Non-thiol farnesyltransferase inhibitors: utilization of an aryl binding site by 5-arylacryloylaminobenzophenones.

Article abstract of DOI:10.1016/S0968-0896(02)00088-3

We recently described a novel aryl binding site of farnesyltransferase. The 2-naphthylacryloyl residue was developed as an appropriate substituent for our benzophenone-based AAX-peptidomimetic capable of occupying this binding site, resulting in a non-thiol farnesyltransferase inhibitor with nanomolar activity. The activity of this inhibitor is readily explained on the basis of docking studies which show the 2-naphthyl residue fitting into the aryl binding site.

Full text of DOI:10.1016/S0968-0896(02)00088-3

Bioorganic & Medicinal Chemistry 10 (2002) 2657–2662
Non-Thiol Farnesyltransferase Inhibitors: Utilization of an Aryl
Binding Site by 5-Arylacryloylaminobenzophenones
Andreas Mitsch,b Markus Bohm,b Pia Wißner,b Isabel Sattlerc and Martin Schlitzera,*
aDepartment fur Pharmazie, Zentrum fur Pharmaforschung, Ludwig-Maximilians-Universitat Munchen,
¨
¨
Butenandtstraße 5-13, D-81377 Munchen, Germany
¨
¨
¨
bInstitut fur Pharmazeutische Chemie, Philipps-Universitat Marburg, Marbacher Weg 6, D-35032 Marburg, Germany
¨
¨
cHans-Knoll-Institut fur Naturstoff-Forschung e.V., Beutenbergstraße 11, D-07745, Jena, Germany
¨
¨
Received 30 January 2002; accepted 8 March 2002
Abstract—We recently described a novel aryl binding site of farnesyltransferase. The 2-naphthylacryloyl residue was developed as an
appropriate substituent for our benzophenone-based AAX-peptidomimetic capable of occupying this binding site, resulting in a non-
thiol farnesyltransferase inhibitor with nanomolar activity. The activity of this inhibitor is readily explained on the basis of docking
studies which show the 2-naphthyl residue fitting into the aryl binding site. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
exact mechanism of the antiproliferative effect remains to
be determined, farnesyltransferase inhibitors are regarded
as a major emerging strategy in cancer therapy.3À12
Over the past years, farnesyltransferase has become a
major target in the development of potential anticancer
drugs. Farnesyltransferase catalyzes the transfer of a
farnesyl residue from farnesylpyrophosphate to the thiol
of a cysteine side chain of proteins carrying the so-called
CAAX-sequence at their C-terminus. C represents the
cysteine to be farnesylated, A reveals amino acids normally
(but not necessarily) carrying aliphatic side chains, and
X mostly represents methionine or serine.1,2
Most inhibitors described in literature are peptidomi-
metics resembling the CAAX-tetrapeptide recognition
sequence of farnesylated proteins. The majority of these
CAAX-peptidomimetics exhibit a free thiol group
which is shown to coordinate the enzyme-bound zinc ion.
However, free thiols are associated with several adverse
drug effects,13 and therefore, the development of farne-
syltransferase inhibitors is clearly directed toward the so-
called non-thiol farnesyltransferase inhibitors. The most
frequently used replacements for cysteine are nitrogen-
containing heterocycles. The ring nitrogen is supposed
to coordinate to the enzyme-bound zinc similarly to the
cysteine thiol group.14 However, it has been shown that
nitrogen heterocycles can be replaced by aryl residues
lacking the ability to coordinate metal atoms without
loosing too much of farnesyltransferase inhibitory
activity.15,16 Therefore, the existence of at least one
hitherto unknown aryl binding region in the farnesyl-
transferase’s active site has been postulated.17,18
Although farnesyltransferase inhibitors have demon-
strated their efficiency in various cancer cell culture assays,
animal models and first clinical studies, it turned out that
their mechanism of action is much more complicated as
initially supposed. Farnesyltransferase inhibitors display
multiple effects, for example inhibition of anchorage
independent cell growth, reversal of the phenotype of
cancer cells back to that of non-transformed cells, cell
cycle arrest and induction of apoptosis. It became obvious
that these multiple effects cannot be attributed solely to
the prevention of Ras farnesylation. Several different far-
nesylated proteins, as for instance Ras, RhoB and cen-
tromere binding proteins seem to be involved in the
action of farnesyltransferase inhibitors. Although the
Using docking studies of model compounds of non-thiol
farnesyltransferase inhibitors as well as GRID analysis
of farnesyltransferase’s active site, we have identified
two different aryl binding clefts in the active site of far-
nesyltransferase which we suggest to be the postulated
aryl binding regions (Fig. 1a).19
*Corresponding author. Tel.: +49-89-2180-7804; e-mail: martin.
schlitzer@cup.uni-muenchen.de
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00088-3

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A. Mitsch et al. / Bioorg. Med. Chem. 10 (2002) 2657–2662
Figure 1. (a) View of the active site of farnesyltransferase. Lipophilic (brown) and hydrophilic (green to blue) properties are displayed on the Con-
nolly surface of the enzyme. The location of the near and far aryl binding sites is indicated by a white and yellow box, respectively. (b) Representative
docking solutions of the cinnamoyl-substituted inhibitor 3a, showing that the terminal phenyl residue only partially fills the far aryl binding site. (c)
Representative docking solutions of inhibitor 3b substituted with 2-naphthylarylic acid, showing that the terminal 2-naphthyl residue occupies a larger
portion of the far aryl binding site. (d) Low energy docking solution of inhibitor 3g, showing the ring nitrogen in the vicinity of Lys 164, enabling a
hydrogen bond. (e) Low energy docking solution of inhibitor 3h, showing the 3-indolyl moiety in the aryl binding side and the carbonyl oxygen of
the acetyl residue in close proximity to Lys 356, enabling a hydrogen bond. The angle of sight of this picture is different from that of the other ones.
In this study, we intended to develop an appropriate
residue for our benzophenone-based AAX-peptidomi-
metic substructure20 which is capable of placing an aryl
moiety into the far aryl binding site.
prepared by knoevenagel condensation from appropriate
aldehydes and malonic acid.
Molecular modeling
The coordinates of the published structure of a ternary
complex of farnesyltransferase, a farnesylpyrophosphate
analogue and N-acetyl-Cys-Val-Ile-selenoMetOH (PDB-
code 1QBQ)21 were used for the modeling studies. The
tetrapeptide was removed from the crystal structure, and
the solvent accessible surface of the farnesyltransferase’s
active site was calculated using the program MOLCAD
as implemented in the molecular modeling software
Chemistry
Synthesis of the target compounds 3a–h was accom-
plished by the acylation of 5-amino-2-(p-tolylacetyl-
amino)benzophenone20 (1) using appropriate 3-arylacrylic
acid chlorides (2) (Scheme 1). These were prepared from
the corresponding 3-arylacrylic acids which in turn were

A. Mitsch et al. / Bioorg. Med. Chem. 10 (2002) 2657–2662
2659
Table 1. Farnesyltransferase inhibitory activity of compounds 3a–h
Compd
R
IC50 (nM)
3a
2400Æ400
Scheme 1. (I) Toluene/dioxane, reflux, 2 h.
3b
3c
3d
3e
3f
115Æ20
5600Æ300
1500Æ200
6300Æ300
10,000Æ500
760Æ105
package SYBYL.22 Farnesylpyrophosphate was included
as part of the enzyme’s molecular surface. The previously
determined position of the benzophenone peptidomi-
metic substructure19 was used as starting fragment for
the docking of inhibitors 3a and 3b. Subsequently, the
docking program FlexX23 places the remaining frag-
ments of the inhibitors in a piece-wise fashion into the
active site searching for favorable hydrophobic and H-
bond interactions while avoiding steric overlaps. The
docking runs provided sets of solutions which were
inspected according to their suggested binding energy.
Obviously unreasonable solutions were excluded, for
example those where major parts of the inhibitors were
exposed merely to the solvent without showing specific
interactions within the active site.
3g
Farnesyltransferase inhibition assay
3h
110Æ40
The inhibitory activity of the inhibitors was determined
using the fluorescence enhancement assay as described by
Pompliano.24 The assay employed yeast farnesyltransfer-
ase (FTase) fused to Glutathione S-transferase at the
N-terminus of the b-subunit.24 Farnesylpyrophosphate
and the dansylated pentapeptide Ds-GlyCysValLeuSer
were used as substrates. Upon farnesylation of the
cysteine thiol the dansyl residue is placed in a lipophilic
environment resulting in an enhancement of fluores-
cence at 505 nm which is use to monitor the enzyme
reaction.
only the smaller part of the far aryl binding site.
Accordingly, a more bulky aryl residue connected to the
3-position of the acrylic acid substructure should fill
more of the aryl binding site. Figure 1c shows repre-
sentative examples of the docking runs of inhibitor 3b
substituted with 2-naphthylarylic acid. In this case, the
bulky 2-naphthyl moiety is more likely to occupy the
aryl binding site than the smaller phenyl residue of
inhibitor 3a. This is mirrored by a 20-fold enhancement
in farnesyltransferase inhibitory activity of 3b (IC50=
115 nM) compared to inhibitor 3a. The inhibitory
activity is proved to be sensitive towards changes in
geometry and electronic properties of the 3-arylacryloyl
moiety. Accordingly, the 1-naphthyl analogue 3c
(IC50= 5600 nM) is markedly less active than its isomer
3b. Docking of this inhibitor (not shown) reveales that
the second annellated ring does not occupy the binding
cleft but is completely exposed to the solvent. Also less
active are the piperonyl derivative 3e (IC50=6300 nM)
and the 2-quinonyl derivative 3d (IC50=1500 nM).
Obviously, the fluorenyl moiety of inhibitor 3f is already
too large to fit into the aryl binding site as indicated by
its poor IC50 of 10 mM. Submicromolar activity is
recorded for the 2-benzothiazolyl derivative 3g (IC50=
760nM) and the 1-acetyl-3-indolyl derivative 3h (IC50=
110nM), the latter one showing an inhibition rate
Results and Discussion
Since we successfully identified the putative location of
the farnesyltransferase’s aryl binding sites,19 we further
set out to develop an appropriate residue for our AAX-
peptidomimetic benzophenone substructure which is
able to place an aryl residue into the far aryl binding site
(Fig. 1a). Docking of several different possible residues
suggested that cinnamic acid might be such an appro-
priate residue. We rationalized that the trans-configured
double bond of this moiety would force the terminal
aryl residue into the aryl binding cleft as shown in Fig-
ure 1b. Indeed, inhibitor 3a carrying this cinnamoyl
residue could inhibit farnesyltransferase with an IC50 of
2.4 mM (Table 1). The representative solutions of the
docking runs (Fig. 1b), however, clearly indicate that
the phenyl residue of the cinnamoyl moiety occupies

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A. Mitsch et al. / Bioorg. Med. Chem. 10 (2002) 2657–2662
comparable to the 2-naphthyl substituted inhibitor 3b.
Docking of these two inhibitors provides a reasonable
explanation for the compatively better activity of these
compounds. Although the benzothiazole moiety of
inhibitor 3g does not fill the aryl binding site better than
the phenyl residue of inhibitor 3a, there is a possible
hydrogen bond between the ring nitrogen and the side
chain of Lys 164a (Fig. 1d) which may account for the
3-fold better activity of the benzothiazole derivative 3g
in comparison to the cinnamoyl derivative 3a. No indi-
cation of an interaction of the thiazole ring of 3g with
the zinc has been found by flexible docking. Regarding
the geometry of the arylacryloyl residues, compound 3h
is somewhere between the 2-naphthyl derivative 3b and
the much less active 1-nahthyl isomer 3c. However,
flexible docking reveals that the 3-indolyl moiety of 3h
nicely fitts into the aryl binding side (Fig. 1e). Further-
more, the carbonyl oxygen of the acetyl residue is placed
in close proximity to the side chain of Lys 356b enabling
a hydrogen bond between these two moieties.
according to general procedure A. Purification: recrys-
tallization from toluene. Yield 214 mg (45%); mp 97 ꢀC.
IR (KBr): n=3440, 3264, 3086, 1668, 1631, 1547, 1504
1
cmÀ1. H NMR (CDCl3): d 2.31 (s, 3H), 3.67 (s, 2H),
6.45 (d, J=16 Hz, 1H), 7.15 (m, 3H), 7.24 (m, 3H), 7.34
(m, 3H), 7.45 (m, 4H), 7.54–7.60(m, 2H), 7.69 (m, 2H),
8.01 (s, 1H), 8.51 (m, 1H), 10.52 (s, 1H). MS (EI): m/z
474 (100) M+, 342 (39), 212 (37), 131 (34). Anal. calcd
for C31H26N2O3: C, 78.46; H, 5.52; N, 5.90; found: C,
78.71; H, 5.52; N, 5.86.
N-3-Benzoyl-4-(p-tolylacetylamino)phenyl-3-naphthalen-
2-yl-acrylamide (3b). From 3-naphtalen-2-yl-acrylic acid
(297 mg, 1.5 mmol) according to general procedure A.
Purification: recrystallization from toluene. Yield 465
mg (88%); mp 97C. IR (KBr): n=3418, 3056, 1674,
1
1634, 1554, 1508 cmÀ1. H NMR (CDCl3): d 2.24 (s,
3H), 3.60(s, 2H), 6.48 (d, J=16 Hz, 1H), 7.09 (m, 2H),
7.17 (m, 3H), 7.36–7.43 (m, 4H), 7.49 (m, 3H), 7.63 (m,
2H), 7.68–7.77 (m, 5H), 7.98 (s, 1H), 8.44 (m, 1H), 10.46
(s, 1H). MS (EI): m/z 181 (100), 212 (18), 105 (73), 154
(43), 344 (37), 524 (28) M+. Anal. calcd for C35H28
N2O3: C, 80.13; H, 5.38; N, 5.34; found: C, 79.80; H,
5.40; N, 5.28.
In summary, the 2-naphthylacryloyl residue was devel-
oped as an appropriate substituent for our benzophe-
none-based AAX-peptidomimetic which is capable of
occupying the aryl binding site of farnesyltransferase.
This resulted in a non-thiol farnesyltransferase inhibitor
with nanomolar activity.
N-3-Benzoyl-4-(p-tolyl-acetylamino)phenyl-3-naphthalen-
1-yl-acrylamide (3c). From 3-naphtalen-1-yl-acrylic acid
(298 mg, 1.5 mmol) according to general procedure A.
Purification: recrystallization from toluene. Yield 522
mg (66%); mp 134 ꢀC. IR (KBr): n=3292, 3055, 2921,
Experimental
1
1655, 1596, 1542, 1507 cmÀ1. H NMR (CDCl3): d 2.25
1H and 13C NMR spectra were recorded on a Jeol
JMN-GX-400 and a Jeol JMN-LA-500 spectrometer.
Mass spectra were obtained with a Vacuum Generators
VG 7070H using a Vector 1 data acquisition system from
Teknivent or a AutoSpec mass spectrometer from Micro-
mass. IR spectra were recorded on a Nicolet 510P FT-IR-
spectrometer. Microanalyses were obtained from a CH
analyzer according to Dr. Salzer from Labormatic and
from a Hewlett Packard CHN-analyzer type 185. Melt-
ing points were obtained with a Leitz microscope and
are uncorrected. Column chromatography was carried
out using silica gel 60 (0.062–0.200 mm) from Merck.
Medium Pressure Liquid Chromatography (MPLC)
was performed using a pump type 688 from Buchi and a
column of 3.5 cm diameter and 45 cm length filled with
silica gel 60 (0.015–0.040 mm) from Merck.
(s, 3H), 3.61 (s, 2H), 6.47 (d, J=16 Hz, 1H), 7.08 (m,
2H), 7.18 (m, 3H), 7.34–7.65 (m, 10H), 7.79 (m, 2H),
8.00 (s, 1H), 8.08 (m, 1H), 8.45 (m, 2H), 10.46 (s, 1H).
MS (EI): m/z 181 (100), 105 (51), 212 (43), 524 (33) M+.
Anal. calcd for C35H28N2O3: C, 80.13; H, 5.38; N, 5.34;
found: C, 79.84; H, 5.43; N, 5.35.
N-3-Benzoyl-4-(p-tolylacetylamino)phenyl-3-quinolin-2-
yl-acrylamide (3d). From 3-quinolin-2-yl-acrylic acid
(199 mg, 1 mmol) according to general procedure A.
Purification: recrystallization from toluene. Yield 220mg
(42%); mp 187 ꢀC. IR (KBr): n=3438, 1641, 1556, 1507
cmÀ1. 1H NMR (CDCl3): d 2.26 (s, 3H), 3.63 (s, 2H), 7.10
(m, 2H), 7.19 (m, 4H), 7.39–7.54 (m, 5H), 7.61–7.65 (m,
3H), 7.71 (m, 1H), 7.75 (d, J=16 Hz, 1H), 7.86 (s, 1H),
7.95 (m, 2H), 8.07 (m, 1H), 8.46 (m, 1H), 10.46 (s, 1H).
MS (EI): m/z 182 (100), 105 (42), 212 (36), 525 (34) M+,
420(34). Anal. calcd for C 34H27N3O3: C, 77.70; H, 5.18;
N, 7.99; found: C, 77.32; H, 5.29; N, 7.71.
General procedure A: amide formation using acids chlorides
Arylacrylic acids were dissolved in dichloromethane and
0.2 mL oxalylchloride per mmol acid and a few drops
DMF were added. The mixture was stirred at room
temperature for 2 h and the volatiles were evaporated in
vacuo. The residue obtained was dissolved in toluene or
dioxane (approx. 10mL) and added to a solution of one
equivalent of 5-amino-2-(p-tolylacetylamino)benzophe-
none in hot toluene (approx. 50mL). The mixture was
heated under reflux for 2 h. Then, the solvent was removed
in vacuo to give the crude products.
3-Benzo[1,3]dioxol-5-yl-N-3-benzoyl-4-(2-p-tolylacetyl-
amino)phenylacrylamide (3e). From piperonyl-acrylic
acid (192 mg, 1.5 mmol) according to general procedure
A. Purification: column chromatography (EtOAc/hex-
ane 3:2) and subsequent recrystallization from toluene.
Yield 106 mg (14%); mp 185ꢀC. IR (KBr): n=3332,
3043, 2907, 1672, 1551, 1506 cmÀ1. 1H NMR (CDCl3): d
2.26 (s, 3H), 3.62 (s, 2H), 5.92 (s, 2H), 6.21 (d, J=16 Hz,
1H), 6.72 (m, 1H), 6.89 (m, 2H), 7.09 (m, 2H), 7.18 (m,
2H), 7.32 (m, 1H), 7.41 (m, 2H), 7.51 (m, 3H), 7.64 (m,
2H), 7.93 (s, 1H), 8.47 (m, 1H), 10.47 (s, 1H). MS (EI):
m/z 175 (100), 145 (51), 212 (50),105 (38), 518 (23) M+.
N-3-Benzoyl-4-(p-tolylacetylamino)phenyl-3-phenylacryl-
amide (3a). From cinnamic acid (148 mg, 1 mmol)

A. Mitsch et al. / Bioorg. Med. Chem. 10 (2002) 2657–2662
2661
Anal. calcd for C32H26N2O5: C, 74.12; H, 5.05; N, 5.40;
found: C, 73.99; H, 5.06; N, 5.46.
The enzyme was purified by standard procedures with
glutathione-agarose beads for selective binding of the tar-
get protein.
N-3-Benzoyl-4-(p-tolylacetylamino)phenyl-3-(9H-fluoren-
2-yl)-acrylamide (3f). From 3-(fluoren-2-yl)acrylic acid
(236 mg, 1 mmol) according to general procedure A.
Purification: MPLC (EtOAc/hexane 1:1). Yield 330mg
(59%); mp 210 ꢀC. IR (KBr): n=3317, 3046, 1682, 1669,
Farnesyltransferase assay
The assay was conducted as described.24 Farnesylpyro-
phosphate (FPP) was obtained as a solution of the
ammonium salt in methanol/10mM aqueous NH 4Cl (7:3)
from Sigma-Aldrich. Dansyl-GlyCysValLeuSer (Ds-
GCVLS) was custom synthesized by ZMBH, Heidelberg,
Germany. The assay mixture (100 L volume) contained 50
1
1648, 1616, 1560, 1510 cmÀ1. H NMR (CDCl3): d 2.30
(s, 3H), 3.67 (s, 2H), 3.81 (s, 2H), 6.49 (d, J=16 Hz,
1H), 7.14 (m, 2H), 7.22 (m, 2H), 7.28–7.37 (m, 2H), 7.43
(m, 3H), 7.49–7.55 (m, 4H), 7.68 (m, 4H), 7.74 (m, 1H),
7.98 (s, 1H), 8.07 (s, 1H), 8.49 (m, 1H), 10.54 (s, 1H).
MS (EI): m/z 219 (100), 43 (87), 41 (36), 73 (35), 562
(19) M+. Anal. calcd for C38H30N2O3: C, 81.11; H,
5.37; N, 4.98; found: C, 80.85; H, 5.50; N, 5.14.
mM Tris/HCl pH 7.4, 5 mM MgCl2, 10 M ZnCl , 5 mM
2
dithiothreitol (DTT), 7 M Ds-GCVLS, 20M FPP and 5
nmol (approx.) yeast GST-farnesyltransferase and 1%
of various concentrations of the test compounds dis-
solved in dimethylsulfoxide (DMSO). The progress of
the enzyme reaction was followed by monitoring the
enhancement of the fluorescence emission at 505 nm
(excitation 340nm). The reaction was started by addi-
tion of the enzyme and run in a Quartz cuvette thermo-
statted at 30 ꢀC. Fluorescence emission was recorded
with a Perkin-Elmer LS50B spectrometer. IC50 values
(concentrations resulting in 50% inhibition) were calcu-
lated from initial velocity of three independent mea-
surements of four to five different concentrations of the
respective inhibitor.
3-Benzothiazol-2-yl-N-3-benzoyl-4-(p-tolylacetylamino)-
phenylacrylamide (3g). From 3-benzothiazol-2-yl-acrylic
acid (92 mg, 0.45 mmol) according to general procedure A.
Purification: reꢀcrystallization from ethanol. Yield 14 mg
(6%); mp 232 C. IR (KBr): n=3431, 1654, 1558, 1508
cmÀ1. 1H NMR (DMSO-d6): d 2.23 (s, 3H), 3.36 (s, 2H),
6.95 (m, 1H), 6.99 (m, 2H), 7.04 (m, 2H), 7.20 (d, J=16
Hz, 1H), 7.49 (m, 3H), 7.55 (m, 1H), 7.62 (m, 2H), 7.68
(m, 2H), 7.78 (m, 1H), 7.88 (m, 1H), 8.04 (m, 1H), 8.13
(m, 1H), 10.04 (s, 1H), 10.56 (s, 1H). MS (EI): m/z 105
(100), 188 (90), 212 (74), 40 (62), 531 (22) M+. Anal.
calcd for C32H25N3O5S: C, 72.30; H, 4.74; N, 7.90;
found: C, 71.91; H, 4.34; N, 8.28.
Acknowledgements
3-(1-Acetyl-1H-indol-3-yl)-N-3-benzoyl-4-(p-tolylacetyl-
amino)phenylacrylamide (3h). From 3-(1-acetyl-3-indo-
lyl) acrylic acid (229 mg, 1 mmol) according to general
procedure A. Purification: MPLC (EtOAc/hexane 1:1).
Yield 333 mg (60%); mp 199 ꢀC. IR (KBr): n=3273, 3136,
3083, 1664, 1623, 1544, 1505 cmÀ1. 1H NMR (CDCl3): d
2.26 (s, 3H), 2.55 (s, 3H), 3.66 (s, 2H), 6.65 (d, J=16
Hz, 1H), 7.05 (m, 2H), 7.21 (m, 3H), 7.33 (m, 1H), 7.40
(m, 2H), 7.49–7.55 (m, 3H), 7.69 (m, 2H), 7.73 (m, 2H),
8.04 (s, 1H), 8.29 (s, 1H), 8.42 (m, 2H), 10.48 (s, 1H).
MS (EI): m/z 212 (100), 170 (69), 344 (61), 555 (37) M+.
Anal. calcd for C35H29N3O4: C, 75.65; H, 5.26; N, 7.56;
found: C, 75.51; H, 5.46; N, 7.56.
The authors thank Prof. Dr. G. Klebe who provided us
with all facilities needed for the modeling and Ms. K.
Burk for technical assistance. The pGEX-DPR1 and
pBC-RAM2 plasmids were kindly provided by Prof. F.
Tamanoi (UCLA). Financial support by the Deutsche
Pharmazeutische Gesellschaft is gratefully acknowledged.
I.S. wishes to thank Prof. Dr. S. Grabley for generous
support and Ms. S. Egner for technical assistance.
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Molecular modeling
Molecular modeling was carried out using SYBYL22 ver-
sion 6.6/6.7 running on a Silicon Graphics O2 (R10000).
Flexible docking was performed using FlexX23 version
1.7.6. The FlexX command MAPREF and the perturbate
mode of the PLACEBAS command were used. Default
parameters were employed except the MAX_ENERGY
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.
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Yeast farnesyltransferase was used as a fusion protein
to Glutathione S-transferase at the N-terminus of the b-
subunit. Farnesyltransferase was expressed in Escher-
ichia coli DH5a grown in LB media containing ampicillin
and chloramphenicol for co-expression of pGEX-DPR1
and pBC-RAM2 for farnesyltransferase production.25

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