP (geranyl pyrophosphate) provides crucial information
for guiding the design of improved inhibitors: the N1 of 13 occupies the same position in the binding
site as the C1 of the GPP (Figure 8) and the rest of the chains align up to the C6 of 13 that overlaps with
C7 of the GPP. C7 of GPP is a tertiary carbon with two methyl groups while the equivalent carbon of
13 (C6) has only one methyl. The terminal methyl of 13 occupies a position
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40
41
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 7. Isothermal titration calorimetry of the reaction between bisphosphonate drugs and TcFPPS.
(a) 11. (b) 12. (c) 10. (d) 13. 13 can be fit either as two identical sites (one per monomer; red) or as two
different sites (two per dimer; blue).
between the positions occupied by the two GPP methyls, rendering this portion of 13 less
complementary to the binding site. Adding a methyl group to the C6 of 13 is likely to result in a better
inhibitor. The same is true of the C3 methyl of GPP: adding a methyl group at the C2 of 13 can fill this
pocket. Furthermore, the bound conformation of 13 is compatible with the double bonds of GPP,
suggesting that introducing double bonds at C1 and C5 of 13 will freeze the compound in the bound
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2
3
4
5
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7
8
9
10
11
12
13
14
15
16
17
18
19
Figure 8. Overlap of GPP and 13. a) Allylic site of 13ꢀTcFPPS complex. 13, amino acids are shown in
stick representation (pink). Portion of the 2mFoꢀDFc electron density corresponding to the inhibitor 13 is
shown in grey color. b) Structural overlap of 13ꢀTcFPPS complex with the chicken FPPSꢀGPP complex
(PDB: 1UBW). TcFPPS is shown in ribbon model (green). The ligands 13 (pink) and GPP (blue) in
stick representation. Only GPP of the chicken FPPS is shown in the figure.
20
21
22
23
24
25
26
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29
30
31
32
33
34
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60
conformation. This modification would reduce the loss of conformational entropy without affecting the
binding enthalpy. The resulting compound with a 2,6ꢀdimethylꢀ1,5ꢀdiene would be an excellent mimic
of the bound GPP; however it would contain a labile enamine functionality that renders the compound
too unstable to be considered a useful inhibitor. These observations point to a 2ꢀalkylaminoethylꢀ1,1ꢀ
bisphosphonate with an (E)ꢀ2ꢀ2,6ꢀdimethylheptaꢀ2,5ꢀdiene chain (compound 21, Figure 9) as a highly
promising lead compound for the next generation of bisphosphonate TcFPPS inhibitors (Figure 8b;
Figure 9). Molecular modeling using MOE (Molecular Operating Environment, Chemical computing
group; Quebec, Canada) showed that the 2,5 diene chain can bind the enzyme in a conformation that still
mimics that of the bound geranyl diphosphate. It appears that the affinity of 10 can also be improved by
an equivalent modification. Replacement of the propyl chain by an isobutylꢀ2ꢀene (compound 20, Figure
9) would result in a compound that binds the enzyme mimicking DMAPP. In addition, the compounds
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equivalent to 20 and 21 but lacking the double bond at the 2ꢀ positions may also show high affinity for
1
2
3
the enzyme.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
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20
21
22
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 9. New proposed bisphosphonates. Compounds labeled as 20 and 21.
EXPERIMENTAL SECTION
Synthesis of inhibitors
Compounds 10–14 were synthesized as reported before40. In brief; they were prepared using tetraethyl
ethenylidene bisphosphonate (compound 15) as a Michael acceptor,53 which in turn was prepared from
tetraethyl methylenebisphosphonate in two steps according to a slightly modified Degenhart protocol.54
Compound 15 was reacted with the corresponding nꢀalkylamine via a 1,4ꢀconjugated addition reaction
to yield the respective Michael adducts (16–20). Once these synthetic precursors were at hand, they were
hydrolyzed with bromotrimethylsilane in methylene chloride55 to afford the free 1,1ꢀbisphosphonic acids
(10–14).39 The purity of the compounds assessed by elemental analysis was greater than 98%40.
Cloning, expression and purification
TcFPPS was cloned and expressed as reported before56. Briefly, DNA coding for TcFPPS with an Nꢀ
terminal HisꢀTag and a thrombin cleaveage site was cloned into a pET28a vector (Novagen).
BL21(DE3) E. coli cells transformed with this plasmid were grown in LB medium until they reached an
o
OD600 of 0.8 and induced with 0.1 mM IPTG at 37.0 C. The cells were harvested 3h after induction,
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washed in buffer A (50 mM NaH2PO4 pH 8.0 300 mM NaCl, 10 mM imidazole, 1 mM TCEP; TCEP:
tris(2ꢀcarboxyethyl) phosphine hydrochloride), and broken with a microfluidizer. The lysate was
centrifuged for 30 mins at 12000 rpm and the supernatant was loaded onto a HisTrap Ni2+ chelate
affinity column equilibrated with buffer A. The protein was eluted using a linear gradient of 0ꢀ100% of
buffer B (50 mM NaH2PO4 pH 8.0 300 mM NaCl, 500 mM Imidazole, 1 mM TCEP). The Hisꢀtag was
cleaved by digestion with thrombin and the sample was loaded into an anion exchange column (binding
buffer: 20 mM Tris pH 8.2, 20 mM NaCl, 1 mM TCEP) and eluted with 20 mM Tris pH 8.2, 1 M NaCl,
1 mM TCEP. The protein, which was more than 95% pure as seen by SDS page gel, was dialyzed
against 20 mM Tris pH 8.2, 150 mM NaCl, 1 mM TCEP and concentrated to 12 mg mLꢀ1.
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Crystallization
Crystals used for data collection were grown by vapor diffusion with the protein and the mother liquor
in a 1:1 ratio. The reservoir consisted of 100 mM sodium acetate, pH 4.6–5.2, 200 mM ammonium
sulfate, and 2–10% PEG 4K. Crystals, which appear within 1ꢀ2 days, belong to the hexagonal space
group P6122. The protein (12.5 mg/ml) inhibitor solution used for coꢀcrystallization contained 250 ꢁM
inhibitor, 250 ꢁM IPP and 1 mM MgCl2.
Data Collection
Diffraction data of all the TcFPPS complexes were collected at beamline X6A of the NSLS,
Brookhaven National Laboratory. Diffraction data collected from a single frozen crystal (100 K) were
processed and scaled using the HKL 2000 suite57 (Table 1).
Structure Determination
The structures of the complexes of TcFPPS with compounds 11ꢀ14 were determined by direct
refinement of the coordinates of the FPPS from Trypanosoma cruzi (1YHM)48 with the program
REFMAC558ꢀ60 of CCP4 suite. The structure of 10 was determined by molecular replacement using the
program AMoRe61 (search molecule PDB id 1YHM).
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Model Building and Refinement
1
2
The initial model was refined using REFMAC5 and rebuilding during refinement was carried out with
the program COOT62. Following the Rꢀvalue monitored progress of the refinement and an Rꢀfree
calculated with a cross validation set containing 5% of the reflections. The overall quality of the final
model was assessed using the programs PROCHECK63 and WHATIF64, 65. Atomic coordinates and
structure factors for the complexes TcFPPS+10, TcFPPS+11, TcFPPS+12, TcFPPS+13 and TcFPPS+14
have been deposited in the Protein Data Bank with accession codes 4DWB, 4DXJ, 4DWG, 4EIE and
4DZW respectively. Structure figures were generated using molscript66 and pymol (The PyMOL
Molecular Graphics System, Version 1.5.0.1 Schrödinger, LLC). Models of the proposed new inhibitors
were built using MOE (Molecular Operating Environment, Quebec, Canada).
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Isothermal Calorimetry
ITC experiments were performed with TcFPPS and each of five ligands: 10ꢀ14. The protein was
diluted to a concentration of 29 ꢁM (in monomers) in a buffer containing 25 mM Hepes pH 7.5, 1 mM
TCEP, 300 mM NaCl, 2 mM MgCl2. The ligands were prepared in the same buffer at a concentration of
250 ꢁM. 1.3 mL of protein in the sample cell were titrated with twenty five 10 ꢁl injections. The data
were analyzed with the Originꢀ5.0 software and fitted to a single binding site per monomer.
Acknowledgments. This work was supported by grants from the National Research Council of
Argentina (PIP 1888), ANPCyT (PICT 2008 #1690), and the Universidad de Buenos Aires
(200201001003801) to J.B.R and Bunge & Born Foundation to S.H.S. Data collection was carried out
at beam line X6A, funded by the National Institute of General Medical Sciences, National Institute of
Health under agreement GMꢀ0080. The NSLS, Brookhaven National Laboratory is supported by the US
Department of energy under contract No. DE AC02ꢀ98CH10886.
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Abbreviations. FPPS: Farnesyl pyrophosphate synthase; FPP: Farnesyl pyrophosphate, GPP: Geranyl
pyrophosphate, BPs: Bisphosphonates, TcFPPS: Trypanosoma cruzi Farnesyl pyrophosphate synthase,
IPP: Isopentanyl pyrophosphate, DMAPP: Dimethylallyl pyrophosphate.
1
2
3
4
5
6
7
8
9
10
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58
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60
ACCESSION CODES
The atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank for
TcFPPS+Mg2+ complexed with 10+IPP (PDB code 4DWB, 11+IPP (PDB code 4DXJ), 12+IPP (PDB
code 4DWG), 13 (PDB codes 4EIE) and 14+IPP (PDB codes 4DZW).
CORRESPONDING AUTHOR INFORMATION
*L. Mario Amzel (email: mamzel@jhmi.edu; Ph no: 410ꢀ955ꢀ3955)
*Sandra B. Gabelli (email: gabelli@jhmi.edu; Ph no: 410ꢀ614ꢀ4146)
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