Amide-substituted farnesylcysteine analogs as inhibitors of human isoprenylcysteine carboxyl methyltransferase

Article abstract of DOI:10.1016/j.bmcl.2006.05.029

N-Acetyl-S-farnesyl-l-cysteine (AFC) is the minimal substrate for the enzyme isoprenylcysteine carboxyl methyltransferase (Icmt). A series of amide-modified farnesylcysteine analogs were synthesized and screened against human Icmt. From a 23-membered libr

Full text of DOI:10.1016/j.bmcl.2006.05.029

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4420–4423
Amide-substituted farnesylcysteine analogs as inhibitors of
human isoprenylcysteine carboxyl methyltransferase
James L. Donelson,a Heather B. Hodges,b Daniel D. MacDougall,a, Brian S. Henriksen,a
Christine A. Hrycynab,* and Richard A. Gibbsa,*
aDepartment of Medicinal Chemistry and Molecular Pharmacology and the Purdue Cancer Center,
Purdue University, West Lafayette, IN 47907, USA
bDepartment of Chemistry and the Purdue Cancer Center, Purdue University, West Lafayette, IN 47907, USA
Received 20 March 2006; revised 8 May 2006; accepted 8 May 2006
Available online 13 June 2006
Abstract—N-Acetyl-S-farnesyl-L-cysteine (AFC) is the minimal substrate for the enzyme isoprenylcysteine carboxyl methyltransfer-
ase (Icmt). A series of amide-modified farnesylcysteine analogs were synthesized and screened against human Icmt. From a 23-mem-
bered library of compounds, six inhibitors were identified and evaluated further. The adamantyl derivative 7c was the most potent
inhibitor with an IC50 of 12.4 lM.
Ó 2006 Elsevier Ltd. All rights reserved.
Numerous proteins are initially synthesized with a C-ter-
minal –CaaX box motif, where -C is cysteine, -aa are
generally two aliphatic residues, and –X is typically S,
M, F, Q, or L. This motif labels the protein for a series
of sequential post-translational modifications (Fig. 1).
First, either a 15-carbon farnesyl or 20-carbon geranyl-
geranyl group is added via a thioether linkage to the cys-
teine by one of two soluble isoprenyltransferases
(protein-farnesyltransferase, FTase, or protein-geranyl-
geranyltransferase I, GGTase I). Following the attach-
ment of the prenyl group, the three –aaX residues are
cleaved by the endoprotease Ras-converting enzyme 1
(Rce1), and subsequently the newly exposed cysteine
carboxyl group is methylated by isoprenylcysteine car-
boxyl methyltransferase (Icmt). It is estimated that at
least 120 mammalian proteins undergo this sequential
three-step post-translational modification sequence,1
the sum of which typically results in increased hydro-
phobicity and enhanced membrane association of an ini-
tially cytosolic protein.
was determined that the oncogenic Ras family of GTP-
ases must be farnesylated in order to properly function.
Importantly, mutations in this family of proteins are
responsible for approximately 20–30% of all human
cancers and 90% of pancreatic cancers. A number of
farnesyltransferase inhibitors (FTIs) are currently
undergoing evaluation in clinical trials.2,3 However,
these agents have not exhibited significant activity in
most patients with Ras-driven tumors4 due to alterna-
tive geranylgeranylation of Ras in FTI-treated cells.5,6
This post-translational pathway became the subject of
intense scrutiny as a target for cancer therapies, as it
Keywords: Farnesylcysteine analogs; Isoprenylcysteine carboxyl
methyltransferase; Post-translational modification.
*
Corresponding authors. E-mail: rag@pharmacy.purdue.edu
Present address: Department of Chemistry, Kalamazoo College,
Kalamazoo, MI 49006, USA.
Figure 1. Post-translational modification of Ras by FTase, Rce1, and
Icmt.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.029

J. L. Donelson et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4420–4423
4421
Rce1 and Icmt have been recognized for a number of
years as potential alternative anticancer targets to
FTase.7 Although originally thought to be too impor-
tant for cell viability due to the embryonic lethal pheno-
type of Rce1À/À and IcmtÀ/À mice,8 a recent publication
by Michaelson et al. showed that proteolysis and meth-
ylation are not as important for the proper functioning
and localization of geranylgeranylated proteins as for
farnesylated proteins.9 Thus, inhibiting Rce1 or Icmt
may result in a phenotype similar to that observed when
inhibiting FTase alone and not like the profound toxic
effects seen with dual FTase/GGTase I inhibitors.10
We have therefore initiated an effort to generate Icmt
inhibitors based on the structure of the minimal Icmt
substrate N-acetyl-S-farnesyl-L-cysteine (AFC, Fig. 2,
1) in hopes of developing potent anticancer agents as
well as molecular tools to study the structure and mech-
anism of Icmt.
Recently reported work from our laboratories has
shown that selective changes in the farnesyl group of
AFC can afford effective inhibitors of yeast Icmt
(Fig. 2, 2; KI = 17 lM).11 However, compound 2 was
not an effective inhibitor of human Icmt. Winter-Vann
et al. have also identified a novel indole-based small
molecule inhibitor of human Icmt from a library screen.
This compound, termed cysmethynil (3 Fig. 2;
IC50 = 2.4 lM in vitro), resulted in an Icmt-dependent
Ras mislocalization, as well as a decrease in cell prolifer-
ation and anchorage-independent growth in soft agar
assays.12 In response to these promising initial reports,
we have expanded our efforts toward the development
of AFC-based Icmt inhibitors, and have now synthe-
sized a series of amide-modified farnesyl cysteine (FC)
analogs (Fig. 3).
The biochemical and cellular effects of other FC analogs
have been previously reported. Farnesyl thiosalicylic
acid (FTS) has been shown to inhibit the growth of
H-Ras-driven Rat1 cells, though it is believed this effect
is not solely due to inhibition of Icmt.13 Perez-Sala et al.
showed that another AFC-based compound, S-farnesyl-
thioacetic acid (FTA), induced apoptosis in HL-60 cells.
However, Ras was not mislocalized away from the cell
membrane, suggesting a non-Icmt-specific effect of this
compound.14 Note that FTS and FTA were reported
to be methyltransferase inhibitors (IC50 = 2.8 and
Figure 2. Structures of N-acetyl farnesylcysteine (AFC) (1), N-acetyl-
3-isobutenylfarnesylcysteine (2), and cysmethynil (3).
Figure 3. Structures of compounds screened against Icmt in SPA.

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J. L. Donelson et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4420–4423
4.6 lM, respectively),13,14 but these studies were per-
formed with crude membrane preparations from tissues
rather than the well-characterized recombinant human
Icmt11 that is now available. In an attempt to distinguish
Icmt inhibition from other non-specific effects, Ma et al.
synthesized a number of amide-modified AFC ana-
logs.15,16 Bulky benzoyl and pivaloyl analogs did not
act as substrates, but the introduction of a flexible gly-
cine spacer between the benzoyl group and farnesylcys-
teine restored substrate activity.16 We propose that a
further exploration of the structure–activity relationship
for AFC analogs could lead to more potent Icmt inhib-
itors. It is our hypothesis that an FC-based inhibitor
that has a higher affinity for the enzyme but is not a sub-
strate will reduce the pleotropic effects associated with
FTS and FTA.
Table 1. Inhibition of human Icmt by selected compounds
% of controla,c
Enzyme inhibition IC50 (lM)
a,d
Compound
None
7c
7e
100.0 ( 19.0)
12.8 ( 7.9)
50.1 ( 3.5)
33.5 ( 4.3)
37.1 ( 4.2)
35.1 ( 2.2)
28.0 ( 2.9)
NAb
12.4 ( 1.0)
16.6 ( 1.3)
23.0 ( 1.9)
14.3 ( 0.8)
17.4 ( 1.6)
20.3 ( 2.6)
7j
7l
7s
7w
a Values are means of three experiments, SEM is given in parentheses.
b NA, not applicable.
c Determined using an SPA procedure.18b
d IC50 values determined using vapor diffusion assay20 and calculated
using GraphPad Prism 4.0.
From our initial screen of twenty-three compounds, six
agents (7c, 7e, 7j, 7l, 7s, and 7w) led to a 50% or greater
decrease in BFC methylation, our criteria for advancing
a compound into additional testing (Fig. 3). In this
study, we found that FC analogs containing larger R-
groups inhibited Icmt more effectively than those with
smaller R-groups. In particular, the adamantyl analog
7c was the most potent inhibitor. Also, compounds con-
taining 3,5-disubstituted phenyl rings (7j and 7l), with
the exception of the difluoro compound 7h, exhibited
inhibitory activity, whereas the corresponding 2,4-disub-
stituted analogs (7i and 7k) did not. These data repre-
sent three independent experiments performed in
duplicate. The assay is robust and reproducible, and sig-
nificantly accelerated the testing of this library.
Using the short synthetic route outlined in Scheme 1, a
library of 23 amide-modified farnesylcysteine analogs
was synthesized. Farnesylcysteine methyl ester 5 was
prepared via the method of Poulter and co-workers.17
Standard peptide coupling conditions were used for
the introduction of the various carboxylic acids. HOBt,
EDC, and R–COOH were dissolved in DMF, followed
by the addition of i-Pr2EtN. In some instances this solu-
tion required heating to dissolve the carboxylic acid
prior to addition of 5. After 2–3 h at room temperature,
work-up and purification gave 6a–w in 55–95% yield.
Hydrolysis of the methyl ester was accomplished by dis-
solving 6a–w in 95% ethanol at zero degrees and adding
NaOH. Reactions were typically complete in one hour
which after work-up and purification gave 7a–w in 40–
70% yield.
We subsequently took the six lead compounds and
determined their IC50 values for human Icmt using the
methanol vapor diffusion assay11,20 and the IC50 values
are shown in Table 1. As in the SPA above, the adaman-
tyl analog 7c was the most potent inhibitor with the low-
The synthesized compounds shown in Figure 3 were
screened for Icmt inhibitory activity using a high-
throughput scintillation proximity assay (SPA).18 In
the SPA, crude membranes from Saccharomyces cerevi-
siae cells overexpressing active human Icmt were
incubated with 20 lM of the methyl donor S-adenosyl-
[14C-methyl]methionine (SAM) and 25 lM of the
est IC50. We are currently synthesizing
a more
comprehensive library to further explore both the sub-
strate and inhibitory requirements for prenylcysteine
binding to Icmt.
substrate
N-biotinyl-(6-aminohexanoic)-S-farnesyl-L-
In summary, we have synthesized a small library of
amide-modified farnesylcysteine analogs and screened
them for inhibitory activity. We have identified inhibi-
tors of Icmt that are both easily synthesized and poten-
tially more useful than AFC for further biochemical and
cellular studies. These compounds are much more po-
tent inhibitors of human Icmt than our previously
reported prenyl-modified analogs such as 2, although
not as potent as inhibitor 3. We are now in a position
to utilize this information to develop more potent inhib-
itors of recombinant human Icmt.
cysteine (BFC).19 The extent of the methylation by hIc-
mt is quantified by the binding of the methylated BFC to
streptavidin SPA beads, and measuring the counts per
minute (cpm).
Acknowledgments
This work was supported in part by the US Army
CDMRP (NF020054; subcontract to R.A..G.), by a
grant from the National Pancreas Foundation to
C.A.H., and by the NIH (P30CA21368; Purdue Cancer
Center Support Grant). D.M.D. (Kalamazoo College)
was supported by an NSF REU in Chemical Biology
grant (NSF 0354097-CHE) at Purdue University.
Scheme 1. Reagents and conditions: (a) farnesyl-Cl, 7 N NH3/MeOH,
0 °C to rt, 82%; (b) R–COOH, HOBt, EDC, DIEA, DMF, rt, 50–95%;
(c) NaOH, 95% EtOH/H2O, rt, 40–75%.

J. L. Donelson et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4420–4423
4423
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G.; Toone, E. J.; Casey, P. J. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 4336.
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