Structural Basis for Binding and Selectivity of Antimalarial and Anticancer Ethylenediamine Inhibitors to Protein Farnesyltransferase

Article abstract of DOI:10.1016/j.chembiol.2009.01.014

Protein farnesyltransferase (FTase) catalyzes an essential posttranslational lipid modification of more than 60 proteins involved in intracellular signal transduction networks. FTase inhibitors have emerged as a significant target for development of antic

Full text of DOI:10.1016/j.chembiol.2009.01.014

Chemistry & Biology
Article
Structural Basis for Binding and Selectivity
of Antimalarial and Anticancer Ethylenediamine
Inhibitors to Protein Farnesyltransferase
Michael A. Hast,1 Steven Fletcher,2 Christopher G. Cummings,2 Erin E. Pusateri,2 Michelle A. Blaskovich,3
Kasey Rivas,4 Michael H. Gelb,5 Wesley C. Van Voorhis,4 Said M. Sebti,3 Andrew D. Hamilton,2,6 and Lorena S. Beese1,
1Department of Biochemistry, Duke University Medical Center, Box 3711, Durham, NC 27710, USA
2Department of Chemistry, Yale University, New Haven, CT 06511, USA
3Department of Drug Discovery, Moffitt Cancer Center and Research Institute, and Department of Oncologic Sciences,
University of South Florida College of Medicine, Tampa, FL 33612, USA
4Department of Medicine
*
5Department of Chemistry
University of Washington, Seattle, WA 98195, USA
6Correspondence concerning inhibitor design and synthesis should be addressed to andrew.hamilton@yale.edu.
*Correspondence: lsb@biochem.duke.edu
DOI 10.1016/j.chembiol.2009.01.014
SUMMARY
(Plasmodium falciparum), and African sleeping sickness (Trypa-
nosoma brucei) (Eastman et al., 2006).
Protein farnesyltransferase (FTase) catalyzes an
essential posttranslational lipid modification of more
than 60 proteins involved in intracellular signal trans-
duction networks. FTase inhibitors have emerged as
a significant target for development of anticancer
therapeutics and, more recently, for the treatment of
parasitic diseases caused by protozoan pathogens,
including malaria (Plasmodium falciparum). We
present the X-ray crystallographic structures of com-
plexes of mammalian FTase with five inhibitors based
on an ethylenediamine scaffold, two of which exhibit
FTase catalyzes the transfer of a 15-carbon isoprenoid lipid
moiety to the C terminus of more than 60 signal transduction
proteins in the eukaryotic cell, including members of the Rho
and Ras superfamily of G proteins (Casey and Seabra, 1996b;
Reid et al., 2004b). Substrate proteins are modified at a car-
boxy-terminal peptide with a Ca1a2X motif, which consists of a
cysteine (the g sulfur is lipid modified), two generally aliphatic
residues (a1a2), and a variable C-terminal (X) residue (Casey
and Seabra, 1996b).
Crystal structures of catalytic reaction intermediates along the
reaction cycle for mammalian FTase have been elucidated (Long
et al., 2002; Taylor et al., 2003) and binding sites for substrates
over 1000-fold selective inhibition of P. falciparum and products have been located (Long et al., 1998, 2000)
(Figure 1). Kinetic analysis of the mechanism reveals that
substrate binding is ordered (Dolence et al., 1995; Hightower
FTase. These structures reveal the dominant determi-
nants in both the inhibitor and enzyme that control
binding and selectivity. Comparison to a homology
model constructed for the P. falciparum FTase
suggests opportunities for further improving selec-
tivity of a new generation of antimalarial inhibitors.
et al., 1998; Tschantz et al., 1997; Zhang and Casey, 1996). The
lipid complex forms first and the Ca1a2X tetrapeptide second.
Binding of the lipid substrate FPP of the next turn of the reaction
cycle is required to displace the isoprenoid moiety of the product
from its original binding site into an ‘‘exit groove.’’ (Long et al.,
2002; Taylor et al., 2003; Tschantz et al., 1997). The new
Ca1a2X substrate binds after the product dissociates from the
INTRODUCTION
active site to continue the new cycle. Kinetic studies reveal that
product release is the rate-limiting step in the reaction (Furfine
Development of protein farnesyltransferase (FTase) inhibitors et al., 1995; Huang et al., 2000; Pickett et al., 2003), an observa-
(FTIs) for treatment of cancer has been a major focus for cancer tion supported by the crystal structures of the displaced preny-
chemotherapeutics research since the early 1990s (Basso et al., lated product complexes in FTase and the related protein
2006). Oncogenic mutant forms of human Ras superfamily geranylgeranyltransferase-I (GGTase-I) (Furfine et al., 1995;
proteins are associated with 30% of all human cancers (Casey Long et al., 2002; Taylor et al., 2003).
and Seabra, 1996a; Konstantinopoulos et al., 2007; Lane and
A series of FTase inhibitors based on the ethylenediamine
Beese, 2006); the transforming ability of these mutants is depen- scaffold has been described recently (Glenn et al., 2006; Glenn
dent upon farnesylation (Casey and Seabra, 1996a). Two FTIs, et al., 2005). Here we also report the synthesis and properties
Lonafarnib (Schering) and Tipifarnib (Johnson & Johnson) have of three new inhibitors in this series (Figures 2 and 3). The series
advanced to late-stage clinical trials (Basso et al., 2006) for the is constructed out of relatively simple, flexible structures and can
treatment of certain cancers. More recently, FTase has emerged be prepared by straightforward chemical syntheses (Glenn et al.,
as a target for development of inhibitors to treat parasitic 2005, 2006). Members of this series are potent inhibitors of
diseases caused by protozoan pathogens, including malaria mammalian FTase ((Glenn et al., 2006) and Table 1) and therefore
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Chemistry & Biology
FTase-Ethylenediamine Inhibitor Complexes
may provide new leads in cancer chemotherapeutic develop- and form a ternary complex with FPP (Figure 2). By contrast,
ment. Several compounds also exhibit nanomolar IC50s for the 5 binds across both the peptide and isoprenoid binding sites,
P. falciparum enzyme and are over 1000-fold selective for P. fal- blocking the binding of both substrates (Figure 4). FPP was
ciparum FTase over the human enzyme (Table 1) (Glenn et al., included with 5 during introduction of inhibitor into FTase crys-
2006).
tals (see Experimental Procedures); however, only the inhibitor
Here we present the crystal structures of five members of the is observed in the active site. All inhibitors share two common
ethylenediamine-scaffold inhibitor series bound to mammalian substituents oriented identically in the active site: an N-methyli-
FTase. These structures reveal two distinct binding modes for midazole moiety, which coordinates the catalytic Zn2+ ion in the
the inhibitors, which are different than originally predicted by active site; and a para-benzonitrile, which is directed toward the
molecular modeling (Glenn et al., 2005, 2006), but consistent exit groove (Figures 2 and 5C). The identity of the other two
with the observed structure-activity relationships. Of particular substituents of the ethylenediamine scaffold appears to direct
interest is a novel binding mode in which the inhibitor occupies the binding mode.
parts of both the isoprenoid and protein substrate binding sites.
This bisubstrate binding mode has not been observed previously Ternary Complexes with 2, 4, and 7
and provides an opportunity to exploit the isoprenoid binding As shown in Figures 1B and 3, 2 and 4 differ by only a single
pocket for inhibitor design. All members of the series coordinate substituent: the benzyl moiety of 2 is exchanged for an N-Boc-pi-
the catalytic Zn2+ via their N-methylimidazole group, contact the peridin-4-ylmethyl in 4. The binding mode of these two inhibitors
a2 residue binding site, and share a common moiety oriented is similar (Figures 2A, 2E, and 2F). As described above, the para-
toward the product exit groove. The structures presented provide benzonitrile substituent is oriented toward the product exit
a framework for refinement of inhibitor design to improve affinity groove and is partially stabilized by a stacking interaction with
for mammalian and parasite FTases. Of note, these inhibitors Y361b. The binding pocket also consists of residues D359b,
bindto two of the most cross-species, structurally divergentareas F360b, Y93b, L96b, and W106b. Both 2 and 4 possess two
of the FTase active site. We propose that the observed selectivity N-methylimidazole moieties: one coordinates the catalytic Zn2+
of the ethylenediamine-scaffold inhibitor series for P. falciparum (Figures 2A, 2E, and 2F); the second (at the sulfonamide position)
FTase is a consequence of inhibitor moieties contacting residues is sandwiched between the first N-methylimidazole and the first
in these divergent regions.
isoprene of FPP. There is a single polar contact made between
the imine nitrogen of the sulfonamide position N-methylimida-
zole and the side chain hydroxyl of Y361b.
RESULTS
The binding of 7 is similar to that of 2 (Figures 2D and 2F). The
two variable substituents, R1 and the R2 sulfonyl group, bind in
Inhibitors
The ethylenediamine-scaffold-based FTIs (2, 4, 5, 7, and 10) opposite orientations relative to 2, with the sulfonamide position
studied in this work are illustrated in Figure 1B, and the IC50 now directed toward the a2 residue binding site. However, the
values for human and P. falciparum FTase are given in Table 1.
positions of the aromatic rings of 7 are similar to the rings at
the R1 and R2 positions of 2, with the phenyl ring stacking next
to the Zn2+-coordinating N-methylimidazole, and the o-methyl-
phenyl group of 7 stabilized in a similar manner to the R1 phenyl
FTI Binding Does Not Induce Conformational
Changes in FTase
The overall structure of the mammalian FTase heterodimer and substituent of 2.
the positions of the substrate (FPP and peptide) and product
The R1 substituents of both 2 and 4 are directed toward the
binding sites are shown in Figure 1. The b subunit (Figure 1A, binding pocket for the Ca1a2X substrate (Figures 2B and 5C).
blue) contains most of the substrate-binding residues and is The phenyl group at this position in 2 occupies the a2 residue
partially enveloped by the crescent shaped a subunit (Figure 1A, binding site (W102b, W106b, and L96b) and makes van der
red). The central cavity of the b subunit accommodates the Waals contact with the lipid chain of FPP. The N-Boc-piperi-
substrates bound in extended conformations side-by-side, with din-4-ylmethyl moiety of 4 traverses the a2 residue-binding site
extensive contact between the binding sites (Long et al., 2000, and reaches into the X-residue binding site, including residues
2002). The Ca1a2X substrate coordinates the catalytic Zn2+ via S99b, W102b, H149b, P152b, A129a, Y131a, and N167a (Fig-
its cysteine SH group at the top of the cavity (Long et al., 2000). ures 2B and 5B). Two specific polar contacts are made between
The product exit groove is a shallow hydrophobic binding site the Boc oxygen atoms and residues in the pocket, one each with
located adjacent to the Ca1a2X binding site, opposite the FPP S99b and W102b.
binding site in the b subunit (Long et al., 2002).
The active site of FTase does not undergo significant confor- Binary Complex with 5
mational changes upon substrate binding or product release Although FPP was included with 5 during introduction of the
(Bell et al., 2002; Long et al., 2002; Reid and Beese, 2004; Reid inhibitor into FTase crystals (see Experimental Procedures), the
et al., 2004a, 2004b). Binding of the ligands (inhibitors and iso- inhibitor binds alone in the active site and occupies both
prenoid diphosphate) observed here also does not induce signif- the FPP binding pocket and the Ca1a2X binding pocket. Com-
icant structural changes in the active site. The all-atom rmsd petitive binding of 5 with respect to isoprenoid was tested using
values for the structures compared with the molecular replace- steady-state kinetics measurements. Double reciprocal plots
ment search models are within the experimental error of the exhibited the pattern expected for competitive binding with an
˚
coordinates (ꢀ0.2A). Inhibitors 2, 4, 7, and 10 all occupy the increase in isoprenoid Km value in the presence of 5 without
portion of the active site normally occupied by protein substrate change in kcat (Figure 4D).
182 Chemistry & Biology 16, 181–192, February 27, 2009 ª2009 Elsevier Ltd All rights reserved

Chemistry & Biology
FTase-Ethylenediamine Inhibitor Complexes
Figure 1. Structural Overview of FTase and
Inhibitors in the Current Study
(A) Adjacent binding sites for the lipid substrate,
Ca1a2X substrate, and displaced product in the
active site of the enzyme. The a subunit is shown
in red, the b subunit is shown in blue, and the cata-
lytic Zn2+ ion is shown in pink.
(B) Chemical structures of the ethylenediamine-
scaffold inhibitors used in the present study.
oriented toward the exit groove, and an
N-methylimidazole substituent coordi-
nates the Zn2+
. The o-methylphenyl
sulfonamide binds in the same place as
the second isoprene of FPP, within van
der Waals contact of G250b and Y251b,
and one of the oxygens of the sulfonamide
makes a water-mediated hydrogen bond
to Y300b. The N-Boc-piperidin-4-yl-
methyl binds in place of isoprene 3 of
FPP, contacting residues Y205b, Y154b,
W303b, C254b, and W102b. Water-medi-
ated hydrogen bondsare formed between
the carbonyl oxygen of the Boc group and
R202b, and also between the piperidine
nitrogenandtheindolenitrogenofW106b.
Derivatives of 5 Designed to
Explore Bisubstrate Binding Mode
The unexpected binding mode of
5
prompted an investigation of the determi-
nants of bisubstrate binding with several
derivatives where the o-methylphenyl
sulfonamide position was altered with
additional hydrophobic and aliphatic
moieties designed to further mimic parts
of the isoprenoid molecule. To this end,
we were successful in obtaining crystal
structures of 7 and 10 bound to FTase.
The biphenyl functionality in 10 was
designed to mimic the lipid chain of FPP
and the attached carboxylic acid group
was intended to interact with the
diphosphate binding pocket. Compound
7 was used to investigate whether both
the N-Boc-piperidin-4-ylmethyl and the
o-methylphenyl sulfonamide substituents
at R1 and R2, respectively (in 5), were
required to direct the bisubstrate binding
mode, or if the o-methylphenyl sulfon-
amide alone was sufficient. As described
above, 7 is a monosubstrate inhibitor
(Figure 2D), suggesting the unique com-
bination of the o-methylphenyl sulfon-
amide and the N-Boc-piperidin-4-yl-
Figure 4 depicts the binding mode of 5 in the active site methyl moiety is required to direct bisubstrate binding.
compared withnaturalsubstrates. Although 5is slightly displaced
The designed isoprenoid mimic 10 reverts to a monosubstrate
toward the isoprenoid site, the para-benzonitrile group remains binding mode and blocks the Ca1a2X binding site. Although 10
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Chemistry & Biology
FTase-Ethylenediamine Inhibitor Complexes
Figure 2. Analysis of Monosubstrate Inhibitor Binding Mode
(A) 4 (blue) bound with FPP (red) in FTase active site.
(B) Superposition of 4:FPP structure with a Ca1a2X peptide CVIM (tan) shows the monosubstrate inhibitors traverse the Ca1a2X substrate binding site. The N-Boc-
piperidin-4-ylmethyl substituent of 4 is able to reach the X-residue binding pocket (residues in green).
(C) Superposition of 4:FPP ternary structure with a displaced CVIM product complex (tan) in the exit groove (residues in orange) illustrates contact between the
inhibitor and exit groove.
(D) Binding mode of 7.
(E) Binding mode of 2.
(F) Superposition of monosubstrate compounds 7, 2, and 4 illustrate similarities in binding mode.
In all panels, the catalytic Zn2+ ion is shown in pink and the FTase protein is shown in gray.
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Chemistry & Biology
FTase-Ethylenediamine Inhibitor Complexes
Figure 3. Synthesis of Ethylenediamine-Scaffold Compounds in the Present Study
(a) 1. 1-Methyl-1H-imidazole-4-sulfonyl chloride, DIPEA, CH3CN, 1 hr, 0ꢁC / rt, 93%. 2. BnBr, Cs2CO3, DMF, 16 hr, rt, 88%.
(b) 1. 2-Methylbenzenesulfonyl chloride, DIPEA, CH3CN, 1 hr, 0ꢁC / rt, 92%. 2. BnBr, Cs2CO3, DMF, 16 hr, rt, 87%.
˚
(c) 1. N-Boc-piperidin-4-ylformaldehyde, AcOH, 4 A molecular sieves, MeOH, 3 hr, rt; 2. NaCNBH3, 16 hr, rt, 78%.
(d) 1-Methyl-1H-imidazole-4-sulfonyl chloride, DIPEA, CH3CN, 16 hr, 0ꢁC / rt, 91%.
(e) 2-Bromobenzenesulfonyl chloride, DIPEA, CH3CN, 16 hr, 0ꢁC / rt, 93%.
(f) 2-Methylbenzenesulfonyl chloride, DIPEA, CH3CN, 16 hr, 0ꢁC / rt, 95%.
(g) 3-(2-Methoxycarbonylethyl)phenylboronic acid, PdCl2(dppf)2, K2CO3, DMF, 16 hr, 110ꢁC, 61%.
(h) LiOH$H2O, THF/MeOH/H2O, 3:1:1, 1 hr, rt, 92%.
did not bind in the designed fashion (Figure 4C), it retained residue of the Ca1a2X substrate. This substituent is within van
potency (Table 1) against the mammalian enzyme. The positions der Waals distance to both the first isoprene of FPP, as well as
of the Zn2+ coordinating moiety and para-benzonitrile in the exit K164a, though in neither case does the interaction appear to
groove are unchanged, underscoring the role of these substitu- contribute significantly to binding.
ents as a dominant pharmacophore in this series. Like 7, the
Selectivity data for 7 and 10 (Table 1) support a model in which
sulfonamide position of 10 is directed toward the a2 binding inhibitor interactions in the divergent Ca1a2X X-residue binding
site, with the first phenyl group stabilized similarly to 7 (Figure 4C). pocket modulate selectivity. Like 2, which differs only at the
The second phenyl group stacks on W102b, while the acid group solvent-exposed sulfonamide position, 7 exhibits only moderate
can form polar contacts with several residues: R202b, N167a, selectivity for the P. falciparum enzyme. 10 is significantly less
and a water-mediated hydrogen bond with H149b. The N-Boc- selective for the P. falciparum enzyme than either 4 or 5, despite
piperidin-4-ylmethyl R1 substituent of 10 extends toward the full exploration of the Ca1a2X X-residue binding site. This dimin-
solvent making no specific polar interactions, similar to the a1 ished selectivity must therefore reflect the chemical nature of the
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Chemistry & Biology
FTase-Ethylenediamine Inhibitor Complexes
arise from amino acid insertions/deletions between helices in
either subunit and as point mutations remote from the active
site (Figure 5A, red surfaces). Although these loops are unlikely
to affect catalysis directly, in aggregate these differences may
alter the protein backbone sufficiently to impact binding of inhib-
itors at the active site. A recently described crystal structure of
a non-mammalian protein prenyltransferase, the GGTase-I
from the human fungal pathogen Candida albicans, demon-
strates that sequence divergence in the exit groove contributes
to significant structural changes to the protein backbone of
this region (Hast and Beese, 2008).
Table 1. IC50 Values and Compound Selectivity
P. falciparum-selective inhibitors
Human FTase
IC50a, nM
P. falciparum
FTase IC50b, nM
Compound
Selectivityc
112
2
4
5
56 29
0.5
3700 790
4050 (n = 2)
1.2
3.0d
3083
1350
Probes of bisubstrate binding
7
160 81
5.0e
32
11
10
800 95
70.0e
a Determined as described in reference (Carrico et al., 2004).
b Value from reference (Glenn et al., 2006).
Homology modeling of the P. falciparum FTase b subunit is
complicated by its size (twice the length of mammalian FTase)
and regions of high divergence. The first 300 amino acids exhibit
no homology to either mammalian FTase or any other protein in
a BLAST search, except FTases of related species of Plasmo-
dium. Consequently, several different alignments can be con-
structed in the region of first three helices of the b subunit, where
the product exit groove is predicted to be located. One alignment
suggests an insertion of ꢀ20 amino acids between helices 2 and
3 in the b subunit, thereby completely remodeling the exit
groove. Even alignments that lack this insertion reveal that the
exit groove is divergent.
c Value calculated by dividing the IC50 for hFTase by the IC50 for PfFTase.
d Value estimated from following values: 76% inhibition at 5 nM; 20%
inhibition at 0.5 nM.
e n = 2. Determined as described in (Glenn et al., 2006).
substituent that probes this pocket (a carboxylate instead of the
N-Boc-piperidin-4-ylmethyl group).
DISCUSSION
All inhibitors presented here contact the product exit groove
The conformational flexibility, potential for introducing chemical with a para-benzonitrile moiety. Residues that form part of the
diversity, and the favorable bioavailability properties of the ethyl- product exit groove in mammalian FTase lie in a 13-residue
enediamine-scaffold inhibitors (Glenn et al., 2006) engender this loop between helices 2b and 3b. The equivalent loop in P. falci-
series with significant potential for optimization as cancer or parum FTase is predicted to share little homology with mamma-
parasitic disease therapeutics. A summary of the structure- lian FTase (Figures 5A and 5C). Biphenyl moieties in place of
activity relationships determined by screening and structural the para-benzonitrile were poorly tolerated in mammalian
studies is presented in Figure 5B. Zn2+ coordination by R4 is FTase, although such derivatives retained potency against the
indispensable, and two substituents (R1 and R3) are largely P. falciparum enzyme (Glenn et al., 2006). Inspection of the
responsible for conferring selectivity profiles for mammalian crystal structures suggests that an additional phenyl ring would
and P. falciparum FTases. The fourth substituent (R2) does not clash with Y93b in the exit groove (Figures 2 and 5C). As
dramatically affect selectivity, but it is critical for directing bisub- described above, the exit groove is predicted to diverge from
strate binding in 5.
mammalian FTase significantly; therefore, the tolerance of
The switch from monosubstrate to bisubstrate binding modes P. falciparum FTase to bulkier substituents at this position
as a consequence of small changes in substituent groups within suggests that the highly diverged exit groove in this enzyme
a single series of FTIs is rarely observed. In the case of the dual is more open, providing greater opportunities for designing
FTase and GGTase-I inhibitor, L-778,123 (Reid et al., 2004a), selective inhibitors.
small structural differences between the enzymes account for
Crystal structures of FTase bound to tetrahydroquinoline
a switch in inhibition mode: in FTase L-778,123 binds only in (THQ)-based FTIs reveal that these compounds adopt a binding
the peptide substrate site, whereas in GGTase-I it blocks both mode similar to the monosubstrate ethylenediamine-scaffold
substrates. In the L-778,123 study, subtle differences in the inhibitors (Eastman et al., 2007; Van Voorhis et al., 2007). Inhib-
active sites of FTase and GGTase-I combined with additional itors based on either scaffold share the N-methylimidazole that
sulfate (or phosphate) ions are sufficient to change the binding coordinates the Zn2+, and have additional moieties that bind in
mode of L-778,123. We observe similar effects in the ethylenedi- the product exit groove. The crystal structure of the THQ-based
amine-scaffold series. Relatively subtle differences in the struc- FTI PB-93 bound to rat FTase reveals that an N-methoxycar-
ture of the inhibitor are sufficient to redirect the binding mode bonyl-piperidiny-4-ylmethyl substituent binds in the pocket
from monosubstrate to bisubstrate, and produce dramatically that binds the X-residue of the Ca1a2X motif, similar to the
different selectivity profiles.
N-Boc-piperidin-4-ylmethyl of 4. Both compounds are selective
In the absence of a crystal structure of P. falciparum FTase, for P. falciparum FTase, highlighting the importance of the
only a homology model can be used to identify structurally diver- X-residue binding pocket and the exit groove in conferring
gent regions in the active sites that potentially can be exploited to compound selectivity (Van Voorhis et al., 2007).
confer inhibitor selectivity. Homology models constructed by
Two mutations in P. falciparum FTase that confer resistance to
two different sequence alignment schemes predict that both THQ-based FTIs have been observed (Eastman et al., 2005; East-
the product exit groove and the pocket that binds the X-residue man et al., 2007). Modeling the G612bA mutation (which corre-
of the Ca1a2X motif diverge across species (Figures 5A and 5C, sponds to G250b in mammalian FTase) suggests a mechanism
blue surfaces). Other major differences between the species by which the mutation disrupts inhibitor binding by narrowing
186 Chemistry & Biology 16, 181–192, February 27, 2009 ª2009 Elsevier Ltd All rights reserved

Chemistry & Biology
FTase-Ethylenediamine Inhibitor Complexes
Figure 4. Analysis of Bisubstrate Inhibitor Binding Mode
(A) Superposition of 5 (blue) and FPP (red; from PDB 1JCR) illustrates binding of two moieties (R1 and R2) of the inhibitor in the isoprenoid binding site.
(B) Superposition of 5 with FPP (red; from PDB 1JCR) and CVIM Ca1a2X peptide (brown; from PDB 1D8D) illustrates the bisubstrate binding mode.
(C) Superposition of 5 (blue) with 10 (gray). 10 was designed to further explore bisubstrate binding by mimicking the lipid chain of FPP and phosphate moiety with
a biphenyl substituent and carboxylic acid moiety. 10 clearly shifts back to monosubstrate binding; however, it does retain sub-micromolar IC50
.
(D) Double reciprocal plot illustrating mode of inhibition of 5. Filled circles represent titration of FPP with no inhibitor; open circles represent the identical exper-
iment where 5 is present at 4 mM. Two independent experiments were performed at each FPP concentration point. Lines intersect near th ey-intercept, suggesting
a change in apparent Km for FPP and therefore an isoprenoid-competitive binding mode. 1/V is given in disintegrations per minute (dpmꢂ1) for radiolabled product
using assay described in (Glenn et al., 2006).
the binding site (Eastman et al., 2007). The fused ring THQ scaf- tural differences between target FTase enzymes for further opti-
fold is rigid and readily disrupted by mutations. It is tempting to mization of inhibitors that are even more selective and potent for
speculate that the flexible nature of the ethylenediamine scaffold mammalian and parasite FTases.
may be more tolerant of perturbations arising from mutations.
These compounds have not yet been tested for activity against SIGNIFICANCE
the THQ-resistant P. falciparum FTase mutants.
The unexpected diversity in structural binding modes ob- Posttranslation lipidation by protein farnesyltransferase
served in this study provides opportunities for exploring struc- (FTase) is required for the transforming ability of oncogenic
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Chemistry & Biology
FTase-Ethylenediamine Inhibitor Complexes
Figure 5. Homology Modeling and Struc-
ture-Activity Summary
(A) Homology model of the b subunit of P. falcipa-
rum FTase (gray and red ribbon) superimposed
upon the b subunit of human FTase (green ribbon,
PDB code 1TN6, chain B). The blue surfaces
represent the Ca1a2X X-residue binding site and
prenylated product exit groove, the two most
divergent areas of the active site. The red surfaces
represent the areas of b subunit predicted to have
amino acid insertions in the P. falciparum enzyme
not present in the mammalian enzyme.
(B) The four substituents of the ethylenediamine
scaffold are correlated with the observed struc-
ture-activity relationship data as determined by
screening (Glenn et al., 2006) and crystal struc-
tures.
(C) FTase active site surface with bound
compound 4 and FPP. The surface is colored to
represent regions of sequence diversity: orange,
product exit groove; green, Ca1a2X X-residue
binding site; gray, homologous residues. 4 is the
most selective compound for P. falciparum FTase
in the present study, and it contacts both regions
of putative divergence in the FTase active site.
values; two have greater than 1000-
fold selectivity toward P. falciparum
FTase. Two different binding modes
have been identified, including one
that has interactions spanning both
the peptide and farnesyl substrate-
binding sites. Of particular importance
to selectivity are interactions within
the farnesylated peptide product ‘‘exit
groove’’ and protein substrate binding
site, the structures of which differ
between species. The ethylenediamine
scaffold lends itself readily to straight-
forward modifications that enable
systematic exploration of such struc-
tural differences, which may ultimately
lead to inhibitors that are sufficiently
mutants of the human Ras superfamily which are associ- inexpensive to synthesize for effective antimalarials to be
ated with 30% of all human cancers. FTase has therefore economically feasible.
been a major focus of anticancer drug development for
more than a decade. High-affinity FTase inhibitors have
also emerged as a significant route for the development
of new antimalarial therapeutics. Malaria currently kills
over one million people each year. Furthermore, response
to increasing mortality rates and emerging resistance to
the current generation of antimalarials requires urgent
development of new potent and selective antimalarial drugs
against Plasmodium falciparum. Ideally such drugs exhibit
high affinity for P. falciparum FTase and selectivity against
the mammalian enzyme. The X-ray crystallographic struc-
tures of complexes of mammalian FTases with five inhibi-
tors based on the ethylenediamine scaffold has revealed
dominant structural determinants for binding and selec-
tivity. Three of these inhibitors exhibit nanomolar IC50
EXPERIMENTAL PROCEDURES
Chemistry, General Methods
Compounds 2 and 4 were resynthesized as described previously (Glenn et al.,
2005, 2006). Synthetic procedures and characterization data for all new
compounds (5, 7, and 10) are given in full below and are summarized in Figure 3.
Generally, starting from 1, inhibitors were accessed either by reaction with the
appropriate sulfonyl chloride, followed by alkylation of the sulfonamide NH, or
by reductive amination with N-Boc-piperidin-4-ylformaldehyde, followed by
reaction with the appropriate sulfonyl chloride (Figure 3). Solvents CH2Cl2,
CH3CN and DMF were dried on an Innovative Technology SPS-400 dry solvent
system. Anhydrous MeOH and was purchased from Sigma-Aldrich and used
directly from its Sure-Seal bottle. Molecular sieves were activated by heating
to 300ꢁC under vacuum overnight. All reactions were performed under an
atmosphere of dry nitrogen in oven-dried glassware and were monitored for
completeness by thin-layer chromatography (TLC) using silica gel (visualized
188 Chemistry & Biology 16, 181–192, February 27, 2009 ª2009 Elsevier Ltd All rights reserved

Chemistry & Biology
FTase-Ethylenediamine Inhibitor Complexes
by UV light, or developed by treatment with KMnO4 stain or Hanessian’s stain).
1H and 13C NMR spectra were recorded on Bruker AM 400 MHz and Bruker AM
500 MHz spectrometers in either CDCl3 or d4-MeOH. Chemical shifts (d) are
reported in parts per million after calibrations to residual isotopic solvent.
Coupling constants (J) are reported in Hertz. Mass spectrometry (MS) was per-
formed using electrospray ionization on either a Varian MAT-CH-5 (HRMS) or
Waters Micromass ZQ (LRMS). Before biological testing and crystallization
experiments, target molecules were subjected to further purification by
reversed-phase high-performance liquid chromatography (rpHPLC). Analysis
and purification by rpHPLC were performed using either Phenonenex Luna
5 mm C18 (2) 250 3 21 mm column run at 15 mL/min (preparative) or a Micro-
sorb-MV 300 A C18 250 mm 3 4.6 mm column run at 1 mL/min (analytical),
using gradient mixtures of (A) water with 0.1% TFA and (B) 10:1 acetonitrile/
water with 0.1% TFA. Appropriate product fractions were pooled and lyophi-
lized to dryness, affording the inhibitors as fluffy white powders as their TFA
salts. Inhibitor purity was confirmed by analytical rpHPLC using linear gradients
from 100% A to 100% B, with changing solvent composition of either (I) 4.5% or
(II) 1.5% per minute after an initial 2 min of 100% A. For reporting HPLC data,
percentage purity is given in parentheses after the retention time for each
condition.
solid (76 mg, 85%): dH (400 MHz, d4-MeOH) 0.93 (qd, J = 12.1, 4.0 Hz, 2H,
CH (piperidinylmethyl)), 1.48 (s, 9H, C(CH3)3), 1.51–1.60 (m, 1H, CH (piperidi-
nylmethyl)), 1.61–1.70 (m, 2H, 2 CH (piperidinylmethyl)), 2.61 (s, 3H, CH3Ar),
2.65–2.78 (m, 2H, 2 CH (piperidinylmethyl)), 3.15 (d, J = 7.2 Hz, 2H, 2 CH
(piperidinylmethyl)), 3.25–3.32 (m, 2H, CH2CH2NSO2), 3.56–3.62 (m, 2H,
CH2CH2NSO2), 3.64 (s, 3H, CH3 (Im)), 3.99–4.06 (m, 2H, 2 CH (piperidinyl-
methyl)), 4.65 (s, 2H, CH2Im), 6.89 (s, 1H, CH (Im)), 6.94 (d, J = 9.2 Hz, 2H,
2 CH (p-cyanoAr)), 7.40–7.46 (m, 2H, 2 CH (Ar)), 7.54 (d, J = 9.2 Hz, 2H, 2
CH (p-cyanoAr)), 7.58 (td, J = 7.4, 1.0 Hz, 1H, CH (Ar)), 7.71 (m, 1H, CH (Im)),
7.90 (dd, J = 8.0, 1.0 Hz, 1H, CH (Ar)); dC (100 MHz, d4-MeOH) 20.8, 28.7,
30.8, 32.1, 36.0, 44.4 (br), 44.6, 45.3, 49.5, 55.2, 81.0, 99.8, 113.8, 121.0,
127.5, 129.0, 129.2, 130.6, 134.2, 134.3, 134.8, 139.0, 139.1, 140.5, 152.3,
156.5; HRMS (ES+) calcd for [C32H42N6O4S + H] 607.3067, found 607.3052;
HPLC (I) tR = 13.02 min (98.7%), (II) tR = 20.32 min (98.6%).
N-{2-[(4-Cyanophenyl)-(3-methyl-3H-imidazol-4-ylmethyl)-amino]-
ethyl} 2-methyl-benzenesulfonamide (6)
o-TsCl (31.2 ml, 0.216 mmol, 1.1 eq) was added dropwise to a solution of 1
(50 mg, 0.196 mmol, 1 eq) and DIPEA (68 ml, 0.392 mmol, 2 eq) in anhydrous
CH3CN (2 ml). After 1 hr, TLC showed the reaction was complete. The reaction
mixture was concentrated, then diluted with CH2Cl2 (50 ml) and washed with
water (3 3 10 ml), brine (10 ml), dried (Na2SO4), filtered, and concentrated.
The residue was chromatographed on silica gel, eluting with CH2Cl2/MeOH/
NH4OH, 192:7:1 to give sulfonamide 6 as a white powder (67 mg, 83%): dH
(400 MHz, d4-MeOH) 2.64 (s, 3H, CH3Ar), 3.15 (t, J = 6.4 Hz, 2H, CH2NHSO2),
3.68 (t, J = 6.4 Hz, 2H, CH2NHSO2), 3.93 (s, 3H, CH3 (Im)), 4.82 (s, 2H, CH2Im),
6.93 (d, J = 9.2 Hz, 2H, 2 CH (p-cyanoAr)), 7.27 (s, 1H, CH (Im)), 7.37–7.43 (m,
2H, 2 CH (Ar)), 7.52–7.57 (m, 3H, 2 CH (p-cyanoAr), CH (Ar)), 7.91 (dd, J = 7.6,
1.0 Hz, 1H, CH (Ar)), 8.92 (s, 1H, CH (Im)); dC (100 MHz, d4-MeOH) 20.7,
34.6, 41.2, 46.2, 51.9, 100.8, 114.4, 121.2, 127.7, 130.3, 130.4, 133.3,8t6
134.2, 134.3, 135.2, 138.2, 138.8, 139.9, 152.1; HRMS (ES+) calcd for
[C21H23N5O2S + H] 410.1651, found 410.1658.
4-[(2-Aminoethyl)-(3-methyl-3H-imidazol-4-ylmethyl)-amino]-
benzonitrile (1)
To a solution of 4-[(2-aminoethyl)-(3-methyl-3H-imidazol-4-ylmethyl)-amino]-
benzonitrile (compound 9c in Glenn et al., 2006) (8.0 g, 30.6 mmol) in CH2Cl2
(30 ml) was added trifluoroacetic acid (30 ml). After stirring for 30 min, the reac-
tion mixture was evaporated to dryness. The crude residue was passed
through a short pad of silica (eluent: CH2Cl2/MeOH/NH4OH, 92:7:1) to furnish
the title compound as the free base (7.6 g, 97%): dH (400 MHz, CDCl3) 1.45 (br,
2H, NH2), 2.88 (t, J = 6.8 Hz, 2H, CH2NH2), 3.44 (t, J = 6.8 Hz, 2H, NH2CH2CH2),
3.55 (s, 3H, CH3 (Im)), 4.55 (s, 2H, CH2Im), 6.78 (d, J = 8.4 Hz, 2H, 2 CH (Ar)),
6.83 (s, 1H, CH (Im)), 7.45 (s, 2H, 2 CH (Ar)), 7.47 (s, 1H, CH (Im)); dC (125 MHz,
CDCl3) 30.1, 37.0, 44.1, 49.5, 99.3, 111.7, 120.4, 127.2, 129.1, 133.0, 139.3,
150.4; HRMS (ESI) m/z calculated for C14H17N5H+ 256.1562, found 256.1559.
N-Benzyl, N-{2-[(4-cyanophenyl)-(3-methyl-3H-imidazol-4-
ylmethyl)-amino]-ethyl} 2-methyl-benzenesulfonamide (7)
4-[(2-(N-tert-Butoxycarbonylpiperidin-4-ylmethyl)-aminoethyl)-
(3-methyl-3H-imidazol-4-ylmethyl)-amino]-benzonitrile (3)
Cs2CO3 (96 mg, 0.294 mmol, 2 eq) was added to a solution of 3 (60 mg, 0.147
mmol, 1 eq) in anhydrous DMF (15 ml). After 10 min, benzyl bromide (19.2 ml,
0.162 mmol, 1.1 eq) was added dropwise to the reaction mixture, and then
allowed to stir overnight at room temperature. The reaction mixture was diluted
with water (100 ml), and the organics were extracted into EtOAc (3 3 15 ml).
The EtOAc extractions were combined, washed with 5% NaHCO3 solution
(10 ml 3 3), water (10 ml), brine (10 ml), dried (Na2SO4), filtered and concen-
trated. The residue was purified by silica gel flash column chromatography
(eluent: CH2Cl2:MeOH:NH4OH, 192: 7:1) to furnish 7 as a white foam (67 mg,
91%): dH (500 MHz, CDCl3) 2.63 (s, 3H, CH3Ar), 3.17–3.23 (m, 2H,
CH2CH2NSO2), 3.30–3.36 (m, 2H, CH2CH2NSO2), 3.45 (s, 3H, CH3 (Im)), 4.29
(s, 2H, CH2Ph), 4.30 (s, 2H, CH2Im), 6.44 (d, J = 9.0 Hz, 2H, 2 CH (p-cyanoAr)),
6.69 (s, 1H, CH (Im)), 7.18–7.22 (m, 2H, 2 CH (Ar)), 7.31–7.36 (m, 7H, 5 CH (Ph),
2 CH (p-cyanoAr)), 7.47–7.51 (m, 2H, CH (Ar)), CH (Im)), 7.85 (dd, J = 7.5,
1.0 Hz, 1H, (Ar)); dC (125 MHz, CDCl3) 20.6, 31.7, 43.7, 44.5, 48.9, 52.9,
99.3, 112.1, 119.9, 126.3, 126.7, 128.4, 128.7, 128.8, 128.9, 129.4, 133.0,
133.1, 133.6, 135.8, 137.1, 137.6, 138.9, 150.0; HRMS (ES+) calcd for
[C28H29N5O2S + H] 500.2120, found 500.2121; HPLC (I) tR = 12.10 min
(100%), tR = 16.78 min (100%).
To a solution of 4-[(2-aminoethyl)-(3-methyl-3H-imidazol-4-ylmethyl)-amino]-
benzonitrile (1; 500 mg, 1.96 mmol, 1 eq) in dry methanol (6.5 ml) was added
N-Boc-piperidin-4-ylformaldehyde (417 mg, 1.96 mmol, 1 eq), followed by
˚
acetic acid (294 ml, 4.90 mmol, 2.5 eq) and powdered 4 A molecular sieves
(approx. 150 mg). The reaction was stirred at room temperature for 3 h, and
then sodium cyanoborohydride (185 mg, 2.94 mmol, 1 eq) was added. The
reaction was stirred for 18 h, then dry-loaded onto silica gel and purified by
silica gel flash column chromatography (eluent: CH2Cl2/MeOH/NH4OH,
192:7:1) to provide the title compound 3 as a colorless gum (693 mg, 78%):
dH (400 MHz, CDCl3) 1.03 (m, 2H, 2 CH (piperidinylmethyl)), 1.40 (s, 9H,
C(CH3)3), 1.45–1.49 (m, 1H, CH (piperidinylmethyl)), 1.58–1.61 (m, 2H, 2 CH
(piperidinylmethyl)), 2.37–2.42 (m, 2H, 2 CH (piperidinylmethyl)), 2.58–2.63
(m, 2H, 2 CH (piperidinylmethyl)), 2.73 (t, J = 5.8 Hz, 2H, NHCH2CH2N(Im)Ar)),
3.44 (m, 2H, NHCH2CH2N(Im)Ar)), 3.52 (s, 3H, CH3 (Im)), 4.03 (br m, 3H, NH,
2 CH (piperidinylmethyl)), 4.51 (s, 2H, CH2Im), 6.73 (s, 1H, CH (Im)), 6.76
(d, J = 7.6 Hz, 2H, 2 CH (Ar)), 7.39–7.41 (m, 3H, CH (Im), 2 CH (Ar)); dC
(125 MHz, CDCl3) 28.6, 30.6, 32.4, 36.8, 44.1, 45.4, 47.3, 49.5, 53.9, 79.6,
99.3, 112.9, 120.5, 127.2, 129.1, 133.7, 139.4, 151.2, 155.2; HRMS (ES+) calcd
for [C25H36N6O2 + H] 453.2933, found 453.2978.
N-(N-tert-Butoxycarbonylpiperidin-4-ylmethyl), N-({2-[(4-
cyanophenyl)-(3-methyl-3H-imidazol-4-ylmethyl)-amino]-ethyl})
2-bromobenzenesulfonamide (8)
N-(N-tert-Butoxycarbonylpiperidin-4-ylmethyl), N-({2-[(4-
cyanophenyl)-(3-methyl-3H-imidazol-4-ylmethyl)-amino]-ethyl})
2-methylbenzenesulfonamide (5)
Toan ice-cold, stirringsolutionof 3(500mg, 1.17 mmol, 1 eq)and DIPEA (310ml,
1.78 mmol, 1.5 eq) in CH3CN (12 ml) was added 2-bromobenzenesulfonyl chlo-
ride (329 mg, 1.29 mmol, 1.1 eq). The reaction was allowed to stir for 16 hr, grad-
ually warming to room temperature. All solvent was evaporated under reduced
pressure, and the residue was then redissolved in CH2Cl2 (200 ml), washed with
water (30 ml), brine (30 ml), dried (Na2SO4), filtered, and concentrated. The
crude material was purified by silica gel flash column chromatography (eluent:
CH2Cl2/MeOH/NH4OH, 192:7:1) to afford the title compound as a white
foam (731 mg, 93%): dH (500 MHz, CDCl3) 0.90 (qd, J = 12.3, 4.0 Hz, 2H, 2
CH (piperidinylmethyl)), 1.33–1.39 (m, 1H, CH (piperidinylmethyl)), 1.42 (s, 9H,
N-Boc-piperidin-4-ylmethyl bromide (61 mg, 0.221 mmol, 1.5 eq) was added to
a solution of 6 (60 mg, 0.147 mmol, 1 eq) and Cs2CO3 (96 mg, 0.294 mmol, 2 eq)
in anhydrous DMF (1.5 ml). The reaction mixture was stirred for 48 hr at room
temperature, then diluted with water (40 ml) and extracted into EtOAc (3 3 10 ml).
The EtOAc extractions were combined and washed with 5% aqueous
NaHCO3 (3 3 10 ml), brine (10 ml), then dried (Na2SO4), filtered, and concen-
trated. The crude residue was purified by silica gel flash column chromatog-
raphy (eluent: CH2Cl2:MeOH:NH4OH, 192:7:1) to give 5 as a colorless, waxy
Chemistry & Biology 16, 181–192, February 27, 2009 ª2009 Elsevier Ltd All rights reserved 189

Chemistry & Biology
FTase-Ethylenediamine Inhibitor Complexes
Table 2. Crystallographic Data Collection and Refinement Statistics
rFTase: FPP: 2
rFTase: FPP: 4
hFTase: 5
rFTase: FPP: 7
rFTase: FPP: 10
(PDB code 3E34)
(PDB code 3E32)
(PDB code 3E30)
(PDB code 3E37)
(PDB code 3E33)
˚
Resolution range (A)
37.5–2.45
42,009/328,517
20.2 (2.4)
40.7–2.45
41,034/283,427
22.5 (2.4)
42.8–1.80
34.0–1.90
85,965/672,359
34.4 (2.6)
32.2–2.05
Reflections (unique/total)
Mean I/sa
101,393/524,635
21.9 (2.8)
68,223/524,533
23.2 (2.9)
a
Rsym
9.4 (44.9)
8.6 (33.4)
7.7 (33.5)
8.1 (55.4)
10.0 (49.5)
99.8 (98.3)
19.2/21.9
Completeness (%)
Rcryst / Rfree
99.6 (95.6)
20.8/23.7
97.6 (76.8)
18.4/22.2
98.2 (93.7)
16.8/19.0
99.2 (98.0)
20.0/22.1
Space group: P61
˚
Unit cell dimensions: a = b (A)
173.4
70.0
173.3
70.2
178.4
64.5
171.0
69.4
170.2
69.5
˚
c (A)
Rmsd from ideal geometry
˚
Bond lenths (A)
0.004
1.30
32
0.007
1.096
35.3
0.009
1.17
20
0.009
1.09
30
0.009
1.26
25
Bond angles (ꢁ)
2
Average B factor (overall, A )
˚
R
sym = (Sj(I- < I >)j)/(SI), where < I > is the average intensity of multiple measurements.
Rcryst and Rfree = (SjFobs-Fcalcj)/(SjFobsj). Rfree was calculated over 5% of the amplitudes not used in refinement.
a Values in parentheses represent those for the highest resolution shell.
C(CH3)3), 1.49–1.56 (m, 2H, 2 CH (piperidinylmethyl)), 2.52–2.64 (m, 2H, 2 CH
(piperidinylmethyl)), 3.02–3.12 (m, 2H, 2 CH (piperidinylmethyl)), 3.21–3.30 (m,
2H, CH2CH2NSO2), 3.47–3.52 (m, 2H, CH2CH2NSO2), 3.53 (s, 3H, CH3 (Im)),
3.96–4.07 (m, 2H, 2 CH (piperidinylmethyl)), 4.46 (s, 2H, CH2Im), 6.72 (d, J =
9.0 Hz, 2H, 2 CH (Ar)), 6.89 (s, 1H, CH (Im)), 7.40–7.48 (m, 5H, 2 CH (Ar)), 2
CH (o-BrPh), CH (Im)), 7.73 (dd, J = 8.0, 1.5 Hz, 1H, CH (o-BrPh)), 8.08 (dd,
J = 8.0, 2.0 Hz, 1H CH (o-BrPh)); dC (125 MHz, CDCl3) 28.4, 29.5, 31.6, 34.8,
43.2 (br), 43.9, 44.3, 47.4, 54.3, 79.4, 99.8, 112.3, 119.8, 120.3, 126.5, 127.7,
129.6, 132.4, 133.8, 133.9, 135.9, 138.6, 139.3, 150.4, 154.6; HRMS (ES+)
127.6, 127.7, 127.8, 127.9, 129.0, 129.9, 130.1, 132.3, 133.0, 133.7, 138.0,
139.0, 139.2, 139.9, 141.1, 150.2, 154.5, 172.9; HRMS (ES+) calcd for
[C41H50N6O6S + H], 755.3591, found 755.3581; HPLC (I) tR = 13.33 min
(100%), (II) tR = 21.74 min (100%).
N-(N-tert-Butoxycarbonylpiperidin-4-ylmethyl), N-({2-[(4-
cyanophenyl)-(3-methyl-3H-imidazol-4-ylmethyl)-amino]-ethyl})
(30-(2-carboxyethyl)-biphenyl)-2-sulfonamide (10).
LiOH.H2O (8.7 mg, 0.207 mmol, 3 eq) was added to a solution of 9 (52 mg,
0.069 mmol, 1 eq) in a 3:1:1 mixture of THF-MeOH-H2O (1 ml). After stirring
for 1 hr at room temperature, the reaction was complete. The mixture was
diluted with water (50 ml), then carefully neutralized by the addition of
1N HCl. The title compound was extracted into EtOAc (10 ml 3 3), dried
(Na2SO4), filtered, and concentrated. The residue was chromatographed
over silica gel (eluent: CH2Cl2/MeOH/NH4OH, 25:7:1) to give pure 10 as a white
powder (47 mg, 92%): dH (500 MHz, d4-MeOH) 0.86 (qd, J = 12.2, 4.0 Hz,
2H, 2 CH (piperidinylmethyl)), 1.42 (s, 9H, C(CH3)3), 1.47–1.55 (m, 3H, 3 CH
(piperidinylmethyl)), 2.50 (br d, J = 7.0 Hz, 2H, 2 CH (piperidinylmethyl)),
calcd for [C31H39N6O4S79Br + H] 671.2015, found 671.2029; HPLC (I) tR
=
12.78 min (100%), tR = 19.92 min (100%).
N-(N-tert-Butoxycarbonylpiperidin-4-ylmethyl), N-({2-[(4-
cyanophenyl)-(3-methyl-3H-imidazol-4-ylmethyl)-amino]-ethyl})
(30-(2-methoxycarbonylethyl)-biphenyl)-2-sulfonamide (9)
To a round-bottomed flask was added 8 (84 mg, 0.130 mmol, 1 eq), 3-(2-me-
thoxycarbonylethyl)phenylboronic acid (41 mg, 0.196 mmol, 1.5 eq) and
K2CO3 (54 mg, 0.390 mmol, 3 eq). The flask was evacuated and purged
with N2 three times, then PdCl2(dppf)2 (27 mg, 25 mol%) was added, followed
by DMF (2 ml), and then the flask was evacuated and purged with N2 three
times again. The reaction mixture was heated to 110ꢁC for 16 hr, then allowed
to cool. Although TLC suggested only the starting aryl bromide, MS suggested
the biphenyl product was present with no remaining aryl bromide, so the reac-
tion was worked up as follows. The DMF reaction mixture was diluted with
water (50 ml), then extracted into EtOAc (10 ml 3 3). The EtOAc extractions
were combined, then washed with water (10 ml 3 3), brine (10 ml), dried
(Na2SO4), filtered, and concentrated. The residue was purified by silica gel
flash column chromatography (eluent: CH2Cl2/MeOH/NH4OH, 25:7:1) to
give the title compound (9) as an off-white film (60 mg, 61%): dH (500 MHz,
CDCl3) 0.82 (qd, J = 12.3, 4.0 Hz, 2H, 2 CH (piperidinylmethyl)), 1.16–
1.20 (m, 1H, CH (piperidinylmethyl)), 1.37–1.45 (m, 11H, C(CH3)3, 2 CH (piper-
idinylmethyl)), 2.34–2.44 (m, 2H, 2 CH (piperidinylmethyl)), 2.49–2.59 (m, 6H,
2.57–2.67 (m, 4H, CH2CH2COOH,
2 CH (piperidinylmethyl)), 2.77–2.82
(m, 2H, CH2CH2NSO2) 2.92 (t, J = 7.5 Hz, 2H, CH2CH2COOH), 3.47–3.53
(m, 2H, CH2CH2NSO2), 3.88 (s, 3H, CH3 (Im)), 3.90–3.96 (m, 2H, 2 CH (piper-
idinylmethyl)), 4.70 (s, 2H, CH2Im), 6.84 (d, J = 9.0 Hz, 2H, CH (p-cyanoAr)),
7.19 (s, 1H, CH (Im)), 7.21–7.32 (m, 4H, 4 CH (Ar)), 7.36 (dd, J = 7.5, 1.5 Hz,
1H, CH (Ar)), 7.54 (d, J = 9.0 Hz, 2H, CH (p-cyanoAr)), 7.61 (td, J = 7.5, 1.0
Hz, 1H, CH (Ar)), 7.69 (td, J = 7.5, 1.0 Hz, 1H, CH (Ar)), 8.16 (dd, J = 8.0, 1.0
Hz, 1H, CH (Ar)), 8.91 (s, 1H, CH (Im)); dC (125 MHz, d4-MeOH) 28.7, 30.7,
31.7, 34.3, 36.4, 36.6, 44.3 (br), 45.3, 46.8, 50.9, 56.0, 81.1, 100.7, 114.1,
119.1, 120.8, 129.0, 129.1, 129.3, 129.4, 131.3, 131.5, 133.0, 133.9, 134.5,
134.9, 137.9, 139.5, 140.9, 142.0, 142.8, 151.5, 156.5, 176.3; HRMS (ES+)
calcd for [C40H48N6O6S + H], 741.3434, found 741.3436; HPLC (I) tR = 12.99
min (100%), (II) tR = 19.68 min (100%).
CH2CH2CO2CH3, CH2CH2NSO2,
2
CH (piperidinylmethyl)), 2.89 (t,
J
=
Protein Preparation and Crystallization
7.8 Hz, 2H, CH2CH2CO2CH3), 3.24–3.31 (m, 2H, CH2CH2NSO2), 3.49 (s, 3H,
CH3 (Im)), 3.64 (s, 3H, CO2CH3), 3.89–4.01 (m, 2H, 2 CH (piperidinylmethyl)),
4.36 (s, 2H, CH2Im), 6.66 (d, J = 8.5 Hz, 2H, CH (p-cyanoAr)), 6.81 (s, 1H,
CH (Im)), 7.10–7.13 (m, 1H, CH (Ar)), 7.16–7.27 (m, 4H, 4 CH (Ar)), 7.43
(d, J = 8.5 Hz, 2H, CH (p-cyanoAr)), 7.47 (td, J = 7.8, 1.0 Hz, 1H, CH (Ar)),
7.51 (s, 1H, Im), 7.56 (td, J = 7.8, 1.0 Hz, 1H, CH (Ar)), 8.07 (dd, J = 8.0,
1.0 Hz, 1H, CH (Ar)); dC (125 MHz, CDCl3) 28.3, 29.4, 30.5, 31.6, 35.2,
35.3, 43.0 (br), 43.8, 44.9, 48.3, 51.6, 54.7, 79.3, 99.5, 112.2, 119.8, 126.6,
Wild-type rat and human protein farnesyltransferases (rFTase and hFTase,
respectively) were expressed and purified as described (Chen et al., 1993;
Long et al., 2001). Crystals of rFTase containing a farnesylated KKKSKTKCVIM
peptide were prepared as described (Long et al., 2002). Similarly, crystals of
wild-type hFTase were prepared containing farnesylated DDPTASACVLS or
DDPTASACNIQ peptides as described (Terry et al., 2006). All compounds
were furnished as lyophilized solids and were solubilized in dimethyl sulfoxide
to prepare a stock solution with a concentration of 30 mM. Inhibitors were
190 Chemistry & Biology 16, 181–192, February 27, 2009 ª2009 Elsevier Ltd All rights reserved

Chemistry & Biology
FTase-Ethylenediamine Inhibitor Complexes
introduced into the FTase active site by soaking preformed product complex
crystals in a stabilizing solution supplemented with 100 mM FPP and 300 mM
inhibitor for 5 to 7 days, followed by cryoprotection and flash cooling in liquid
nitrogen as described (Reid and Beese, 2004; Reid et al., 2004a).
REFERENCES
Basso, A.D., Kirschmeier, P., and Bishop, W.R. (2006). Lipid posttranslational
modifications. Farnesyl transferase inhibitors. J. Lipid Res. 47, 15–31.
Bell, I.M., Gallicchio, S.N., Abrams, M., Beese, L.S., Beshore, D.C., Bhimnath-
wala, H., Bogusky, M.J., Buser, C.A., Culberson, J.C., Davide, J., et al. (2002).
3-Aminopyrrolidinone farnesyltransferase inhibitors: design of macrocyclic
compounds with improved pharmacokinetics and excellent cell potency.
J. Med. Chem. 45, 2388–2409.
Data Collection, Model Building, and Refinement
X-ray diffraction data were collected at the Southeast Regional Collaborative
Access Team (SER-CAT) 22-BM beamline at the Advanced Photon Source,
Argonne National Laboratory. Data reduction and scaling were performed
with HKL2000 (Otwinowski and Minor, 1997). Phases were calculated using
molecular replacement as implemented by PHASER in the CCP4 suite (Storoni
et al., 2004). Iterative model building and refinement were performed using
Coot and REFMAC5, respectively (Emsley and Cowtan, 2004; Murshudov
et al., 1997). MOLPROBITY was used to calculate a Ramachandran plot, iden-
tify and correct rotamer outliers, and identify any potential steric clashes in the
models (Davis et al., 2004). Figures illustrating the determined structures and
superpositions were prepared with Pymol (Delano, 2002). Data collection and
refinement statistics are contained in Table 2. The atomic coordinates have
been deposited with the RCSB Protein Data Bank with the following PDB ID
codes: 3E30, 3E32, 3E33, 3E34, and 3E37. Multiple homology models of
P. falciparum FTase were prepared with web-based servers Swiss Model
(Guex and Peitsch, 1997) and Phyre (Bennett-Lovsey et al., 2008) using align-
ments prepared by the modeling program based upon input crystal structure
(PDB code 1TN6) or a user-defined alignment prepared in ClustalW (Thomp-
son et al., 1994).
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Activity Assays
We determined the effect of the ethylenediamine-based compounds on their
abilities to inhibit human FTase activity of 60,000 3 g supernatants of human
Burkitt’s lymphoma (Daudi) cells (American Type Culture Collection, Rockville,
MD) as described (Carrico et al., 2004). Inhibition studies were performed to
determine the ability of hFTase to transfer [3H]farnesyl from [3H]farnesylpyro-
phosphate (GE Biosciences, Piscataway, NJ) to recombinant human H-Ras.
After a 30-min incubation in the presence of the inhibitor, H-Ras protein was
precipitated with trichloroacetic acid, filtered onto glass fiber filters, and filters
were washed thoroughly to remove unbound [3H]farnesylpyrophate from the
filter. Activity of dried filters was counted on a scintillation counter, and activ-
ities of samples treated with compound were compared with those treated
with vehicle alone to determine IC50 values. Results shown are the mean/stan-
dard deviation of at least three independent experiments, except for 5, for
which the value was the mean of two replicates. The IC50 determinations for
P. falciparum FTase 2 and 4 listed in Table 1 were obtained from reference
(Glenn et al., 2006). For 7 and 10 we calculated the IC50 according to the
methods in (Glenn et al., 2006). For 5, we titered the inhibitory activity of the
compound and determined the IC50 by interpolation from points above and
below the 50% inhibition of enzyme activity (76% and 20% inhibitory activity,
respectively). The activity assay used to estimate the IC50 of 5, 7, and 10 was
performed as described (Glenn et al., 2006).
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Assays to determine the mode of competition of 5 were conducted as
described above for human FTase, except that the source of enzyme was puri-
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substrate and inhibitor concentrations. Data were analyzed using a standard
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ACKNOWLEDGMENTS
This work was supported by NIH Grants GM52382 (to L.S.B), CA067771 (to
S.M.S and A.D.H.), and AI054384 (to M.H.G and W.C.V.). Use of the Advanced
Photon Source was supported by the U.S. Department of Energy, Office of
Science, Office of Basic Energy Sciences under Contract No. W-31-109-
Eng-38. We thank Matthew Glenn and Sung-Yoon Chang for their early work
on the design and synthesis of several compounds.
Glenn, M.P., Chang, S.Y., Horney, C., Rivas, K., Yokoyama, K., Pusateri, E.E.,
Fletcher, S., Cummings, C.G., Buckner, F.S., Pendyala, P.R., et al. (2006).
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Received: September 1, 2008
Revised: January 21, 2009
Accepted: January 26, 2009
Published: February 26, 2009
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