Inhibition of geranylgeranyl diphosphate synthase by bisphosphonates: A crystallographic and computational investigation

Article abstract of DOI:10.1021/jm800325y

We report the X-ray structures of several bisphosphonate inhibitors of geranylgeranyl diphosphate synthase, a target for anticancer drugs. Bisphosphonates containing unbranched side chains bind to either the farnesyl diphosphate (FPP) substrate site, the geranylgeranyl diphosphate (GGPP) product site, and in one case, both sites, with the bisphosphonate moiety interacting with 3 Mg²⁺ that occupy the same position as found in FPP synthase. However, each of three V-shaped bisphosphonates bind to both the FPP and GGPP sites. Using the Glide program, we reproduced the binding modes of 10 bisphosphonates with an rms error of 1.3 ?. Activities of the bisphosphonates in GGPPS inhibition were predicted with an overall error of 2x by using a comparative molecular similarity analysis based on a docked-structure alignment. These results show that some GGPPS inhibitors can occupy both substrate and product site and that binding modes as well as activity can be accurately predicted, facilitating the further development of GGPPS inhibitors as anticancer agents.

Full text of DOI:10.1021/jm800325y

5594
J. Med. Chem. 2008, 51, 5594–5607
Inhibition of Geranylgeranyl Diphosphate Synthase by Bisphosphonates: A Crystallographic
and Computational Investigation†
Cammy K.-M. Chen,‡,§,| Michael P. Hudock,‡, Yonghui Zhang,‡,3 Rey-Ting Guo,|,# Rong Cao, Joo Hwan No,
Po-Huang Liang,§,| Tzu-Ping Ko,| Tao-Hsin Chang,|,# Shiou-chi Chang,3 Yongcheng Song,3 Jordan Axelson,3 Anup Kumar,3
,3
Andrew H.-J. Wang,*,§,|,# and Eric Oldfield*,
Institute of Biochemical Sciences, National Taiwan UniVersity, Taipei 106, Taiwan, Institute of Biological Chemistry, Academia Sinica,
Taipei 115, Taiwan, Center for Biophysics and Computational Biology, UniVersity of Illinois at Urbana-Champaign, 607 South Mathews
AVenue, Urbana, Illinois 61801, Core Facility for Protein Crystallography, Academia Sinica, Taipei 115, Taiwan, Department of Chemistry,
UniVersity of Illinois at Urbana-Champaign, 600 South Mathews AVenue, Urbana, Illinois 61801
ReceiVed March 21, 2008
We report the X-ray structures of several bisphosphonate inhibitors of geranylgeranyl diphosphate synthase,
a target for anticancer drugs. Bisphosphonates containing unbranched side chains bind to either the farnesyl
diphosphate (FPP) substrate site, the geranylgeranyl diphosphate (GGPP) product site, and in one case,
both sites, with the bisphosphonate moiety interacting with 3 Mg2+ that occupy the same position as found
in FPP synthase. However, each of three “V-shaped” bisphosphonates bind to both the FPP and GGPP
sites. Using the Glide program, we reproduced the binding modes of 10 bisphosphonates with an rms error
of 1.3 Å. Activities of the bisphosphonates in GGPPS inhibition were predicted with an overall error of 2×
by using a comparative molecular similarity analysis based on a docked-structure alignment. These results
show that some GGPPS inhibitors can occupy both substrate and product site and that binding modes as
well as activity can be accurately predicted, facilitating the further development of GGPPS inhibitors as
anticancer agents.
Introduction
inhibiting protein prenylation, cell signaling, and cell survival
pathways, Figure 1.
Geranylgeranyl diphosphate synthase (GGPPS,a EC 2.5.1.30)
catalyzes the formation of geranylgeranyl diphosphate (1) from
one molecule of farnesyl diphosphate (2) and one molecule of
isopentenyl diphosphate (3) (Chart 1):1The GGPP product is
used in the biosynthesis of many natural products, such as
taxanes and gibberellins, and is also used to prenylate proteins
such as Rho, Rap, and Rac, involved in cell signaling pathways,2,3
Figure 1. It can be further elongated by some polyprenyl
synthases4 to produce the long chain isoprenoids used in quinone
biosynthesis, and in plants and some bacteria, two GGPP
molecules can condense to form phytoene, the precursor for
many carotenoids.5 GGPPS is inhibited by a variety of
bisphosphonates,6–9 and is of current interest in the context of
the development of anticancer drugs,7,8 which function by
In earlier work,6 we found that n-alkyl bisphosphonates
such as 4 (Chart 2) had quite potent activity against GGPPS,
and more recently, Weimer et al. have reported7,8 that novel
diprenyl methylenebisphosphonates, such as digeranyl me-
thylene bisphosphonate (5), have potent activity against
GGPPS as well as against a K562 tumor cell line, but the
structures of neither the n-alkyl nor any dialkenyl bisphos-
phonate inhibitor-GGPPS complexes have been reported.
The structure of human GGPPS is now known, however, with
recent work of Kavanagh et al.10 finding the presence of the
“isoprene fold” found in other prenyl synthases such as
farnesyl diphosphate synthase (FPPS, EC 2.5.1.10).11–14
These workers also showed10 that the GGPP product bound
to a central “inhibitor” binding site, and in more recent work,9
we have found that other GGPPS inhibitors such as 6 (which
is too large to inhibit FPPS) also bind to this site and are
potent inhibitors of GGPPS activity. We also found that
GGPPS substrates and diphosphate and bisphosphonate
inhibitors can bind in four distinct ways to GGPPS, with their
polar (diphosphate, bisphosphonate) groups binding to either
the FPP or IPP diphosphate binding sites, and their more
hydrophobic fragments binding to the (human) GGPP
(inhibitor) site or to the FPP (substrate) site.9 Here, we report
the first structures of a series of n-alkyl and dialkenyl
bisphosphonates bound to GGPPS. We also show that the
binding modes seen crystallographically can be well predicted
computationally, facilitating the development of quantitative
structure-activity models. Given the widespread use of
bisphosphonates in treating bone resorption diseases and the
current interest in them as anticancer agents,15–17 these results
are of broad general interest because they lay the foundation
†
Crystal structure coordinates have been deposited in the Protein Data
Bank and will be released upon publication (2z4w, 2z4z, 2z78, 2z4x, 2z4y,
2z50, 2z52, 2z4v, 2z71).
* To whom correspondence should be addressed. For A. H.-J. W.:
phone, +886-2-2788-1981; fax, +886-2-2788-2043; E-mail, ahjwang@
gate.sinica.edu.tw. For E.O.: phone, 217-333-3374; fax, 217-244-0997;
E-mail: eo@chad.scs.uiuc.edu.
‡
These authors contributed equally to this work.
Institute of Biochemical Sciences, National Taiwan University.
Institute of Biological Chemistry, Academia Sinica.
§
|
Center for Biophysics and Computational Biology, University of Illinois
at Urbana-Champaign.
#
Core Facility for Protein Crystallography, Academia Sinica.
Department of Chemistry, University of Illinois at Urbana-Champaign.
3
a Abbreviations: GGPPS, geranylgeranyl diphosphate synthase; GGPP,
geranylgeranyl diphosphate; FPPS, farnesyl diphosphate synthase; FPP,
farnesyl diphosphate; CoMSIA, comparative molecular similarity index
analysis; QSAR, quantitative structure activity relationship; rmsd, root-mean-
square deviation; CoMFA, comparative molecular field analysis; SP,
standard precision; XP, extra precision; rms, root-mean-square; PLS, partial-
least-squares; MLR, multiple linear regression; DMF, dimethylformamide;
TFA, trifluoroacetic acid; TMS, tetramethylsilane.
10.1021/jm800325y CCC: $40.75
2008 American Chemical Society
Published on Web 08/23/2008

Geranylgeranyl Diphosphate Synthase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5595
Chart 1
Chart 2
Chart 3
These observations clearly pose a challenge for structure
based inhibitor design because it is by no means clear how
a given, novel inhibitor might bind. We elected, therefore,
to investigate the structures of the following series of
inhibitors (Chart 4): which, when combined with previous
structures,9 might provide a database with which to test the
results of computational docking calculations. If successful,
it might then be possible to use docked structures of a much
larger range of inhibitors (whose crystallographic structures
are not known) to develop quantitative structure activity
relationships using, in particular, the comparative molecular
similarity index analysis (CoMSIA) method.18 For the simpler
bisphosphonates, it seemed possible that several of the four
binding poses seen previously (Figure S1, Supporting Infor-
mation) might be possible, while for the branched chain
species (5, 12, 13), it seemed likely that more than one site
might be needed in order to accommodate these “V-shaped”
molecules.
for the further development of, in particular, the novel
disubstituted bisphosphonates.
Results and Discussion
GGPPS is a highly R-helical protein and diphosphates (IPP,
FPP, and GGPP) as well as bisphosphonates such as 7, 8
(Chart 3) have previously been shown9 to bind to GGPPS in
four distinct ways. In the first, the polar (diphosphate or
bisphosphonate) groups (of e.g. 7) bind in the FPP substrate
or GGPP product binding site, with the large hydrophobic
side chain occupying the GGPP side chain site first reported
by Kavanagh et al.10 (Figure S1A, Supporting Information).
In the second mode, the FPP substrate site is occupied (by,
e.g., FPP, zoledronate, minodronate, or 8) with the long side
chain (when present) occupying the FPP side chain site
(Figure S1B, Supporting Information). In the third binding
mode, seen only so far with 8, the IPP polar site is occupied
by the polar bisphosphonate and the large hydrophobic side
chain site resides in the hydrophobic GGPP (human) site
(Figure S1C, Supporting Information). And in the fourth
binding mode, seen so far only with GGPP in the Saccha-
romyces cereVisae (yeast) GGPPS structure, the polar diphos-
phate of GGPP occupies the polar IPP site, while ): the
hydrophobic side chain occupies the FPP (substrate) hydro-
phobic side chain site (Figure S1D, Supporting Information).
To investigate the numerous possibilities for n-alkyl
bisphosphonate binding, we first determined the structure of
4 bound to GGPPS, obtaining two structures. In the first, we
find that 4 (yellow) binds to the FPP site (FPP-FPP) (Figure
2A,B; PDB 2z4x). The bisphosphonate fragment coordinates
to three Mg2+ (yellow), Figure 2B, which in turn are
coordinated to the DDXXD repeats (Figure 2A). The C8 side-
chain is closely aligned to the thiolo-farnesyl diphosphate
(FsPP) side chain seen in the FsPP-IPP-GGPPS structure
(PDB 2e8t) and, as can be seen in the superposition shown
in Figure 2C, the three Mg2+ are closely aligned to the three
Mg2+ seen in many FPPS structures,11–14,19 with a ∼0.32 Å
rmsd between the Mg2+ positions in the GGPPS (PDB 2z4x)
and FPPS/IPP/zoledronate (PDB 2f8z) structures. Essentially
the same binding pattern is seen with the longer chain species
11 (Figure S2, Supporting Information). These structures
present the first observation of 3 Mg2+ bound to GGPPS and,
as can be seen in Figure 2C, the similarity in metal binding
in GGPPS and FPPS is clear. So, at least for medium-length
side chains (about the length of GPP), the FPP-binding site
is an important target for bisphosphonates, as is binding to
Mg2+. Surprisingly, however, in a second structure of 4, we
find evidence for two binding sites for 4, in chain A (1 in
chain B). In this structure (PDB 2z4y, chain 1A), Figure 2D,
one inhibitor molecule binds to the FPP site while a second
binds to the IPP-GGPP product/inhibitor site, the same
binding pattern as seen previously with 8.9 There is, therefore,
some variability in how bisphosphonates bind to GGPPS and,
indeed, this variability is also seen with binding of the GGPP
product where in earlier work,9 we found GGPP bound to
the IPP-FPP site (Figure S1D, Supporting Information) in
Figure 1. GGPP biosynthesis pathway. GGPP is formed by condensa-
tion of FPP and IPP by the enzyme GGPPS. The GGPPS product can
then be used prenylate cell signaling proteins such as Ras, Rac, and
Rap and is also the precursor of many other isoprenoids.

5596 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Chen et al.
Chart 4
contrast to the FPP-GGPP binding mode seen with the human
enzyme.10 However, in a new structure (PBD 2z4v), we find
the same binding pattern as seen in human GGPPS (Figure
2E, pink), due presumably to the slight changes in crystal-
lization conditions (higher [Mg2+]) employed in the current
investigation. Ligand interaction diagrams for all of the new
structures are shown in Figure S3, Supporting Information.
We find that bisphosphonate groups of both 4 (P21) and 11
coordinate to three Mg2+, which in turn are coordinated to
the DDXXD repeats. The bisphosphonate backbones form
H bonds with Arg84, Lys169, and Lys233. In a second
structure of 4, 4 can bind to two sites; one in the FPP site,
interacting with the side chains of Arg84, Lys169, Gln206
and Lys233, the other one in the GGPP product site,
interacting with Arg39, His68, Arg84, and Arg85. In the case
of 10, there is no Mg2+ observable, and the bisphosphonate
backbones interact with the side chains of Asp75, Arg84,
and Lys233 via hydrogen bonds.
The wide range of binding modes observed experimentally
suggests the possibility that two sites might be occupied by
the 1,1-disubstituted bisphosphonates, such as 5, 12, which
could bind in a “V-shaped” conformation that could easily
occupy both the FPP-FPP and FPP-GGPP sites (Figure
S1A,B, Supporting Information) or the IPP-GGPP and IPP-
FPP sites (Figure S1C,D). The former turns out to be the
case as can be seen in Figure 3A, where we see the location
of the two geranyl chains in 5 (plus Mg2+) bound to the
GGPPS dimer, illustrating binding to the FPP and GGPP sites.
Close-up views of 5, 12, and 13 (PDB 2z4w, 2z4z, and 2z78)
superimposed on the FsPP and GGPP ligands in GGPPS
(PDBs 2e8t, 2z4v) are shown in Figure 3B-D (ligand
interaction plots are provided in Figures S4A-C, Supporting
Information) and clearly indicate that each of these “V-
shaped” molecules bind to both the FPP and GGPP (substrate,
inhibitor) sites. A major difference between the three
structures is, however, that the number of Mg2+ varies (Figure
3B-D). The 2 Mg2+ (green) that are seen in the 5 structure
occupy the same positions as those seen in the 3 Mg2+
structures (Figures 2B,C), but there are no Mg2+ seen in the
two other structures, which might, however, simply reflect
the relatively low pH (∼4.8) required for crystallization,
making Mg2+-binding relatively weak. Data collection and
refinement statistics for 5, 12, and 13 bound to GGPPS are
shown in Table 4.
the Supporting Information. We find that the long chain
phosphonium bisphosphonate binds to the GGPP inhibitor
site while the short chain n-alkyl species binds to the FPP
site, as illustrated in Figure S5A,B of the Supporting
Information. Electron densities for all structures reported here
are shown in Figure S6 of the Supporting Information.
These results are clearly of interest in the context of deducing
structure-activity relationships for GGPPS inhibition, not least
because there are clearly several different types of binding that
might be anticipated for a given compound, which would be
expected to complicate the use of purely ligand-based predic-
tions of activity. We thus next consider how these crystal-
lographic results might be used to guide QSAR investigations.
Bisphosphonate Activities and QSAR. We investigated the
activities in GGPPS inhibition of the 60 bisphosphonates
(plus 1 phosphosulfonate, 61) shown in Figure 4. The most
potent inhibitors (Table 1) all contained long alkyl chains,
with short chain species, conventional bisphosphonates such
as pamidronate (62), risedronate (64), and alendronate (65),
having essentially no activity (>100 µM). Two phosphonate
groups were essential for activity (e.g., 8, IC50 ) 4 µM; the
phosphonosulfonate analogue 61 had no detectable activity),
but unlike FPPS inhibition, a cationic center was not essential
for good activity, e.g., 15, IC50 ) 0.3 µM; 19, IC50 ) 0.6
µM. Likewise, the neutral side chain species 5 had an IC50
) 1 µM. So the cationic species are not acting as carboca-
tionic transition state/reactive intermediate analogues as they
do in FPPS inhibition.20
To interpret the activity results (Table 1), it seemed likely
that it would be necessary to deduce the most probable binding
site for each inhibitor because the activity of a given compound
would be expected to depend on which site it occupied. To do
this, we first investigated the docking poses (using Glide21) of
12 inhibitors whose crystallographic structures were known in
order to validate this approach. Using a superposition of 12
bisphosphonate-GGPPS complexes (Figure 5A), we selected
five different structures to serve as target receptors. They
contained zero, one, two (2 structures), or all three Mg2+ ions
and had a variety of protein side chain (Leu138, Arg39, and
Arg85) conformations (Figure 5B and Table S2 of the Sup-
porting Information). Of the five structures, the GGPPS contain-
ing compound 8 yielded the best docking results (Table S2,
Supporting Information), most likely because it had two large
bound ligands, with the protein (PDB 2e93) side chain orienta-
tions (Figure 5B, cyan) permitting binding to many different
inhibitors.
In addition to these structures, we also determined the
structures of a shorter (C6) n-alkyl inhibitor (10) as well as
that of a potent long chain phosphonium inhibitor (9), bound
to GGPPS (Figure S5A,B, Supporting Information). Data
collection are refinement statistics are shown in Table S1 in
Twelve inhibitors whose active site conformations were
known crystallographically were then docked into each target
receptor in a cross docking approach, specifying no constraints,

Geranylgeranyl Diphosphate Synthase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5597
Figure 3. GGPPS dimer structure and binding motifs of branched
bisphosphonates. (A) GGPPS dimer structure with 5 bound (PDB
2z4w). (B,C) Disubstituted of “V-shape” bisphosphonate GGPPS
inhibitors. The three inhibitors (5, 12, 13) bind in a very similar manner,
despite the varying numbers of Mg2+ ions present. The hydrophobic
tails extend into both the FPP as well as the GGPP sites. (B) 5 (green)
binds with two Mg2+ ions (green) (PDB 2z4w). (C) 12 (yellow) binds
without any Mg2+ apparent (PDB 2z4z). (D) 13 (pink) likewise binds
to both the FPP and GGPP side chain sites and no Mg2+ is apparent
(PDB 2z78).
to the GGPPS 8 structure. A surface representation of all X-ray
and predicted binding poses is presented in Figure 6A and
clearly shows the presence of two different binding sites. In all
cases, the lowest energy poses of each inhibitor were those that
might be deduced based on chemical similarity to known
inhibitors. However, despite the good predicted binding poses,
the docking score (G Score in Glide) was only weakly correlated
with GGPPS inhibition (r2 ) 0.3, Figure 6B). A possible
explanation for this is that, although the calculations were
performed using the highest level of accuracy available in Glide
4.521 (extra-precision mode), the receptor is still treated rigidly,22
so the accuracy of the scoring function might be compromised.
To improve the docking score, we next investigated the use of
the linear interaction approximation (LIE)23 method in the
Liaison24 program. This performs molecular mechanics simula-
tions on the bound and free states of the enzyme-ligand
complex using a continuum-solvent model24 and takes into
account receptor flexibility. However, improvements were small:
r2 ) 0.4, Figure 6C. Finally, we constructed a reparameterized
energy function based on the experimentally determined activi-
ties, computed energies, and the lipophilicity molecular descrip-
tor, SlogP.25 This resulted in a further improvement with
experiment: r2 ) 0.6, q2 ) 0.6, F ) 85, Figure 6D, Table 1,
and by using a training and test set approach, we found that
IC50 values could be predicted within a factor of 3× (over a
range of 4000× in activity). Full output results are shown in
Table S4 of the Supporting Information. So, while promising,
these results are clearly worse than those we have obtained
previously using CoMSIA or CoMFA (comparative molecular
field analysis) methods for bisphosphonate inhibition of a variety
of enzymes in which only a single site is occupied.16,26,27
We thus next investigated the use of CoMSIA methods to
predict activity. Structurally similar compounds were aligned
to their closest X-ray structure, followed by a flexible
common feature superposition, as described previously.16 The
alignment and fields, Figure 7A-C, resulted in a q2 ) 0.59,
Figure 2. GGPPS dimer structure and binding motifs. (A) GGPPS
dimer structure with 4 bound to Asp-rich active site (PDB 2z4x). (B)
4 (yellow, PDB 2z4x) shown bound in the FPP site chelating 3 Mg2+
ions (yellow). Also shown are IPP + FsPP (PDB 2e8t), FPP (PDB
2e90), and GGPP (PDB 2z4v). (C) Close overlap is observed between
locations of Mg2+ ions in FPPS and GGPPS structures (PDB 2f8z and
2z4x). (D) 4 (yellow, PDB 2z4y) observed in two sites in the absence
of Mg2+. (E) GGPP can bind in two distinct orientations, one (reported
earlier, PDB 2e8v) structure extending from the IPP site to the FPP
site (yellow), the second orientation (magenta, PDB 2z4v) is that
reported in this work and is the same as that found with human GGPPS
(PDB 2q80).
using Glide.21 The results obtained were then compared with
the known crystallographic results, with the GGPPS 8 “Mg2+
”
BC
structure (Figure 5B, PDB 2e93) providing the best overall
results (Table S2, Supporting Information). Using this structure,
we found, on average, a 1.3 Å rmsd between the X-ray and
docked poses, with the correct binding site predicted in all cases.
We observed slightly worse results for the other receptor
containing 2 Mg2+ and very poor results for each of the other
receptors (full details shown in Supporting Information Tables
S2 and S3). Interestingly, the GGPPS 8 structure (which gave
the best docking results) contains two bulky bisphosphonates
and can presumably accommodate ligands having diverse
structures.
We next docked 51 additional GGPPS inhibitors, having a
large range of activity (∼0.1-150 µM, Figure 5 and Table 1)

5598 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Chen et al.
Table 1. Glide, Liaison and Scoring Function Training and Test Set Predictions for GGPPS Inhibition
computed values
test set pIC50 predictionsb
predicted residual
IC50
pIC50
(M)
glide
score
liaison
score
liaison liaison
liaison
Uele
compda (µM)
Uvdw
Ucav
SlogP training
0.20
1
2
3
4
5
9
0.1
0.3
0.3
0.3
0.4
0.5
0.6
0.7
0.7
0.7
0.8
0.9
0.9
1.0
1.2
1.2
1.3
1.4
1.5
1.8
1.9
2.1
2.1
2.3
2.3
2.5
2.7
2.7
2.7
3.0
3.3
4.0
4.1
4.2
4.5
4.7
6.8
8.8
8.9
7.0
6.6
6.5
6.5
6.4
6.3
6.2
6.2
6.2
6.1
6.1
6.1
6.0
6.0
5.9
5.9
5.9
5.9
5.8
5.8
5.7
5.7
5.7
5.6
5.6
5.6
5.6
5.6
5.6
5.5
5.5
5.4
5.4
5.4
5.3
5.3
5.2
5.1
5.0
5.0
5.0
5.0
4.8
4.6
4.3
4.2
4.2
4.1
4.1
4.1
4.0
4.0
4.0
4.0
3.9
3.8
3.8
3.7
3.5
3.5
3.4
-11.26 -15.52
-13.94 -15.59
-12.25 -16.60
-11.24 -14.59
-11.13 -14.84
-12.60 -15.02
-11.18 -16.79
-11.11 -14.31
-10.94 -13.03
-13.40 -17.46
-14.50 -19.69
-10.04 -14.75
-11.34 -13.58
-12.97 -16.97
-11.54 -17.07
-11.08 -14.66
-11.43 -16.75
-11.32 -13.74
-13.30 -14.90
-10.83 -15.60
-12.18 -15.74
-10.50 -13.77
-13.32 -15.81
-15.81 -18.12
-12.15 -14.46
-10.92 -18.05
-12.40 -15.19
-14.14 -18.65
-12.24 -14.27
-12.34 -15.57
-9.40 -14.24
-16.72 -18.09
-14.23 -17.76
-11.76 -15.42
-12.27 -15.37
-14.05 -15.94
-12.71 -14.89
-10.46 -12.08
-12.06 -15.93
-10.26 -13.80
-12.50 -14.99
-8.60 -11.85
-8.02 -12.84
-12.29 -15.61
-8.17 -11.91
-7.66 -12.43
-7.43 -12.88
-9.73 -12.21
-9.97 -14.74
-11.57 -13.04
-10.77 -15.85
-8.33 -11.90
-7.98 -13.63
-11.02 -13.21
-5.81 -11.60
-8.40 -12.09
-7.82 -12.89
-6.64 -11.86
-10.36 -12.91
-8.62 -12.23
-11.97 -11.82
25.97
23.47
21.84
30.16
27.86
23.07
10.20
33.90
52.57
14.85
3.81
37.02
14.00
26.97
19.71
29.11
21.15
30.78
29.51
22.78
34.93
31.69
23.22
17.53
36.16
16.50
27.07
1.95
43.79
25.75
38.18
47.42
32.46
40.03
34.33
24.00
34.08
48.46
27.08
42.22
17.59
56.25
44.04
38.97
47.88
54.44
49.63
63.30
34.87
43.21
22.98
59.35
44.16
50.27
52.98
54.71
51.06
53.71
46.65
58.53
64.49
-0.92 -588.38
6.2
5.8
5.8
5.8
5.9
5.8
6.1
5.5
4.7
5.9
6.7
5.6
5.9
6.1
5.9
5.8
5.6
5.4
5.9
5.4
5.8
5.4
5.6
6.7
6.0
5.3
5.3
5.6
5.2
5.9
5.3
5.0
5.1
4.5
5.5
6.0
5.4
4.7
5.5
4.8
5.4
4.5
4.5
5.6
4.6
4.1
4.5
3.2
5.1
4.9
5.3
3.9
4.7
4.3
4.5
4.3
4.5
3.8
4.1
4.0
3.8
5.9 6.2 6.3 6.3 6.2
5.7 5.8 5.8 5.8 5.8
5.6 5.9 5.8 5.9 5.9
5.2 5.9 5.8 6.1 5.9
5.6 5.9 5.9 5.9 5.9
5.9 5.8 5.9 5.7 5.8
6.0 6.2 6.0 6.2 6.2
5.3 5.5 5.5 5.5 5.5
5.1 4.6 4.9 4.6 4.6
5.9 6.0 5.9 6.0 6.0
6.4 6.7 6.6 6.7 6.8
5.5 5.6 5.7 5.7 5.6
5.8 6.0 5.8 6.1 6.1
6.3 6.0 6.2 5.9 6.0
5.6 6.0 5.9 6.0 6.0
5.6 5.8 5.8 5.8 5.8
5.7 5.6 5.6 5.6 5.7
5.3 5.4 5.4 5.5 5.5
5.9 5.8 6.0 5.8 5.8
5.4 5.5 5.4 5.5 5.6
5.9 5.7 5.9 5.6 5.7
5.3 5.4 5.4 5.4 5.4
6.1 5.6 5.7 5.6 5.7
6.6 6.6 6.8 6.5 6.6
6.1 5.9 6.2 5.9 5.9
5.4 5.4 5.3 5.4 5.5
5.3 5.3 5.4 5.3 5.4
6.2 5.7 5.7 5.5 5.9
5.3 5.1 5.3 5.1 5.1
5.8 5.9 6.0 5.9 5.9
5.2 5.2 5.3 5.3 5.3
5.6 4.9 5.3 4.8 4.9
5.6 5.0 5.2 4.9 5.1
4.7 4.5 4.6 4.6 4.7
5.6 5.4 5.6 5.4 5.5
5.8 6.0 6.1 6.0 6.0
5.1 5.4 5.4 5.5 5.4
4.8 4.6 4.8 4.7 4.7
5.5 5.5 5.6 5.5 5.6
5.0 4.8 4.9 4.8 4.9
5.7 5.5 5.4 5.4 5.6
4.7 4.3 4.6 4.4 4.4
4.9 4.5 4.6 4.5 4.6
5.9 5.4 5.8 5.4 5.5
4.6 4.5 4.6 4.6 4.6
4.3 4.0 4.1 4.1 4.1
4.6 4.5 4.6 4.5 4.5
3.2 3.1 3.2 3.3 3.3
5.0 5.1 5.1 5.2 5.2
4.3 4.9 4.8 5.2 5.0
5.0 5.4 5.3 5.6 5.5
4.2 3.8 4.0 3.9 3.9
4.8 4.6 4.7 4.7 4.7
4.5 4.2 4.4 4.3 4.3
4.6 4.4 4.6 4.6 4.5
4.3 4.2 4.3 4.4 4.3
4.5 4.4 4.6 4.6 4.5
3.9 3.8 3.9 4.0 3.9
4.4 4.0 4.2 4.1 4.2
4.4 3.9 4.2 4.0 4.0
3.6 3.7 3.9 3.9 3.8
6.2
5.8
5.9
5.6
5.8
5.8
6.0
5.5
4.8
6.0
6.8
5.6
5.8
6.0
6.0
5.8
5.6
5.5
5.9
5.4
5.8
5.4
5.7
6.6
6.2
5.4
5.4
5.8
5.1
5.9
5.2
5.2
5.2
4.6
5.6
6.0
5.5
4.6
5.6
4.9
5.5
4.4
4.6
5.9
4.6
4.0
4.5
3.1
5.1
5.2
5.5
4.0
4.7
4.3
4.6
4.2
4.5
3.9
4.3
4.3
3.9
0.8
0.7
0.7
0.9
0.6
0.5
0.3
0.6
1.4
0.2
-0.6
0.4
0.3
0.0
-0.1
0.1
0.3
0.4
0.0
0.3
0.0
0.3
-0.1
-1.0
-0.5
0.2
0.2
-0.2
0.5
-0.4
0.3
0.2
0.2
0.8
14
15
16
17
18
19
20
4
21
13
22
23
5
24
25
26
27
28
29
12
30
31
32
33
34
35
6
11
36
37
8
38
39
40
41
42
43
44
45
7
10
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
62
63
64
65
0.97 -550.66 -1.05
0.72 -582.70 -1.23
-1.64 -541.97 -2.56
-0.16 -541.31 -1.06
1.49 -498.16 -0.39
1.35 -499.39 -0.92
0.27 -554.07 -1.84
0.86 -391.39 -2.93
1.62 -476.31 -1.10
1.07 -622.40
1.26
-0.61 -524.48 -1.36
0.73 -317.90 -2.54
0.87 -546.14
1.54
0.49 -541.89 -1.52
0.23 -565.45 -0.83
2.17 -551.14 -1.19
0.56 -473.26 -2.52
0.33 -552.59 -0.05
1.79 -529.85 -2.42
0.39 -557.13
0.42
0.90 -513.18 -2.40
1.77 -326.88 -1.10
0.13 -631.08
2.88
0.87
-0.58 -496.32
3.32 -548.30 -2.64
2.08 -600.40 -1.90
5.59 -534.20 -0.22
0.50 -507.04 -1.76
0.37 -565.66 -0.45
0.45 -517.54 -2.39
1.40 -467.20 -0.87
3.16 -484.01 -1.27
2.22 -438.34 -4.75
1.11 -573.12 -0.84
0.41 -635.98 -0.06
0.26 -571.84 -2.62
1.10 -488.47 -3.56
1.46 -579.42 -1.51
1.52 -478.65 -3.08
3.07 -444.31 -2.09
0.98 -504.43 -3.71
2.38 -476.51 -3.76
-0.2
-0.6
-0.3
0.5
-0.5
0.1
-0.5
0.5
0.2
-1.3
-0.4
0.2
10
11
11
14
28
54
66
66
74
0.97 -526.22
0.57
1.04 -478.59 -4.44
1.73 -443.12 -5.80
1.19 -500.59 -4.34
2.65 -484.91 -9.02
0.96 -500.12 -3.49
-0.90 -515.34 -5.59
1.21 -548.60 -3.76
2.13 -461.76 -5.52
1.51 -505.70 -3.91
2.06 -483.23 -4.74
0.48 -404.20 -4.77
0.88 -445.35 -5.43
0.81 -506.09 -4.75
2.12 -455.27 -7.05
3.31 -508.64 -5.04
1.87 -433.67 -5.04
1.00 -637.28 -6.66
-0.4
1.0
79
83
94
-1.0
-1.1
-1.5
0.0
-0.7
-0.4
-0.6
-0.4
-0.7
-0.2
-0.8
-0.8
-0.5
100
107
108
118
141
169
180
329
350
440
a Structures of all inhibitors shown in Figure . b bold values indicated compounds not included in training set.
r2 ) 0.8, F ) 73, n ) 61, Figure 7D. Full results are shown
in Tables S5 and S6 of the Supporting Information. Although
these results are quite promising, it seemed logical to see to
what extent they might be improved upon by using a receptor-
guided alignment28 based on the Glide docking results
discussed above. The CoMSIA fields so obtained are shown
in Figure 7E-G, and using this receptor-guided alignment,
we found that all three parameters, q2, r2, and F, improved.
The q2 parameter increased from q2 ) 0.6 to q2 ) 0.7; r2
from 0.8 to 0.9, and F from 73 to 166, Figure 7H, with
predictions now within a factor of 2× (over a 4000× range
in activity). Full results are shown in Table 2 and Table S7
in the Supporting Information. Also of importance, the
receptor-guided alignment could be generated rapidly, and

Geranylgeranyl Diphosphate Synthase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5599
Figure 4. GGPPS inhibitors investigated, rank-ordered in terms of decreasing activity from top left to bottom right.
in a uniform manner, for all compounds. Despite the slight
differences in the predictivity of the models obtained from
the two alignments, the CoMSIA fields produced are very
similar, with a steric-favorable region (green) coincident with
the end of the FPP-GGPP sites in the protein, consistent with
the observation that long alkyl species typically provide
maximum inhibitory effects. Additionally, the hydrophobic-
favorable CoMSIA fields (orange) are very prominent,

5600 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Chen et al.
Table 2. Receptor-Guided Alignment CoMSIA Training and Test Set Predictions for GGPPS Inhibitiona
GGPPS pIC50 predictions
test sets
experimental
compd
IC50 (µM)
pIC50 (M)
training
1
2
3
4
5
predicted
residual
9
0.10
0.28
0.28
0.35
0.40
0.51
0.59
0.66
0.71
0.72
0.76
0.89
0.89
0.98
1.15
1.23
1.26
1.38
1.48
1.74
1.86
2.14
2.14
2.34
2.51
2.51
2.51
2.69
2.69
3.02
3.24
3.98
4.07
7.0
6.6
6.6
6.5
6.4
6.3
6.2
6.2
6.2
6.1
6.1
6.1
6.1
6.0
5.9
5.9
5.9
5.9
5.8
5.8
5.7
5.7
5.7
5.6
5.6
5.6
5.6
5.6
5.6
5.5
5.5
5.4
5.4
5.4
5.3
5.3
5.2
5.1
5.1
5.0
5.0
5.0
4.9
4.6
4.3
4.2
4.2
4.1
4.1
4.1
4.0
4.0
4.0
4.0
3.9
3.9
3.8
3.7
3.5
3.5
3.4
6.6
6.5
6.3
6.7
6.3
6.3
6.4
6.3
5.6
6.0
6.1
5.9
6.0
5.8
5.9
5.6
6.2
5.9
5.7
5.5
5.8
5.7
5.7
5.7
6.0
5.8
5.3
5.3
5.6
5.5
5.8
5.2
5.6
5.7
5.3
5.4
4.8
5.1
5.0
5.0
5.3
4.8
4.6
4.4
4.3
4.3
4.1
4.0
4.5
4.3
3.7
3.7
4.0
3.9
4.5
4.5
4.2
3.9
3.4
3.4
3.3
0.68
0.93
5
6.4
6.1
6.0
6.3
6.3
6.6
6.3
6.2
5.6
6.0
6.3
5.9
5.0
5.6
5.8
5.6
5.9
5.7
5.9
5.2
6.0
5.5
5.9
5.5
6.3
5.8
5.0
5.8
5.3
5.8
5.5
4.9
5.8
5.7
5.8
5.8
4.6
4.9
5.2
4.9
5.2
4.6
4.2
4.6
4.0
4.3
4.0
3.9
4.7
4.2
4.4
4.0
4.0
4.2
4.4
4.3
4.1
3.7
3.4
3.6
3.6
0.66
0.89
3
6.7
6.6
6.4
6.5
6.2
6.3
6.5
6.2
5.7
5.9
6.1
6.0
6.0
6.0
5.9
5.9
5.9
6.0
5.6
5.5
5.9
5.8
5.8
5.9
6.9
5.6
5.4
5.5
5.8
5.5
6.0
5.3
5.5
5.5
5.4
5.4
4.7
5.1
5.3
4.8
5.1
5.0
4.6
4.6
4.4
4.4
4.4
4.0
6.0
4.1
3.8
3.7
4.1
4.0
4.7
4.4
4.2
3.8
3.6
3.5
3.3
0.78
0.96
5
6.5
6.5
6.3
6.7
6.3
6.4
6.4
6.3
5.7
6.0
6.1
5.8
6.1
5.8
6.0
5.6
6.1
5.9
5.8
5.6
5.9
5.7
5.7
5.7
5.9
5.8
5.3
5.2
5.6
5.5
5.8
5.3
5.6
5.8
5.3
5.5
4.9
5.1
5.0
5.1
5.7
4.8
4.6
4.5
4.3
4.3
4.1
3.4
4.4
4.2
3.8
3.9
4.1
4.2
4.6
4.4
4.4
3.9
3.4
3.5
3.2
0.67
0.94
4
6.3
6.5
6.1
5.8
6.2
6.4
6.5
6.2
5.8
6.1
6.2
5.6
6.0
5.7
6.1
5.5
6.3
5.4
5.7
5.6
5.9
5.5
5.6
5.8
6.2
5.7
4.9
5.5
5.7
5.7
5.5
5.2
5.5
5.5
5.8
5.6
4.6
4.9
5.0
4.8
5.1
4.6
4.4
4.4
4.3
4.4
4.1
4.0
4.6
4.3
3.8
3.9
4.0
4.1
4.4
4.4
4.3
3.7
3.4
3.6
3.2
0.64
0.91
4
6.5
6.5
5.9
6.7
6.4
6.3
6.4
6.3
5.6
6.0
6.0
5.9
6.0
5.9
6.0
5.5
6.2
5.7
5.6
5.5
5.8
5.8
5.7
5.7
6.0
5.8
5.4
5.3
5.6
5.5
5.7
5.3
5.6
5.6
5.3
5.4
5.0
5.0
5.0
5.2
5.3
4.7
4.7
4.4
4.2
4.5
4.2
4.1
4.4
4.2
3.8
3.8
4.1
3.9
4.4
4.5
4.1
3.8
3.3
3.4
3.3
0.62
0.94
5
6.5
6.1
5.9
5.8
6.2
6.4
6.4
6.2
5.7
5.9
6.0
5.6
5.0
5.8
6.0
5.6
5.9
5.4
5.6
5.2
5.9
5.7
5.7
5.8
6.9
5.7
5.0
5.8
5.6
5.7
6.0
5.3
5.6
5.8
5.5
5.8
4.6
5.1
5.3
5.2
5.7
4.7
4.2
4.5
4.3
4.5
4.4
3.4
6.0
4.3
4.4
3.9
4.1
4.2
4.7
4.4
4.4
3.8
3.4
3.6
3.3
0.5
0.4
0.7
0.6
0.2
-0.1
-0.2
0.0
0.5
0.2
0.1
0.5
1.0
0.2
-0.1
0.4
0.0
0.4
14
15
16
17
18
19
20
4
21
13
22
23
5
24
25
26
27
28
29
12
30
31
32
33
34
35
6
11
36
37
8
38
39
40
41
42
43
44
45
7
10
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
62
63
64
65
0.2
0.5
-0.2
-0.1
-0.1
-0.2
-1.3
-0.1
0.6
-0.3
-0.1
-0.1
-0.6
0.1
-0.2
-0.5
-0.2
-0.5
0.6
-0.1
-0.2
-0.2
-0.8
0.3
4.17
4.57
5.01
6.31
8.71
8.91
10.00
11.22
11.22
14.13
27.54
53.70
66.07
66.07
74.13
79.43
79.43
93.33
100.00
107.15
107.15
117.49
141.25
169.82
181.97
316.23
331.13
436.52
q2
0.7
0.1
0.0
-0.3
-0.2
0.7
-1.9
-0.2
-0.4
0.1
-0.1
-0.2
-0.8
-0.5
-0.6
0.0
0.1
-0.2
0.0
r2
N
F
n
166
61
0.22
0.36
0.23
0.20
122
51
0.25
0.38
0.20
0.18
207
51
0.24
0.38
0.22
0.17
148
51
0.21
0.35
0.23
0.20
117
51
0.22
0.35
0.22
0.21
146
51
0.20
0.35
0.24
0.21
% steric
% hydrophobic
% donor
% acceptor
a Structures of all inhibitors shown in Figure 1. Bold values indicated compounds not included in training set. N ) number of components; n ) number
of training set compounds.

Geranylgeranyl Diphosphate Synthase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5601
Figure 6. Docking and linear interaction approximation results. (A)
van der Waals surface representation of the docked poses for all of the
GGPPS inhibitors investigated, showing two distinct hydrophobic
binding pockets but only a single polar (bisphosphonate) binding domain
(magenta). (B) Correlation of Glide score with GGPPS pIC50 showing
r2 ) 0.3. (C) Correlation plot of Liaison score against GGPPS pIC50
showing r2 ) 0.4. (D) GGPPS pIC50 experiment vs predicted activity
for reparameterized scoring function showing q2 ) 0.6, r2 ) 0.6, F )
85.
tion produced models with varying degrees of predictive
utility. Docking scores performed worst (r2 ) 0.3), a linear
interaction approximation (LIE) method had moderate pre-
dictivity (q2 ) 0.6, r2 ) 0.6), while a receptor-guided
CoMSIA alignment method performed best overall (q2 ) 0.7,
r2 ) 0.9). Given the selective GGPPS inhibition of the
dialkenyl bisphosphonates, and their activity in cell-based
assays, the availability of these new crystal structures is of
broad general interest in the context of the development of
potent and specific tumor cell growth inhibitors, where
GGPPS inhibition offers a potentially interesting alternative
to conventional FPPS-based cell growth inhibition by bis-
phosphonates, not least because such hydrophobic species
are expected to bind only weakly to bone mineral.29
Figure 5. GGPPS binding site organization. (A) Superimposition of
bisphosphonate inhibitors showing chelation of up to three Mg2+ ions,
and several distinct binding modes. (B) Highly variable residues
surrounding the GGPPS inhibitors bound in the active site. Yellow
residues have large deviations in side chain conformation; gray residues
are representative of those seen other all other structures. The cyan
shading and arrows indicate the side chain conformations that gave
the best docking results.
especially in the receptor-guided alignment (Figure 7G),
consistent again with the high potency of the long alkyl (and
dialkyl) bisphosphonates in GGPPS inhibition.
Experimental Section
Conclusions
Chemicals. All reagents used were purchased from Aldrich
The results we have obtained above are of interest because
they show how n-alkyl and dialkenyl bisphosphonates bind
to, and inhibit, the GGPPS enzyme. All three n-alkyl
bisphosphonates bind to the FPP site. Surprisingly, however,
in one case we find that the GGPP site is also occupied, and
in two structures (PDBs 2z4x and 2z52) of the 24 GGPPS
structures now investigated, we see for the first time the
presence of three Mg2+, located in essentially the same
positionasfoundinmanyFPPS-bisphosphonatestructures.10–13
The dialkenyl bisphosphonates investigated both bind with
their polar groups in the FPP/GGPP polar binding site, but
one side chain occupies the FPP hydrophobic site, while the
second occupies the GGPP inhibitor site, basically as seen
with the doubly occupied n-alkyl bisphosphonate structure.
A similar arrangement is seen with another, biphenyl-
containing, “V-shaped” inhibitor. A computational investiga-
(Milwaukee, WI). The purities of all compounds were routinely
1
monitored by using H and 31P NMR spectroscopy at 400 or 500
MHz on Varian (Palo Alto, CA) Unity spectrometers using, in some
instances, absolute spin-count quantitative analyses. The elemental
analysis results for all new compounds are provided in the
Supporting Information (Table S8). Samples of bisphosphonates
7, 11, 21, 23, 25, 28, 31, 37, 39, 43, 45, 46, 48, 49, 50, 52, 53, 55,
56, 57, 58, 59, 60, 62, 64, 65 were available from previous work.16
Pyridinium-1-yl bisphosphonates 13, 15, 17, 18, 20, 24, 26, 27,
29, 34, 35, 36, 42, 44, 54, 63 were synthesized based on our
published procedures.27
2-(N-dodecyl, N-methylaminoethyl)ethylidene-1,1-bisphospho-
nic Acid (16). 16 was prepared using the following scheme (Chart
5 Dodecyl methylamine (1 g, 5 mmol) was mixed with tetraethyl
vinylidene bisphosphonate (1.5 g, 5 mmol) and stirred for 7 days.
CH2Cl2 (5 mL) was added, followed by TMSBr (6 g, 40 mmol).
Water workup afforded 16 as a white powder. Anal. (C15H33-

5602 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Chen et al.
Figure 7. CoMSIA fields and results for common feature and receptor-guided alignments. (A-D) Common feature alignment, fields, and prediction
statistics. (A) Common feature alignment. (B) CoMSIA steric fields showing favorable regions in green and disfavored in yellow. (C) CoMSIA
hydrophobic fields showing favorable regions in orange and disfavored in white. (D) Correlation plot of experimental GGPPS pIC50 vs predicted
activity, showing q2 ) 0.6, r2 ) 0.8, and F ) 73. (E-H) Receptor-guided alignment, fields, and prediction statistics. (E) Receptor-guided alignment.
(F) CoMSIA steric fields showing favorable regions in green and disfavored in yellow. (G) CoMSIA hydrophobic fields showing favorable regions
in orange and disfavored in white. (H) Correlation plot of experimental GGPPS pIC50 vs predicted activity, showing q2 ) 0.7, r2 ) 0.9, and F )
166.
Table 3. Data Collection and Refinement Statistics for GGPPS-Bisphosphonate Crystals
GGPPS-Mg-4 2z4x
GGPPS-Mg-11 2z52
GGPPS-Mg-4 2z4y
GGPPS-Mg-GGPP 2z4v
Data Collection
space group
P21
30-1.90
(1.97-1.90)
P212121
30-2.13
(2.21-2.13)
P212121
30-2.10
(2.18-2.10)
P212121
30-1.86
(1.93-1.86)
resolution (Å)a
unit cell dimensions
a (Å)
b (Å)
82.41
47.87
91.8
46.09
116.29
128.41
45.95
116.21
126.02
47.16
115.94
128.4
c (Å)
ꢀ (deg)
110.86
no. of reflections
observed
unique
completeness (%)
Rmerge (%)
I/s(I)
166199 (15414)
51962 (5138)
98.4 (98.4)
6.1 (41.8)
264434 (25207)
39542 (3878)
99.9 (99.7)
6.9 (32.5)
221099 (20940)
40023 (3951)
99.4 (99.7)
5.7 (45.3)
313204 (30826)
59802 (5928)
99.4 (99.9)
3.5 (10.2)
42.4 (17.9)
18.8 (3.1)
26.0 (7.6)
29.1 (4.3)
Refinement
no. of reflections
Rwork (%)
Rfree (%)
49646 (4064)
19.6 (28.5)
24.6 (31.8)
38397 (3627)
17.2 (17.9)
21.0 (23.0)
38785 (3538)
18.1 (19.7)
23.2 (25.6)
59160 (5770)
17.3 (18.6)
21.7 (23.4)
geometry deviations
bond lengths (Å)
bond angles (deg)
no. of all protein atoms
mean B values (Å2)
no. of all cofactor atoms
mean B values (Å2)
no. of water molecules
mean B values (Å2)
Ramachandran plot (%)
most favored
0.018
1.7
4659
32.5
42
23.1
667
52
0.019
1.7
4848
28.7
48
28.2
547
43.3
0.019
1.7
4670
37
0.021
1.8
5129
27
55
63
48.7
391
49.3
28.5
819
45.5
95.5
4.5
0
96.8
3.2
0
95.7
4.3
0
96.5
3.5
0
additionally allowed
generously allowed
a Values in the parenthesis are the highest resolution shells.

Geranylgeranyl Diphosphate Synthase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5603
Chart 5
Chart 6
Chart 7
Phosphonium-1-yl bisphosphonates (9, 22) were synthesized from
the corresponding dimethylalkylphosphines via Michael addition
in TFA (Chart 7):
2-(Dodecyl-dimethyl phosphonium-1-yl)ethylidene-1,1-bispho-
sphonic Acid (9). Dodecyl dimethylphosphine (1.2 mmol) and
vinylidene-1, 1-diphosphonic acid (1.1 mmol) were dissolved in
TFA (5 mL) and refluxed overnight under N2. Upon removal of
the solvent under reduced pressure, the residue was washed with
hexane, ether, and then acetone to afford 9 as a white powder. Anal.
1
NNa2O6P2 ·NaBr) C, H, N. 1H NMR (D2O, 400 MHz): δ 2.86-3.12
(4H, m), 2.57 (3H, s), 1.95-2.20 (1H, m), 1.41-1.49 (m, 2H),
0.91-1.18 (18H, m), 0.65 (3H, t, J ) 6.5 Hz). 31P NMR (D2O,
162 MHz): δ 19.9.
(C16H35Na2O6P3) C, H. H NMR (D2O, 400 MHz): δ 2.36-2.45
(2H, m), 2.00-2.18 (2H, m), 1.68 (6H, d, J ) 13.5 Hz), 1.65-1.69
(m, 1H), 1.36-1.45 (2H, m), 1.25-1.27 (2H, m), 1.00- 1.20 (16H,
m) 0.66 (3H, t, J ) 6.5 Hz). 31P NMR (D2O, 162 MHz): δ 31.1 (t,
J ) 22 Hz), 17.9 (d, J ) 22 Hz).
2-(5-Decyloxypyridin-3-yl)ethylidene-1,1-bisphosphonic Acid (40).
40 was prepared using the following scheme (Chart 6 To a
suspension of NaH (480 mg, 60%, 12 mmol) in THF at 0 °C was
added tetraethyl methylene bisphosphonate (2.88 g, 10 mmol). The
mixture was allowed to stir at room temperature for 30 min. Then
3-decyloxy-5-chloromethyl-pyridine (2.83 g, 10 mmol) followed
by NaI (12 mmol) were added, and the reaction mixture stirred for
2 h at 80 °C. The mixture was quenched with saturated aq NH4Cl.
After extraction with ether, the pyridine-bisphosphonate was purified
by flash chromatography as a colorless oil (50% yield). Direct
dealkylation with TMSBr afforded 40 as a white powder. Anal.
2-(Octyl-dimethyl phosphonium-1-yl)ethylidene-1,1-bisphospho-
nic Acid (22). 22 was prepared in the same way as 9. Anal.
(C12H29O6P3) C, H. 1H NMR (D2O, 500 MHz): δ 2.36-2.46 (2H,
m), 2.00-2.18 (2H, m), 1.68 (6H, d, J ) 14 Hz), 1.68-1.79 (m,
1H), 1.36-1.47 (2H, m), 1.25-1.27 (2H, m), 1.00- 1.20 (16H,
m) 0.70 (3H, t, J ) 6.5 Hz). 31P NMR (D2O, 202 MHz): δ 32.1 (t,
J ) 23 Hz), 17.9 (d, J ) 23 Hz).
1-Octyl-1-(3-decyloxybenzyl)-1, 1-bisphosphonic Acid (BPH-
804). BPH-804 was made using the following scheme (Chart 8):
To a suspension of NaH (480 mg, 60%, 12 mmol) in THF at 0 °C
was added tetraethyl methylenebisphosphonate (2.88 g, 10 mmol).
The mixture was allowed to stir at room temperature for 30 min.
Then, 1-bromo-2-octane (1.9 g, 10 mmol) was added dropwise.
After 6 h, more NaH (480 mg, 60%, 12 mmol) was added, followed
by 1-bromoethyl-3-decyloxy-benzene (3.59 g, 11 mmol). The
mixture was stirred overnight and quenched with aqueous NH4Cl.
Chromatography (5% methanol in ethyl acetate) afforded the
disubstituted intermediate as a colorless syrup (35%). Hydrogenation
in methanol in the presence of 10% Pd/C, followed by dealkylation
with TMSBr (8 equivalent), provided 32 as a white powder. Anal.
(C26H50Na4O10P2 ·NaBr) C, H. 1H NMR (D2O, 400 MHz) 6.6-7.0
(4H, m), 3.75 (3H, t, J ) 6.8 Hz), 2.81-3.00 (2H, m), 0.69-1.60
(36H, m). 31P NMR (D2O, 162 MHz): δ 24.25.
1
(C17H31NO7P2 ·H2O). H NMR (D2O, 400 MHz): δ 7.91 (1H, s),
7.82 (1H, s), 7.28 (1H, s), 3.95 (3H, t, J ) 6.4 Hz), 2.90 (2H, td,
J ) 14.8 Hz, 6.8 Hz), 1.93 (1H, tt, J ) 21 Hz, 6.8 Hz), 1.55-1.63
(2H, m), 1.10-1.23 (2H, m), 1.00-1.11(12H, m), 0.63 (3H, t, J )
6.0 Hz). 31P NMR (D2O, 162 MHz): δ 19.6.
3-(2,2-Bisphosphono-ethyl)-5-ethoxy-1-methyl-pyridinium Io-
dide (14). The pyridine-bisphosphonate 66 (1 mmol) was treated
with MeI (5 mmol) in ether (5 mL) overnight. Upon removal of
the solvent, the residue was hydrolyzed in refluxing concentrated
HCl (37%). After removal of the volatile solvent, the concentrated
residue was washed with hexane, ether, and then acetone to afford
1
14 as a gray powder. H NMR (D2O, 400 MHz): δ 8.17 (1H, s),
8.00 (1H, s), 7.83 (1H, s), 4.17 (3H, s), 3.95 (3H, t, J ) 6.4 Hz),
3.01 (2H, td, J ) 15 Hz, 6.8 Hz), 1.89 (1H, tt, J ) 21 Hz, 6.8 Hz),
1.54-1.64 (2H, m), 1.10-1.22 (2H, m), 1.00-1.11(12H, m), 0.63
(3H, t, J ) 6.0 Hz). 31P NMR (D2O, 162 MHz): δ 18.6.
1, 1-(2-Octenyl)ethylidene-1,1-bisphosphonic Acid (12). 12 was
made using the following scheme (Chart 9): To a suspension of
NaH (1 g, 60%, 25 mmol) in THF (15 mL) at 0 °C was added
tetraethyl methylenebisphosphonate (2.88 g, 10 mmol), and the
mixture was allowed to stir at room temperature for 30 min.
1-Bromo-oct-2-enyl (5.7 g, 30 mmol) was then added dropwise
and the mixture stirred overnight and then quenched with aqueous
NH4Cl. Chromatography (2% methanol in ethyl acetate) afforded
the dialkylated intermediate in 65% yield. Direct dealkylation with
2-(5-Dodecyloxypyridin-3-yl)ethylidene-1,1-bisphosphonic Acid
(41). 41 was prepared in the same way as for 40. Anal.
1
(C19H35NO7P2 ·H2O), C, H, N. H NMR (D2O, 400 MHz): δ 7.98
(1H, s), 7.80 (1H, s), 7.38 (1H, s), 3.94 (3H, t, J ) 6.4 Hz), 2.90
(2H, td, J ) 14.8 Hz, 6.8 Hz), 1.99 (1H, tt, J ) 21 Hz, 6.8 Hz),
1.55-1.63 (2H, m), 1.10-1.23 (2H, m), 1.00-1.11(14H, m), 0.63
(3H, t, J ) 6.0 Hz). 31P NMR (D2O, 162 MHz): δ 20.6.
TMSBr provided 12 as
Na2O6P2 ·0.25H2O). H NMR (D2O, 500 MHz): δ 5.5-5.7 (2H,
a
white powder. Anal. (C17H32-
1

5604 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Chen et al.
Chart 8
Chart 9
Chart 10
Chart 11
m), 5.2-5.35 (2H, m), 2.18-2.32 (4H, m), 1.82-1.95 (4H, m),
0.98-1.21 (12H, m), 0.64 (6H, t, J ) 6.0 Hz). 31P NMR (D2O,
202 MHz): δ 23.7. Hydrogenation of the tetraethyl ester in methanol
in the presence of Pd/C (10%), followed by dealkylation with
TMSBr, gave 1,1-octylethylidene-1,1-bisphosphonic Acid (33) as
a white powder. 1H NMR (D2O, 400 MHz): δ 1.40-1.60 (4H, m),
1.18-1.35 (4H, m), 0.98-1.18 (20H, m), 0.64 (6H, t, J ) 6.5 Hz).
31P NMR (D2O, 162 MHz): δ 25.2.
1-Hydroxy-2-[3′-(naphthalene-2-sulfonylamino)-biphenyl-3-yl]-
ethylidene-1,1-bisphosphonic Acid (6). 6 was made as follows
(Chart 10): 3-Aminophenyl boronic acid (6 mmol), methyl 3-bro-
mophenyl acetate (5 mmol), Na2CO3 (15 mmol), Pd(PPh3)4 (50 mg)
in toluene (20 mL), H2O (3 mL), and ethanol (3 mL) were heated
at 110 °C under N2 overnight. After extraction with diethyl ether,
the (3′-amino-biphenyl-3-yl)-acetic acid methyl ester product was
purified by column chromatography (56% yield). (3′-Amino-
biphenyl-3-yl)-acetic acid methyl ester (5 mmol) and 2-naphthalene
sulfonyl chloride (5 mmol) were then dissolved in anhydrous
CH2Cl2 (10 mL), followed by addition of pyridine (10 mmol),
dropwise. After washing with HCl (3N, 3 mL), the sulfonamide
was isolated after chromatography as a syrup (78%). The ester (1
mmol) was then hydrolyzed with 3 N NaOH (1 mL) in methanol
(5 mL) at room temperature for 1 h. After acidification with 2 N
HCl, methanol was removed, and the resulting carboxylic acid
filtered and then washed with water. The dried acid was dissolved
in benzene (5 mL) and oxalyl chloride (2 mmol) added, followed
by one drop of DMF. The reaction mixture was then stirred for
1 h. Upon removal of solvent, the crude acid chloride so obtained
was dissolved in dry THF (5 mL) and P(OTMS)3 (2 mmol) was
added. After 3 h at room temperature, solvent was removed,
methanol-H2O (2 mL, 1:1) was added, and the mixture was stirred
for 30 min. Concentrated aqueous NaOH was then added to
precipitate the target compound, which was washed thoroughly with
methanol, then ether, and dried to afford the bisphosphonic acid as
its sodium salt. Anal. (C24H20NO9P2SNa3) C, H, N. 1H NMR (D2O,
400 MHz): δ 8.18 (1H, s), 6.72-7.80 (14H, m), 3.16 (2H, t, J )
12.5 Hz). 31P NMR (D2O, 162 MHz): δ 20.3.
1-Hydroxy-2-([1′,1′;3′1′′]terphenyl-3′′-yloxy)-ethylidene-1,1-bis-
phosphonic Acid (38). 38 was made according to the following
scheme (Chart 11): 3-Biphenyl boronic acid (6 mmol), 3-bro-

Geranylgeranyl Diphosphate Synthase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5605
Table 4. Data Collection and Refinement Statistics for
GGPPS-bisphosphonate Crystals
Crystallization and Data Collection for GGPPS Complexes.
Native GGPPS crystals for soaking were obtained by using the
hanging drop method (Hampton Research; Laguna Niguel, CA) by
mixing 2 µL of a GGPPS solution (5-10 mg/mL GGPPS in 25
mM Tris-HCl, pH 7.5 and 150 mM NaCl) with 2 µL of mother
liquor (0.08 M CH3COONa, pH 4.6, 16% PEG 4000, 6-10%
glycerol, and 6-10% 1,2-propanediol), and equilibrating with 500
µL of the mother liquor. Crystals grew to 0.5 mm × 0.2 mm × 0.2
mm in 7 days, at room temperature, and then were soaked in
cryoprotectant solution containing 2.5 mM MgCl2, 2.5 mM GGPP
product, or bisphosphonate (4, 5, 10, 11, 12), 0.08 M CH3COONa,
pH 4.6, 20% PEG 4000, 10% glycerol, and 10% 1,2-propanediol,
for 3-12 h.
GGPPS-Mg-5 GGPPS-Mg-12 GGPPS-Mg-13
PDB 2z4W
PDB 2z4z
(PDB) 2z78
Data Collection
space group
P212121
30-2.45
P212121
30-2.09
(2.54-2.45) (2.16-2.09)
P212121
30-2.10
(2.18-2.10)
resolution (Å)a
unit cell dimensions
a (Å)
47.52
47.61
48.33
116.64
129.78
b (Å)
c (Å)
116.55
129.07
116.34
129.03
no. of reflections
observed
134955
(13120)
224260
(19223)
197140
(18388)
For preparing GGPPS-Mg-4 (P21) crystals, 2 µL of GGPPS-
substrate solution (5-10 mg/ml GGPPS, 2.5 mM Tris-HCl, 150
mM NaCl, 2.5 mM MgCl2, 2.5 mM BPH252, pH 7.5) were mixed
with 2 µL of mother liquor (0.08 M CH3COONa, pH 4.6, 12-16%
PEG 4000, 8-10% glycerol, and 10% 1,2-propanediol) and then
were equilibrated with 500 µL of mother liquor by using the hanging
drop method at room temperature. Crystals appeared in 5-7 days
and grew to 0.5 mm × 0.5 mm × 0.1 mm and then were soaked
unique
26937 (2624) 42400 (4090) 42730 (4179)
completeness (%)
Rmerge (%)
I/s(I)
99.6 (99.6)
10.3 (53.2)
14.8 (2.9)
97.8 (95.3)
4.4 (27.4)
40.6 (6.9)
97.5 (97.9)
11.5 (33.6)
16.3 (6.5)
Refinement
24802 (2066) 40792 (3658) 41719 (3940)
no. of reflections
Rwork (%)
Rfree (%)
20.5 (33.3)
26.0 (37.6)
18.7 (24.6)
23.7 (26.9)
18.1 (21.9)
23.7 (28.0)
with cryoprotectant (containing 2.5 mM MgCl2, 0.08
M
CH3COONa, pH 4.6, 20% PEG 4000, 10% glycerol, and 10% 1,2-
propanediol) for 3 s, then frozen in liquid nitrogen. GGPPS-Mg-4
(P21) was the only structure obtained by cocrystallization. All other
crystals were obtained by soaking the native crystals with the
bisphosphonates (using the same soaking method as described
above).
X-ray diffraction data were collected at beamline BL13B1 of
the National Synchrotron Radiation Research Center (NSRRC,
Hsinchu, Taiwan) and Taiwan Contract BL12B2 station at Spring-8
(Hyogo, Japan). Diffraction data were processed and scaled by using
the program HKL2000.31 GGPPS-Mg-GGPP and eight GGPPS-
bisphosphonate crystals belonged to the P212121 space group and
had typical unit cell parameters of a ) 46-48 Å, b ) 116-119
Å, and c ) 126-130 Å. The monoclinic crystal (GGPPS-Mg-
BPH252-p21) had unit cell parameters of a ) 82 Å, b ) 48 Å, c
) 92 Å, and ꢀ ) 111°. Each asymmetric unit contained a dimeric
GGPPS molecule. Prior to use in structural refinements, 5%
randomly selected reflections were set aside for calculating Rfree as
a quality monitor.32
Structure Determination and Refinement. The structures of the
GGPPS-complexes were determined by using the native GGPPS
structure (2DH4) solved previously because the new crystals were
isomorphous. For GGPPS, the 2Fo - Fc difference Fourier map
showed clear electron densities for most amino acid residues,
including those in the substrate binding site(s), but several loops
and the C-terminal segments were disordered. Most product and
bisphosphonate electron densities were obvious. Subsequent refine-
ment with incorporation of the cofactors and water molecules at a
1.0σ map level yielded R and Rfree values of 0.17-0.21 and
0.21-0.26, respectively, at 1.86-2.45 Å resolution. Statistics for
the final models are listed in Tables 3 and 4 and Table S1 of the
Supporting Information. All manual modifications of the models
were performed using the XtalView33 program. Structure refine-
ments, which included maximal likelihood and simulated-annealing
protocols, were carried out by using CNS.34 The PyMol (http://
pymol.sourceforge.net/) program was used in creating figures.
GGPPS Inhibition. GGPPS inhibitor screening was carried out
basically as described previously.6,9
Molecular Docking. Docking calculations were performed for
all crystallographically determined protein-ligand complexes using
a cross-docking approach to a series of GGPPS-bisphosphonate
complex structures (5, PDB 2z4w; 8, PDB 2e93; 9, PDB 2z71; 10,
PDB 2z50; 11, PDB 2z52). The target proteins were prepared using
the protein preparation wizard in Maestro 8.0.35 Hydrogen atoms
were added and a +2 charge assigned to Mg ions present in the
active site. In one case, Mg ions were modeled into the active site
by initially placing them into locations observed in other GGPPS-
bisphosphonate complexes. A full energy minimization in Macro-
Model36 was then run on the entire protein to optimize the geometry
geometry deviations
bond lengths (Å)
bond angles (deg)
0.01
1.4
0.015
1.6
0.018
1.7
5214
29.9
70
48.4
711
47.3
no. of all protein atoms 4816
mean B values (Å2) 39.6
no. of all cofactor atoms 62
mean B values (Å2) 36.9
no. of water molecules 314
mean B values (Å2) 44.4
Ramachandran plot (%)
5104
40.7
51
47
619
55
most favored
additionally allowed 4.9
generously allowed 0.4
94.7
95.2
4.8
0
94.1
5.9
0
a Values in the parenthesis are the highest resolution shells.
mophenol (5 mmol), K2CO3 (15 mmol), and Pd(PPh3)4 (50 mg) in
toluene (10 mL) and H2O (3 mL) were heated at 110 °C under N2
overnight. Upon extraction with diethyl ether, [1,1′;3′,1′′]terphenyl-
3′′-ol was purified by column chromatography as a white powder
(78% yield). This compound (1 mmol) and BrCH2COOMe (1.2
mmol) and K2CO3 (2 mmol) were then dissolved in acetone (5 mL)
and refluxed overnight. Upon filtration, ([1,1′;3′,1′′]terphenyl-3′′-
yloxy)-acetic acid methyl ester was isolated after chromatography
(75% yield). After hydrolysis with NaOH-H2O and bisphosphory-
lation with P(OTMS)3, as described above for 6, 38 was obtained
as a white powder. Anal. (C20H18Na2O8P2 ·0.5H2O) C, H. 1H NMR
(D2O, 400 MHz): δ 7.8 (1H, s), 7.6 (1H, d, J ) 8.5 Hz), 6.9-7.7
(12H, m), 4.8 (2H, t, J ) 12 Hz). 31P NMR (D2O, 162 MHz): δ
17.0.
1-Hydroxy-2-guanidino-ethylidene-bisphosphonic Acid (51). 51
was prepared from 2-guanidine acetic acid by bisphosphorylation
using the PCl3-H3PO3-pyridine system, as reported previously.16
Anal. (C3H9N3Na2O7P2 ·0.5CH3OH) C, H, N. 1H NMR (D2O, 400
MHz): δ 3.56 (2H, t, J ) 11 Hz). 31P NMR (D2O, 162 MHz): δ
16.5.
1-Hydroxy-2-(3-decyloxyphenyl)ethylidene-1,1-bisphosphonic Acid
(19). 19 was prepared from (3-decyloxy-phenyl)-acetic acid methyl
ester by hydrolysis with NaOH and bisphosphorylation with
P(OTMS)3, as described for 6. Anal. (C18H31NaO8P2 ·0.5H2O) C,
H, N. 1H NMR (D2O, 500 MHz): δ 7.07 (1H, t, J ) 7.2 Hz),
6.80-6.95 (2H, m), 6.68 (1H, d, 8 Hz), 3.89 (2H, t, J ) 6.0 Hz),
3.05 (2H, tt, J ) 22.5 Hz, 12 Hz), 1.55-1.62 (2H, m), 1.00-1.29
(14H, m), 0.64 (3H, t, J ) 6.4 Hz). 31P NMR (D2O, 202 MHz): δ
18.8.
Protein Expression and Purification. Yeast and human GGPPS
were expressed and purified as described previously.6,30 The
molecular weights of the purified enzymes were verified by mass
spectrometry, and purities (>95%) were determined by SDS/PAGE.

5606 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Chen et al.
using default protein parameters. For all other structures, hydrogen
bonds were optimized to the default value. A receptor grid large
enough to encompass all crystallographically observed binding sites
was then generated from the prepared target protein. Constraints
for the Mg2+ ions were created but not actually used subsequently.
Water and heteroatoms >5 Å from the active site region were
removed. Geometry optimized ligands were prepared using Lig-
Prep,37 specifying a target pH 7.0 with tautomer and stereoisomer
generation. For the docking calculations, standard-precision (SP)
was specified for preliminary calculations, and the extra-precision
(XP) mode specified for the final calculations. Crystallographically
determined ligand poses from each structure were then compared
with the top 5 poses obtained from Glide; the rms errors are reported
in the text and in Tables S2 and S3 of the Supporting Information.
Docking poses were exported to Sybyl38 for CoMSIA analysis and
also into Liason24 for scoring function parametrization.
Docking Scoring Function Parameterization. Predicted docked
poses of all ligands investigated (generated above) were imported
into Liaison24 for molecular-mechanics energy calculations. Default
options were specified including a minimization sampling method
using a truncated Newton algorithm. Ensemble averages of van
der Waals, electrostatic, and cavity (solvent exposed ligand surface
area) energies were computed for the ligand-bound and ligand-
free states using an implicit solvation model. The computed energies
for each inhibitor complex and SlogP25 (computed in MOE) were
then imported into Strike,39 where partial-least-squares (PLS) and
multiple linear regression (MLR) methods were applied, to construct
a linear equation representing binding affinity. The optimal number
of components was automatically selected, and outliers identified.
All molecules were used to construct an initial training set, then
five test sets were selected at random from the data set. Each test
set compound was removed from the subsequent training set, with
binding affinities being predicted by using the constructed linear
equation. Coefficients for each energy term, fitting statistics and
predictions were reported, and are shown in Tables S2 and S5 of
the Supporting Information.
should remain constant through the series of perturbations, with
the optimum value of 1, for stable models.
Acknowledgment. We thank H. Sagami (Institute of Mul-
tidisciplinary Research for Advanced Materials, Tohoku Uni-
versity, Sendai, Japan) for providing the human GGPPS
expression system. Portions of this research were carried out at
the National Synchrotron Radiation Research Center, a national
user facility supported by the National Science Council of
Taiwan, Republic of China. This work was supported by
Academia Sinica and the National Core Facility of High-
throughput Protein Crystallography grant NSC95-3112-B-001-
015-Y (to A.H.-H.W.) and by the U.S. Public Health Service
(NIH grants GM-65307 and GM-073216, to E.O.). Y.Z. was
supported by an American Heart Association, Midwest Affiliate,
Postdoctoral Fellowship. Y.S. was supported by a Leukemia
and Lymphoma Society Special Fellowship.
Supporting Information Available: Data deposition: The
atomic coordinates and structure factors have been deposited in
the RCSB Protein Data Bank, www.pdb.org. Additional data
collection and refinement information, protein expression, crystal-
lization, inhibition and cell growth inhibition results. This material
is available free of charge via the Internet at http://pubs.acs.org.
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