Targeting Tumour Proliferation with a Small-Molecule Inhibitor of AICAR Transformylase Homodimerization

Article abstract of DOI:10.1002/cbic.201200279

Aminoimidazole carboxamide ribonucleotide transformylase/ inosine monophosphate cyclohydrolase (ATIC) is a bifunctional homodimeric enzyme that catalyzes the last two steps of de novo purine biosynthesis. Homodimerization of ATIC, a protein-protein intera

Full text of DOI:10.1002/cbic.201200279

DOI: 10.1002/cbic.201200279
Targeting Tumour Proliferation with a Small-Molecule
Inhibitor of AICAR Transformylase Homodimerization
Ian B. Spurr,[a] Charles N. Birts,[b] Francesco Cuda,[a] Stephen J. Benkovic,[c]
Jeremy P. Blaydes,[b] and Ali Tavassoli*[a, b]
Aminoimidazole carboxamide ribonucleotide transformylase/
inosine monophosphate cyclohydrolase (ATIC) is a bifunctional
homodimeric enzyme that catalyzes the last two steps of de
novo purine biosynthesis. Homodimerization of ATIC, a pro-
tein–protein interaction with an interface of over 5000 ꢀ2, is
required for its aminoimidazole carboxamide ribonucleotide
(AICAR) transformylase activity, with the active sites forming at
the interface of the interacting proteins. Here, we report the
development of a small-molecule inhibitor of AICAR transfor-
mylase that functions by preventing the homodimerization of
ATIC. The compound is derived from a previously reported
cyclic hexapeptide inhibitor of AICAR transformylase (with a Ki
of 17 mm), identified by high-throughput screening. The active
motif of the cyclic peptide is identified as an arginine-tyrosine
dipeptide, a capped analogue of which inhibits AICAR transfor-
mylase with a Ki value of 84 mm. A library of nonnatural ana-
logues of this dipeptide was designed, synthesized, and as-
sayed. The most potent compound inhibits AICAR transformy-
lase with a Ki value of 685 nm, a 25-fold improvement in activi-
ty from the parent cyclic peptide. The potential for this AICAR
transformylase inhibitor in cancer therapy was assessed by
studying its effect on the proliferation of a model breast
cancer cell line. Using a nonradioactive proliferation assay and
live cell imaging, a dose-dependent reduction in cell numbers
and cell division rates was observed in cells treated with our
ATIC dimerization inhibitor.
Introduction
The activity of many enzymes is
controlled by their quaternary
structure, through active sites
that form at the interface of ho-
modimeric or oligomeric com-
plexes. These enzymes may not
only be targeted with substrate
analogues, but also by protein–
Scheme 1. The final two steps of the de novo purine biosynthesis pathway catalyzed by ATIC.
protein interaction inhibitors, en-
abling an alternative approach
to modulating their activity.[1] One such enzyme is aminoimida-
zole carboxamide ribonucleotide transformylase/inosine mono-
phosphate cyclohydrolase (ATIC), a bifunctional, highly con-
served, homodimer that catalyzes the last two steps of de
novo purine biosynthesis (Scheme 1).[2] Structural studies have
shown the ATIC homodimer to have two distinct domains, and
a relatively large protein–protein interaction interface of over
5000 ꢀ2.[3] The C-terminal aminoimidazole carboxamide ribo-
nucleotide transformylase (AICAR Tfase) domain catalyzes the
transfer of a formyl group from N10-formyl-tetrahydrofolate
(10-f-THF) to aminoimidazole carboxamide ribonucleotide
(AICAR), and the N-terminal inosine monophosphate cyclohy-
drolase (IMPCH) domain catalyzes the subsequent dehydrative
ring closure to give inosine monophosphate (IMP). The AICAR
Tfase active sites are formed at the interface of two interacting
ATIC molecules, with each monomer contributing residues to
the folate and AICAR binding pockets.[3] Homodimerization is
therefore a requisite for the AICAR Tfase activity of ATIC, but
not for its IMPCH activity. The significant reliance of rapidly
dividing cells on de novo purine biosynthesis for nucleotide
production[4] has resulted in much interest and effort towards
the development of inhibitors of this enzyme and pathway for
use as potential antineoplastic agents.[5]
ATIC inhibition has also been recently demonstrated to in-
directly inhibit cell proliferation and cell cycle progression in
[a] Dr. I. B. Spurr,+ Dr. F. Cuda, Dr. A. Tavassoli
School of Chemistry, University of Southampton
Southampton, SO17 1BJ (UK)
E-mail: a.tavassoli@soton.ac.uk
[b] Dr. C. N. Birts,+ Dr. J. P. Blaydes, Dr. A. Tavassoli
Faculty of Medicine, University of Southampton
Southampton SO16 6YD (UK)
[c] Prof. S. J. Benkovic
Department of Chemistry, Pennsylvania State University
University Park, PA 16802 (USA)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201200279.
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AICAR Transformylase Homodimerization Inhibitor
human carcinomas by indirectly activating the AMP activated
protein kinase (AMPK), highlighting the potential significance
of ATIC inhibitors for use in cancer therapy.[6] The development
of substrate and cofactor analogues that specifically inhibit
ATIC activity has proven challenging however, with folate-
based compounds displaying nonspecific activity and undesira-
ble side effects.[7] The dimerization requisite for AICAR Tfase ac-
tivity provides a mechanism by which ATIC may be selectively
targeted in the presence of other folate-utilizing enzymes.[8] To
this end, we previously reported the screening of a library of
3.2 million cyclic hexapeptides[9] for inhibitors of ATIC homodi-
merization.[8a] The most potent inhibitor identified was the
cyclic peptide CRYFNV (1), which inhibits AICAR Tfase activity
with a Ki value of 17Æ4 mm by preventing ATIC homodimeriza-
tion.[8a] Although a relatively modest inhibitor for direct use as
a therapeutic, a six-membered cyclic peptide that blocks the
interaction of the ATIC homodimer holds much potential for
further development and evolution into a more potent small
molecule.
Table 1. AICAR Tfase inhibition by CRYFNV alanine-scanning analogues.
Compound
Sequence
Ki [mm]
1
2
3
4
5
6
7
cyclo-CRYFNV
cyclo-ARYFNV
cyclo-CAYFNV
cyclo-CRAFNV
cyclo-CRYANV
cyclo-CRYFAV
cyclo-CRYFNA
17Æ3
651Æ25
>2.5
>2.5
38Æ5
368Æ29
252Æ22
acting as a backbone to present the RY motif to ATIC. A trun-
cated analogue of cyclo-CRYFNV, in which the FNVC backbone
had been removed, was therefore designed (compound 8,
Scheme 2), synthesized (Scheme 3) and assayed (Table 2) for its
ability to inhibit AICAR Tfase.
The capped RY dimer 8 was found to inhibit the AICAR
Tfase activity of ATIC with a Ki value of 84Æ7 mm, suggesting
Results and Discussion
Identification of the active motif of cyclo-CRYFNV
The development of small molecules from peptides is consid-
ered a laborious and time-consuming process.[10] Compared to
its linear counterpart, a cyclic hexapeptide has limited confor-
mational freedom, with substantially fewer possible structures.
This conformational restriction, combined with the sequence
homology observed in the nine selected ATIC inhibitors,[8a] indi-
cated that some residues of the cyclic peptide ATIC inhibitor
are key to its potency, with others simply functioning as a back-
bone. To validate this hypothesis
Scheme 2. The capped RY dipeptide 8 was designed to mimic the active RY
motif (highlighted) of CRYFNV 1. RY analogues 9–17 containing unnatural
amino acids were synthesized in an attempt to improve inhibitor activity.
and identify the critical motif, we
synthesized six analogues of
cyclo-CRYFNV, in which each
amino acid was sequentially re-
placed with alanine.
Each alanine-scanning ana-
logue was assayed for inhibition
of the AICAR Tfase activity of
ATIC. The initially ordered bind-
ing of 10-f-THF and AICAR to
ATIC[11] result in a dimerization
inhibitor effectively acting as
a competitive inhibitor with re-
Scheme 3. Synthesis of RY dipeptides. Reagents and conditions (R=H, Me). a) EDC, HOBt, Et2NH, CH2Cl2; b) TFA/
CH2Cl2; c) Fmoc-Arg(Pbf)-OH, EDC, HOBt, CH2Cl2; d) DBU, EtOAc; e) Ac2O, Et3N, CH2Cl2; f) 10% Pd/C, H2, MeOH;
g) TFA/TIS/H2O.
spect to 10-f-THF, and a noncom-
petitive inhibitor with respect to
AICAR;[8a] we therefore used Mi-
chaelis–Menten
kinetics
to
assess the potency of each analogue.
The data (Table 1) indicated that the neighboring arginine
and tyrosine residues are critical for the activity of cyclo-
CRYFNV, as replacing either residue resulted in an inactive ATIC
inhibitor. In contrast, replacing the other residues (FNVC) with
alanine resulted in 2- to 40-fold loss of activity, suggesting that
these amino acids are less critical for inhibitor activity, possibly
that the RY motif of cyclo-CRYFNV is responsible for its AICAR
Tfase activity. The fivefold loss in activity may be attributed to
the increase in conformational freedom of the active motif
upon removal of the cyclic backbone; linear-CRYFNV inhibits
AICAR Tfase with a Ki value of 142Æ22 mm,[8a] an eightfold loss
compared to the cyclic counterpart, due to the same reason.
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A. Tavassoli et al.
upon reintroduction of the cyclic peptide backbone remain un-
clear.
Table 2. AICAR Tfase inhibition by RY analogues.
Compound
R1
R2
Ki [mm]
8
9
H
H
H
H
H
H
H
H
OH
H
F
OMe
CN
B(OH)2
NO2
NO2
OH
84Æ7
Deciphering the mode of action of compound 14
339Æ37
135Æ12
87Æ9
10
11
12
13
14
15[a]
16
17
We previously demonstrated by progress rate analysis that
cyclo-CRYFNV is a competitive inhibitor of ATIC with respect to
10-f-THF, and that its inhibition of AICAR Tfase is due to dis-
ruption of the ATIC dimer.[8a] To assess if the most potent di-
peptide uncovered in this study maintains this mechanism of
action, or inhibits AICAR Tfase through a nonspecific aggregate
mechanism,[14] 14 was assayed in the presence of 1 and
10 mgmLÀ1 of bovine serum albumin (BSA). Inhibition by non-
specific aggregators is prevented by the addition of milligram
per milliliter concentrations of BSA (prior to the addition of the
enzyme and inhibitor);[14] a nonspecific inhibitor would there-
fore be expected to lose activity under these conditions. The
56Æ5
59Æ5
0.685Æ0.035
52Æ4
Me
Me
145Æ14
2.5Æ0.2
NO2
[a] Lysine in place of arginine.
Improving the activity of the RY dipeptide with unnatural
amino acids
To improve the activity of the capped RY dimer 8, chemical
spaces not accessible to our original cyclic peptide library
(which is synthesized in vivo using split inteins[9,12] and there-
fore limited to the 20 canonical amino acids) were explored
using unnatural amino acids. A series of related compounds
containing electron-donating or electron-withdrawing para-
substituted tyrosine analogues were synthesized (9–14) to
probe the electronic and steric requirements of the tyrosine-
binding pocket. These compounds were assayed for AICAR
Tfase inhibition (Table 2). Removing the hydroxyl group of tyro-
sine (compound 9) resulted in a fourfold loss of activity. Re-
placing the hydroxyl group with fluorine (compound 10) also
resulted in activity loss (1.6-fold), while the p-methoxy deriva-
tive (compound 11) had similar levels of activity as the RY
dimer. Introducing an electron-withdrawing p-nitrile (com-
pound 12), or a p-borono substitution (compound 13) caused
a 1.5-fold improvement in activity, while a p-nitro group (com-
pound 14) caused a 120-fold improvement in activity, yielding
an AICAR Tfase inhibitor with a Ki value of 685Æ35 nm. Re-
placing the arginine residue of 14 with lysine (compound 15)
resulted in a 75-fold loss of activity.
The effect of N-methylation of the peptide backbone was
also explored, as this would be expected to restrict the confor-
mational freedom of the compound, and had been shown to
improve the activity of other cyclic peptides.[13] N-methylated
analogues of the capped RY dimer 8 and its p-nitro analogue
14 were therefore synthesized and assayed. N-methylation of 8
diminished its activity by 1.7-fold, while N-methylation of 14
resulted in a 3.6-fold loss of activity. The observed loss of activ-
ity on methylation may be a result of the steric hindrance asso-
ciated with the methyl group affecting binding to the active
site. The p-nitro-tyrosine analogue of cyclo-CRYFNV was also
synthesized and assayed to determine if the increase in poten-
cy observed for the RY dipeptide upon introduction of p-nitro
group, would also be observed in the parent cyclic peptide.
The p-nitro analogue of cyclo-CRYFNV was found to inhibit
AICAR Tfase with a Ki value of 36Æ4 mm, a surprising twofold
reduction in activity compared to the parent compound. With-
out structural data to identify the binding site of these com-
pounds, the reasons for the observed reduction in activity
Figure 1. A) The effect of mgmLÀ1 quantities of BSA on AICAR Tfase inhibi-
tion by 14. The compound continues to inhibit the enzyme regardless of
BSA presence, indicating it does not function through nonspecific aggrega-
tion. B) and C) Size-exclusion chromatography. B) The ratio of monomer to
dimer is shown in a 50 mm solution of ATIC C) The same mixture with 10 mm
of compound 14 shows the presence of monomeric ATIC at 85 mL.
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AICAR Transformylase Homodimerization Inhibitor
rate of the AICAR Tfase reaction was not affected by the pres-
ence of 1 mgmLÀ1 of BSA in the assay buffer, but was slowed
by around 40% in the presence of 10 mgmLÀ1 BSA (Figure 1A).
This may be due to the high BSA concentration affecting the
ATIC monomer–dimer equilibrium by stabilizing the monomer-
ic form of ATIC, and therefore reducing AICAR Tfase activity. In
both cases however, compound 14 at 10 and 50 mm continued
to inhibit the reaction to a similar extent as observed in the
absence of BSA (Figure 1A). This indicated that 14 does not
function using a nonspecific aggregate mechanism of inhibi-
tion.
radioactive proliferation assay. A dose-dependent reduction in
viability was observed in cells treated with compound 14 (Fig-
ure 2A), reaching 40% inhibition at the maximum dose of
500 mm. The effect of ATIC inhibition on cell proliferation was
further analyzed in cells treated with 250 mm of 14 using time-
lapse microscopy. Images were captured every 45 min over
72 h and analyzed. These images showed that cells treated
with 250 mm 14 exhibited a 35% reduction in proliferation
compared to control-treated cells (Figure 2B and C). These
data were in agreement with that from the cell viability assays
(Figure 2A). Further analysis of the videos indicated that the
effect of 14 on cell numbers was through a reduction in cell
division rates, rather than an increase in cell death. The MCF-7
breast cancer cell line used retains active cell cycle checkpoints
that protect from stress-induced cell death,[17] which may pro-
vide a possible explanation for the above observations. Inter-
estingly, indirect inhibition of AICAR Tfase by the anti-folate
methotrexate has been proposed as the principal mode of
action for its cytotoxicity in MCF-7 cells.[4a] Further cell-based
studies are necessary to clarify the role of de novo purine bio-
synthesis in the survival and growth of various cancer cells.
Non-folate inhibitors of various enzymes in this pathway, such
as compound 14, will be valuable chemical tools for these
studies.
To further assess if the inhibition of AICAR Tfase by com-
pound 14 is due to its disruption of ATIC homodimerization,
size-exclusion chromatography was utilized. The large change
in molecular mass of ATIC upon homodimerization (from
64 kDa to 129 kDa) enables the ratio of monomer to dimer to
be monitored by this technique. ATIC has a Kd value of 240Æ
50 nm in the absence of its substrates, with a 50 mm solution
containing mainly dimeric species.[15] Size-exclusion chroma-
tography of a 50 mm solution of ATIC showed a single peak at
72 mL, corresponding to a mass of ~130 kDa (see Figure S17 in
the Supporting Information for column calibration), indicating
the presence of only dimeric species (Figure 1B).
The experiment was repeated with a 50 mm solution of ATIC
treated with 10 mm of compound 14. An additional peak at
85 mL was observed, corresponding to a mass of ~65 kDa, in-
dicating the presence of monomeric ATIC (Figure 1C). As a con-
trol, a 50 mm solution of ATIC was treated with 10 mm of the
least potent dipeptide inhibitor identified in this study (com-
pound 9, Table 2). As 9 is a significantly weaker ATIC inhibitor
than 14, it should not disrupt ATIC dimerization at the concen-
tration used in this experiment. As expected, only a single
peak corresponding to dimeric ATIC was observed (Figure S18).
The presence of ATIC monomer in the solution treated with
14, but not in the untreated ATIC solution or that treated with
a weak inhibitor, suggests that 14 inhibits AICAR Tfase by per-
turbing ATIC homodimerization.
In addition to the above, ATIC inhibition has also recently
been proposed to be the primary mode of action for the anti-
cancer drug pemetrexed;[6a] the rise in unmetabolized AICAR
proposed to allosterically activate AMPK by mimicking AMP,
causing inhibition of cell proliferation. It is therefore possible
that the effect of 14 on cell proliferation is due to AMPK acti-
vation, as well as inhibition of de novo purine biosynthesis.
This possibility is currently being investigated in our laborato-
ry.
Conclusions
The development of a small-molecule inhibitor of ATIC homo-
dimerization with a Ki value of 685 nm from a cyclic hexapep-
tide is reported. The finding that four of the six amino acids of
cyclo-CRYFNV can be eliminated, and that the RY dipeptide is
sufficient to disrupt the interaction of a 5000 ꢀ2 interface raises
several interesting questions about the possible binding site of
the compound, and may be significant for the peptidomimetic
evolution of other cyclic peptide inhibitors of protein–protein
interactions.[18]
Inhibition of ATIC homodimerization in breast cancer cells
Inhibition of purine metabolism has been long proposed as
a possible approach to targeting tumor growth.[16] Two path-
ways operate in cells for purine production: the purine salvage
pathway operates in normal cells, whilst de novo purine pro-
duction is thought to predominate in rapidly dividing cancer
cells. Inhibition of de novo purine biosynthesis has therefore
been proposed as a possible strategy for the selective inhibi-
tion of growth in rapidly dividing cancer cells. The majority of
current inhibitors of enzymes in the de novo purine biosyn-
thetic pathway are folate analogues that inhibit multiple en-
zymes in the cell; the significance of de novo purine produc-
tion in cancer therapy is therefore difficult to decipher. We
therefore used 14 to assess the effect of inhibiting ATIC dimeri-
zation on the viability of a model breast cancer cell line; MCF-7
cells were dosed by addition of 100, 250 or 500 mm of com-
pound 14 to their growth media. The viability of treated versus
untreated cells was determined 48 h after dosing, using a non-
We are currently developing nonpeptidic analogues of 14
with the aim of further improving the potency of this com-
pound. Structural studies are also underway to determine the
binding pocket targeted on ATIC, and to enable further optimi-
zation of the inhibitor using traditional medicinal chemistry
approaches. Experiments are also underway to decipher the
mechanism by which 14 inhibits cell proliferation and to deter-
mine the effect of 14 on the proliferation of other cancer cell
lines and in vivo tumor models. Whilst the potency of 14 may
be further improved as a result of these efforts, the compound
is sufficiently active in its current form to serve as a tool for
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A. Tavassoli et al.
investigations into the role of de novo purine biosynthesis in
cells.
Experimental Section
General: Amino acids and peptide coupling reagents were ob-
tained from Merck (Novabiochem). All other chemical reagents
were obtained from Sigma–Aldrich or Fisher Scientific and used
without further purification. Thin-layer chromatography (TLC) was
carried out on Merck silica gel plates with a fluorescence indicator
(254 nm). Visualization of TLC plates was carried out by UV light,
then by using either a cerium sulfate/ammonium molybdate or po-
tassium permanganate stain. Flash chromatography was performed
with 35–70 mm silica gel (Fisher Davisil). Infrared spectra were re-
corded using a FTIR spectrometer fitted with an attenuated total
reflectance (ATR) accessory. 1H NMR and 13C NMR were recorded
using a Bruker DPX400 (400 and 100 MHz respectively). Chemical
shifts (d) are given in ppm. All enzymatic assays were performed
using a Varian Cary 100 Spectrometer. All peptides were purified
on a Waters HPLC instrument, using a Waters Atlantis Prep T3 OBD
5 mm 19ꢂ100 mm column using a water/acetonitrile mix at a flow
rate of 17 mLminÀ1. For cell culture experiments, all reagents were
obtained from Invitrogen. MCF-7 cells were maintained in Dulbec-
co’s modified Eagle’s medium supplemented with 10% fetal
bovine serum (Autogen Bioclear), penicillin (100 UmLÀ1), strepto-
mycin (100 mgmLÀ1), and l-glutamine (292 mgmLÀ1).
Cyclic peptide synthesis: The alanine-scanning analogues of
CRYFNV were synthesized and purified as previously described for
CRYFNV.[8a]
Synthesis of capped dipeptides: The dipeptides in Table 2 were
synthesized as outlined for compound 8 in Scheme 3 on a 500 mg
scale. Further experimental details and analytical information for
compounds 8–13 and 15–17 are provided as Supporting Informa-
tion. The synthesis of compound 14 is detailed below:
Synthesis of Boc-Phe(4-NO2)-diethylamide: Dry CH2Cl2 (8 mL) and
Boc-4-nitro-l-phenylalanine (500 mg, 1.61 mmol) were added to
a three-neck flask under an inert atmosphere, with vigorous stir-
ring. To this mixture, EDC hydrochloride (309 mg, 1.61 mmol) and
HOBt (218 mg, 1.61 mmol) were added, and the solution was
stirred for 30 min at room temperature. Diethylamine (0.34 mL,
3.38 mmol) was added and the resulting yellow solution was left
to stir for 4 h. The progress of this reaction was checked by TLC,
and was shown to be complete in 4 h. The reaction was stopped,
diluted with CH2Cl2 (30 mL) and washed with citric acid (10 mL),
NaHCO3 saturated solution (10 mL) and brine (10 mL). The organic
phase was dried with anhydrous Na2SO4, filtered and evaporated in
vacuo to give a pale yellow oil that solidified upon standing. The
crude material was dried under an argon stream and reprecipitated
using cold hexane to give a white solid that was collected in
a Buchner funnel (483 mg, 82% yield). Rf =0.82 (EtOAc/Hex 2:1).
m.p. 90–90.58C. 1H NMR (300 MHz, [D6]DMSO): d=8.17 (d, J=
8.8 Hz, 2H, Ar), 7.57 (d, J=8.8 Hz, 2H, Ar), 7.31 (d, J=8.4 Hz, 1H,
NH), 4.57 (q, J=7.7 Hz, 1H, CH2); 2.97–3.41 (m, 6H, CH), 1.30 (s,
9H, tBu), 1.08 (t, J=6.9 Hz, 3H, CH3), 1.01 ppm (t, J=6.9 Hz, 3H,
CH3); 13C NMR (75 MHz, [D6]DMSO): d=169.7, 154.9, 146.3, 130.8,
123.7, 78.1, 51.1, 41.1, 37.5, 27.9, 14.2, 12.7 ppm; HRMS/ES+ (m/z):
calcd for C18H27N3O5Na: 388.1843 [M+Na]+; found: 388.1854
[M+Na]+.
Figure 2. Assessing the proliferation of MCF-7 cells treated with 14. A) MTS
assays show a dose-dependent reduction in viable cells 48 h after treatment.
B) Analysis of cell growth by live-cell imaging. C) Representative images
from live-cell imaging showing a population of untreated cells over 72 h,
compared to a population of cells treated with 14. The reduction in prolifer-
ation in cells treated with 14 is due to reduced rate of division rather than
increased cell death.
Synthesis of Fmoc-Arg(Pbf)-CONH-Phe(4-NO2)-l-diethylamide:
Dry CH2Cl2 (6 mL) and Boc-4-nitro-l-phenyldiethylamide (350 mg,
1.32 mmol) were added to a flask under an inert atmosphere with
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AICAR Transformylase Homodimerization Inhibitor
vigorous stirring. Trifluoracetic acid (6 mL) was added to this mix-
ture and stirred for 1 h. The reaction was monitored by TLC. After
this time, the solvent was removed in vacuo and the orange oil co-
evaporated with EtOAc (3ꢂ10 mL) to give a yellow oil (350 mg,
100% yield) whose identity was confirmed by mass spectrometry.
The deprotected amine was added to stirred dry CH2Cl2 (15 mL) in
a flask under an inert atmosphere. Fmoc-Arg(Pbf)-OH (1.88 mg,
3.0 mmol) was added to this solution and the reaction was cooled
to À208C. HOBt (535 mg, 3.96 mmol) and EDC (380 mg,
1.98 mmol) were added, and the reaction was left to stir overnight
and allowed equilibrate to room temperature. The reaction was di-
luted with CH2Cl2 (100 mL), washed with citric acid (20 mL), saturat-
ed NaHCO3 solution (20 mL) and brine (20 mL). The yellow solution
was evaporated in vacuo to leave a yellow solid, which was puri-
fied using column chromatography with CH2Cl2/MeOH (95:5) to
give the product (Rf =0.45) as an off-white solid (520 mg, 44%
168.9, 157.3, 155.9, 147.1, 146.7, 138.1, 135.1, 135.1, 133.3, 131.7,
125.2, 124.0, 117.1, 87.2, 50.3, 43.4, 42.0, 38.5, 30.4, 29.1, 26.3, 23.3,
22.0, 18.5, 15.2, 13.6, 13.2 ppm; LRMS/ES+ (m/z): 716 [M+H]+
100%, 1431 [2M+Na]+ 26%; HRMS/ES+ (m/z): calcd for
C34H50N7O8S: 716.3436 [M+H]+; found: 716.3434 [M+H]+.
Synthesis of acetyl-Arg-CONH-Phe(4-NO2)-l-diethylamide: To
a flask, a cocktail of TFA/TIS/H2O (9.5:0.25:0.25, 22 mL) and acetyl-
Arg(Pbf)-CONH-Phe(4-NO2)-diethylamide (386 mg, 0.54 mmol) were
added, and stirred at room temperature for 2 h. The reaction was
monitored by TLC, and the solvent evaporated in vacuo to leave
a viscous yellow oil. This was co-evaporated with CH2Cl2 (3ꢂ5 mL)
and MeOH (3ꢂ5 mL) and the residue dried with an argon stream
for ~20 min. Diethyl ether (20 mL) was then added and the white
precipitate collected by filtration (263 mg, 100% crude yield). The
crude product was purified by reversed-phase HPLC (tR =7.63 min)
and lyophilized to obtain the product as a white solid (125 mg,
1
yield). m.p. 1108C decomp. H NMR (300 MHz, [D6]DMSO): d=8.25
1
50% yield). m.p. 1138C decomp. H NMR (300 MHz, [D6]DMSO): d=
(d, J=8.4 Hz, 1H), 8.03 (d, J=8.4 Hz, 2H), 7.84 (d, J=7.3 Hz, 2H),
7.65 (d, J=8.8 Hz, 2H), 7.46–7.34 (m, 7H), 4.96–4.89 (m, 1H), 4.34–
4.22 (m, 3H), 4.04–4.00 (m, 1H), 3.35–2.98 (m, 12H), 2.47 (s, 3H),
2.04 (s, 3H), 1.43 (brs, 10H), 1.07–0.97 ppm (m, 6H); 13C NMR
(75 MHz, [D6]DMSO): d=172.3, 170.1, 158.4, 157.0, 156.7, 147.2,
146.6, 144.8, 144.6, 141.7, 138.2, 132.4, 131.7, 131.6, 128.6, 128.0,
126.1, 125.2, 124.0, 123.9, 121.0, 117.2, 87.2, 66.5, 60.7, 55.1, 50.3,
47.6, 43.4, 42.1, 38.6, 29.2, 19.9, 18.5, 15.2, 13.7, 13.2 ppm; LRMS/
ES+ (m/z): 896 [M+H]+ 5%, 918 [M+Na]+ 100%, 1813 [2M<M+
>Na]+ 9.6%. HRMS/ES+ (m/z): calcd for C47H58N7O9S: 896.4011
[M+H]+; found: 896.4010 [M+H]+.
8.31 (d, J=8.7 Hz, 1H), 8.10 (d, J=8.6 Hz, 2H), 7.91 (d, J=8.1 Hz,
2H), 7.65 (t, J=5.6 Hz, 1H), 7.50 (d, 8.6 Hz, 2H), 7.17 (brs, 3H),
4.90–4.82 (m, 1H), 4.32–4.23 (m, 1H), 3.37–3.00 (m, 7H), 2.95 (dd,
1H), 1.82 (s, 3H), 1.61–1.50 (m, 3H), 1.00 (t, 7.12 Hz, 3H), 0.95 ppm
(t, 7.13 Hz, 6H); 13C NMR (75 MHz, [D6]DMSO): d=171.1,169.1,
169.1, 156.8, 146.3, 145.8, 130.8, 123.1, 51.8, 49.4, 41.4, 40.4, 39.8,
37.6, 29.2, 25.0, 22.4, 14.2, 12.7 ppm; LRMS/ES+ (m/z): 464 [M+H]+
100%; HRMS/ES+ (m/z): calcd for C21H33N7O5: 464.2616 [M+H]+;
found: 464.2610 [M+H]+.
Protein expression and enzyme kinetics: Avian and human ATIC
fused to an N-terminal His6 tag were expressed from pET28 vectors
in BL21(DE3) strains of Escherichia coli, and purified as previously
described.[8a,19] AICAR Tfase assays were conducted with avian ATIC
and carried out in 1 cm path length quartz cuvettes at 258C as pre-
viously described.[8a,19] Each inhibitor was dissolved in DMSO to
give a 100 mm stock solution that was further diluted for use in
each assay. The data was analyzed using KaleidaGraph (Synergy
Software) and Microsoft Excel, by assuming competitive inhibition
with 10-f-THF as previously described.[8a] Errors in the Ki values
were calculated by linear regression. Compounds 1, 8, and 14 were
also assayed with human ATIC, with the same Ki value (within
error) being observed for each compound as for the avian enzyme.
Synthesis of amino-Arg(Pbf)-CONH-Phe(4-NO2)-l-diethylamide:
Dry EtOAc (14 mL) was added to Fmoc-Arg(Pbf)-CONH-Phe(4-NO2)-
diethylamide (520 mg, 0.58 mmol) in a flask under an inert atmos-
phere and stirred vigorously. To this mixture, DBU (93 mg, 0.1 mL,
0.61 mmol) was added; the solution was stirred for 1 h, and the
reaction was monitored using TLC. The solvent was removed in
vacuo to give a brown solid, which was purified using column
chromatography with CH2Cl2/MeOH (9:1) to give the product (Rf =
0.35) as a white solid (363 mg, 93% yield). m.p. 1218C decomp.
1H NMR (300 MHz, [D6]DMSO): d=8.31 (d, J=8.4 Hz, 1H), 8.15 (d,
J=8.4 Hz, 2H), 7.51 (d, J=7.3 Hz, 2H), 4.96–4.91 (m, 1H), 3.43–2.94
(m, 13H), 2.54–2.46 (m, 8H overlap [D6]DMSO), 2.03 (s br, 5H), 1.44
(s br, 10H), 1.07–0.97 ppm (m, 6H); 13C NMR (75 MHz, [D6]DMSO):
d=174.3, 169.3, 157.3, 156.0, 146.2, 145.7, 137.2, 134.2, 131.3,
130.7, 124.2, 123.0, 116.2, 86.2, 54.1, 48.7, 42.4, 41.1, 37.9, 32.1,
28.2, 25.3, 18.9, 17.5, 14.3, 12.7, 12.2 ppm; LRMS/ES+ (m/z): 674
[M+H]+ 39%, 696 [M+Na]+ 100%, 1869 [2M<M+ >Na]+ 11 %.
HRMS/ES+ (m/z): calcd for C32H47N7O7S: 674.3330 [M+H]+; found:
674.3319 [M+H]+.
Size-exclusion chromatography: Three 50 mm solutions of human
ATIC were prepared as above, in buffer [20 mm Tris·HCl, pH 7.5,
NaCl (150 mm), KCl (50 mm), EDTA (5 mm), dithiothreitol (5 mm),
1 mL]. Compound 14 or 9 (negative control; 10 mm) was added to
two of these solutions. Each solution was filtered through a Millex
GP 0.22 mm filter (Millipore), and loaded onto a HiLoad 16/60 Su-
perdex 200 gel filtration column (GE Healthcare) pre-equilibrated
with the above buffer. The flow rate was 1 mLminÀ1 and the flow
monitored by UV absorbance on an AKTAprime FPLC instrument
(GE Healthcare). The column was calibrated using a HMW gel filtra-
tion calibration kit (GE Healthcare).
Synthesis of acetyl-Arg(Pbf)-CONH-Phe(4-NO2)-l-diethylamide:
Dry ethyl acetate (7 mL) and Fmoc-Arg(Pbf)-CONH-Phe(4-NO2)-di-
ethylamide (363 mg, 0.54 mmol) were added to flask under an
inert atmosphere and stirred vigorously. Triethylamine (58 mg,
0.08 mL, 0.57 mmol), and acetic anhydride (58 mg, 0.05 mL,
0.57 mmol) were added to this mixture and stirred overnight. The
solvent was removed in vacuo giving a sticky yellow oil that was
purified by column chromatography (short path) using CH2Cl2/
MeOH 9:1 to give a white solid (Rf =0.52) in quantitative yield
(386 mg, 100% yield). m.p. 1138C decomp. 1H NMR (300 MHz,
[D6]DMSO): d=8.36 (d, J=8.4 Hz, 1H), 8.14 (d, J=8.8 Hz, 2H), 7.89
(d, J=8.2 Hz, 2H), 7.56 (d, 8.8 Hz, 2H), 4.92–4.84 (m, 1H), 4.28–4.24
(m, 1H), 3.36–2.99 (m, 16H), 2.54–2.51 (s br, 8H overlap [D6]DMSO),
2.45 (s, 3H), 1.94 (s, 3H), 1.84 (s, 3H), 1.55–1.28 (brs, 11H), 1.05–
0.96 ppm (m, 6H); 13C NMR (75 MHz, [D6]DMSO): d=172.1, 170.1,
Cell proliferation assays: Cells were plated at 2000 cells per well
in 96-well plates 24 h prior to dosing with appropriate drug in
fresh medium (100 mL); the final concentration of solvent DMSO in
each well was 0.5%. MTS-based cell proliferation assays were per-
formed 48 h post-dosing according to the manufacturer’s instruc-
tions (CellTitre Aqueous One Cell Proliferation assay, Promega).
Live-cell imaging: Cells were plated at 10000 cells per well in 24-
well plates. Time-lapse microscopy[17] was initiated on dosing, with
images captured every 45 min for 72 h. Analysis was carried out
using Image J software. All conditions were assayed in triplicate,
and expressed as means ÆS.E.M. Statistical significance was calcu-
ChemBioChem 2012, 13, 1628 – 1634
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www.chembiochem.org
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bration data. This work was funded by National Institutes of
Health grant GM24129-28, and Cancer Research UK grant 10263.
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Keywords: AICAR transformylase · ATIC · cancer · peptides ·
protein–protein interaction inhibitors
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Received: April 25, 2012
Published online on July 4, 2012
1634
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