Small molecule inhibitors of Myc/Max dimerization and Myc-induced cell transformation

Article abstract of DOI:10.1016/j.bmcl.2009.09.044

The preparation and evaluation of a series of inhibitors of Myc/Max dimerization and Myc-induced cell transformation are described providing mycmycin-1 (3) and mycmycin-2 (4).

Full text of DOI:10.1016/j.bmcl.2009.09.044

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6038–6041
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Small molecule inhibitors of Myc/Max dimerization and Myc-induced
cell transformation
Jin Shi a, James S. Stover b, Landon R. Whitby b, Peter K. Vogt a, Dale L. Boger b,
*
a Departments of Molecular and Experimental Medicine, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
b Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The preparation and evaluation of a series of inhibitors of Myc/Max dimerization and Myc-induced cell
transformation are described providing mycmycin-1 (3) and mycmycin-2 (4).
Ó 2009 Elsevier Ltd. All rights reserved.
Received 3 August 2009
Revised 12 September 2009
Accepted 14 September 2009
Available online 17 September 2009
Keywords:
Myc
Max
Cell transformation
The c-myc proto-oncogene is involved in the progression of a
wide range of human tumors.1–5 Proteins in the immediate Myc
network are essential regulators of differentiation and prolifera-
tion. Oncogenic transformation occurs mainly through elevated
expression of its gene product Myc, a short-lived nuclear protein
and an important member of the basic helix–loop–helix leucine
zipper (bHLHLZ) transcription factors (Fig. 1).6 The oncogenic po-
tential7,8 of c-myc has been demonstrated in transgenic animals
and cell culture, and typically requires the action of at least one
additional oncogene such as ras or bcl-2 (inhibits apoptosis). In
normal cells, Myc is required for cell proliferation and prevents dif-
ferentiation. It is transiently expressed in response to mitogenic
stimuli and has a short half life (t1/2 = 20–30 min). Its aberrant
expression in the absence of such growth factors drives cells into
cell cycle but also induces apoptosis unless co-oncogenic mecha-
nisms are present (i.e., inhibition by constitutive bcl-2 overexpres-
sion). All known activities of Myc require heterodimerization with
Max,9–12 a constitutively expressed bHLHLZ protein that is stable
(t1/2 > 14 h). The Myc/Max heterodimer binds the DNA sequence
CACGTG (E box element) and activates transcription through the
transactivation domain of Myc. DNA binding occurs through the
basic region while both the HLH and the leucine zipper form the
dimerization interface.6 Max, but not Myc, forms homodimers that
bind the same DNA site and inhibit transcription. In part, this re-
sults from the competition for Max and for the common DNA site.
In addition, Max heterodimerizes with another family of bHLHLZ
proteins that include Mad (Mad-1), Mxi-1, Mad-3, and Mad-4 that
are also transiently expressed (t1/2 = 15–30 min) and signal growth
arrest and differentiation.13 Their association behavior is similar to
that of Myc in that they do not homodimerize or interact with Myc
family members, but readily form heterodimers with Max that
bind the CACGTG core consensus sequence. Mad and Myc compete
for Max with approximately equal affinities and the heterodimers
are formed in preference to Max homodimerization. Mad/Max het-
erodimers repress Myc/Max transcriptional activation. Thus, Myc/
Max induces proliferation and apoptosis in nontransformed cells
while Max/Max and Mad/Max are involved in growth arrest, differ-
entiation, and cell survival. In transformed cells, when accompa-
nied by oncogenic events inhibiting apoptosis, constitutive Myc
expression maintains the cell proliferation state and prevents
growth arrest and differentiation.
With the recognition that the aberrant expression of Myc lead-
ing to transformation may be addressed through inhibition of Myc/
Max heterodimerization and its subsequent transcriptional activa-
tion, we screened our library of candidate modulators of protein–
protein interactions for inhibitors of Myc/Max dimerization using
a novel FRET assay.14 This led to the identification of the first pro-
totypical inhibitors of Myc/Max dimerization whose behavior was
not only confirmed in more classical ELISA and EMSA assays, but
two (1 and 2) of which were also shown to inhibit Myc-induced
oncogenic transformation in chicken embryo fibroblast cultures
(CEF), providing the first validation of the approach for small mol-
ecule therapeutic intervention (Fig. 2).14 Not only did this serve as
the first example of inhibiting the (aberrant) function of a tran-
scription factor by small molecule inhibition of an integral pro-
tein–protein interaction, but it inspired our examination of a
number of additional protein–protein interaction targets.15–24
* Corresponding author. Tel.: +1 858 784 7522; fax: +1 858 784 7550.
E-mail address: boger@scripps.edu (D.L. Boger).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.044

J. Shi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6038–6041
6039
which is disordered and undergoes coupled folding and binding
upon heterodimerization, assumes a more rigid and defined con-
formation with the small molecule binding and limiting its ability
to interact with Max.28
In a continuation of our studies and in efforts to improve on the
modest potency of our original lead compounds, we have prepared
two complementary 120-membered libraries to those that led to
the discovery of 1 and 2.29 These were assembled using a symmetri-
cal and racemic trans-3,4-dicarboxylic acid template enlisting acid–
base liquid–liquid extractions30 for isolation of the pure intermedi-
ates or final products and employed the identical appended substit-
uents (A1ÀA6, B1ÀB20, where B3ÀB20 are the single (S)-
enantiomers)29 used in the libraries that provided 1 and 2, Scheme 1.
Since Myc induces the formation of oncogenically transformed
cell foci in cultures of chicken embryo fibroblasts (CEF) and be-
cause this transforming activity of Myc depends on dimerization
with Max, we examined all members of the candidate libraries di-
rectly for inhibition of this oncogenic transformation in this cell-
based focus assay. Although laborious in requiring the mainte-
nance of the cell culture over the three weeks of its conduct and
a manual visual counting of the individual foci in each well, the
use of the focus assay as the primary screen for activity ensured
the identification of all members that display the desired func-
tional activity in a cell-based assay.14 Thus, all 240 compounds pro-
duced were examined for inhibition of Myc-induced cell
transformation in the focus assay at 20 lM enlisting CEF infected
with the RCAS viral vector expressing the transforming chicken
cellular Myc.14 In these efforts, 40, 16, and finally 9 of the candidate
compounds were examined more carefully in iterative rounds of
MeO2C
NH2
Me3SiCCH2Cl, 89%
HCHO, MeOH, 94%
N
SiMe3
OMe
CO2Me
LiF, MeCN, 96%
R1CO2H, EDCI
R2NH2, EDCI
CO2Me
CO2R
CO2R
O
R
N
N
CO2Me
R1
H2, Pd(OH)2
R = Bn
LiOH
R = Me
R = H
R = H
O
O
O
O
MeNH2, EDCI
NHR2
NHR2
N
N
R1
CO2H
R1
CONHMe
Library I, 120 compounds
Library II, 120 compounds
R1CO2H
CO2H
BocHN
CO2H
CO2H
BocHN
BocHN
CO2H
CO2H
A1
A4
A2
A3
A6
CO2H
Boc
N
Boc
N
Boc N
A5
Figure 1. Myc–Max–Mad network and structure of the Max dimer bound to DNA.
R2NH2
CO2tBu
CO2tBu
B3−B20
H2N
CO2tBu
H2N
H2N
B1
B2
HN
Since this disclosure, additional inhibitors of Myc/Max dimeriza-
tion have been reported and their therapeutic potential further val-
idated.25–27 In addition, the examination of their interaction with
Myc has revealed that the bHLHLZ interacting domain of Myc,
X
X =
MeSO2
B3
CF3SO2
B4
EtSO2
B5
CF3CH2SO2
B6
PrSO2
B7
BuSO2
B8
SO2
SO2
SO2
SO2
SO2
B9
SO2
O
N
O
O
B10
B11
SO2
B12
B14
B13
SO2
CO2tBu
SO2
CO2tBu
O
O
N
H
N
H
N
N
SO2
SO2
SO2
N
SO2
HN
HN
SO2
Br
CONHMe
CONHMe
N
N
Cl
Cl
Cl
Br
Cl
O
S
2
1
N
Boc
N
N
S
S
N
N
Boc
S
Cl
Cl
Cl
B15
B16
B17
B18
B19
B20
NMe2
NMe2
Figure 2. Lead compounds.
Scheme 1.

6040
J. Shi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6038–6041
Of the final nine compounds examined in detail, eight exhibited
significant inhibition of Myc-induced oncogenic transformation in
CEF, two were found to completely inhibit foci formation at the
tested concentration of 20 lM (EOT = 0.05), all had no effect on
Jun- or Src-induced oncogenic transformation in CEF, and each dis-
played substantial inhibition of Myc/Max dimerization. The inhib-
itors fall into two classes being derived from Library I or II. Those
derived from Library I including 3 exhibited the most effective
inhibition of Myc/Max dimerization, but bear a free carboxylic acid
that may impede cellular uptake limiting their inhibition of Myc-
induced oncogenic transformation. Even with this potential limita-
tion, compound 3 (mycmycin-1) displayed effective inhibition of
the Myc-induced oncogenic transformation (EOT = 0.05) consistent
with its inhibition of Myc/Max dimerization. The remaining five
compounds including 4 (mycmycin-2) were derived from Library
II and exhibited less effective inhibition of Myc/Max dimerization
that, in part, correlated with their effectiveness for inhibiting the
Myc-induced oncogenic transformation. The two exceptions,
which include 4 (EOT = 0.05), displayed more potent inhibition of
the oncogenic transformation than might be predicted simply from
their relative inhibition of Myc/Max dimerization established in
the FRET assay, yet bear structures that most closely resemble
the original lead 2. Significantly, both mycmycin-1 and -2 (3 and
4) provide additional effective probes with which to continue
examining this proteinÀprotein interaction target.33
Acknowledgments
We gratefully acknowledge the financial support of the National
Institutes of Health (CA78045). L.R.W. is a Skaggs Fellow.
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J. Shi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6038–6041
6041
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33. For 3 (mixture of two diastereomers): 1H NMR (600 MHz, CDCl3, 25 °C) d 9.03
(s, 1H), 7.44–7.26 (m, 1H), 6.90 (s, 2H), 6.21–6.05 (m, 1H), 4.21–3.17 (m, 12H),
2.68 (s, 1H), 2.57 (s, 6H), 2.24 (s, 3H), 2.22–2.13 (m, 2H), 1.98 (s, 1H), 1.69 (d,
J = 11.2 Hz, 2H), 1.43 (s, 9H), 1.19 (d, J = 7.8 Hz, 9H), 1.10 (d, J = 11.8 Hz, 2H);
13C NMR d 177.5, 176.05, 176.02, 175.81, 175.77, 174.9, 174.8, 173.20, 173.16,
172.3, 172.1, 171.7, 169.59, 169.57, 169.55, 169.4, 155.79, 155.78, 155.77,
143.54, 143.49, 143.48, 140.13, 140.12, 140.11, 140.08, 140.06, 140.05, 84.07,
84.05, 83.93, 83.91, 80.4, 80.3, 68.87, 68.86, 67.54, 67.49, 65.47, 65.42, 56.39,
56.36, 56.21, 56.16, 49.94, 49.90, 49.50, 49.47, 49.2, 49.1, 49.0, 47.5, 47.38,
47.35, 47.2, 46.8, 46.4, 46.2, 45.77, 45.74, 45.1, 44.3, 42.6, 42.5, 41.77, 41.75,
33.8, 33.7, 32.9, 30.6, 29.3, 28.4, 23.8, 21.7, 15.94, 15.92, 15.0; ESI-TOFMS (m/z)
calcd for [C34H52N4O10S+H]+ 709.3477; found: 709.3473. For 4 (mixture of two
diastereomers): 1H NMR (400 MHz, DMSO-d6, 25 °C) d 8.64 (s, 1H), 8.24–8.14
(m, 1H), 7.96–7.88 (m, 1H), 7.27–7.25 (m, 1H), 6.78–6.70 (m, 1H), 4.04–3.88
(m, 1H), 3.74–3.60 (m, 2H), 3.54–3.44 (m, 1H), 3.42–3.34 (m, 1H), 3.30–3.14
(m, 3H), 3.14–3.02 (m, 1H), 2.88 (dd, J = 12.9, 6.6 Hz, 2H), 2.57 (dd, J = 4.5,
2.6 Hz, 3H), 2.20–2.14 (m, 2H), 1.65–1.56 (m, 1H), 1.50–1.41 (m, 2H), 1.40–1.32
(m, 11H), 1.31–1.27 (m, 9H), 1.26–1.17 (m, 2H); 13C NMR d 171.79, 171.75,
171.71, 171.69, 171.24, 171.22, 170.7, 170.6, 168.88, 168.85, 168.84, 156.0,
154.3, 137.79, 137.75, 137.70, 137.66, 129.35, 129.33, 129.28, 129.26, 127.3,
126.5, 81.98, 81.93, 81.90, 79.08, 79.04, 77.8, 56.2, 56.0, 55.4, 49.44, 49.38,
49.35, 49.09, 49.03, 49.01, 46.93, 46.91, 46.86, 46.75, 46.72, 45.4, 45.3, 45.22,
45.17, 41.60, 41.58, 41.49, 41.47, 33.99, 33.91, 29.9, 28.8, 28.6, 28.1, 27.8,
26.57, 26.52, 26.18, 26.16, 24.48; ESI-TOFMS (m/z) calcd for
[C29H45Cl2N5O9S2+H]+ 742.2108; found: 742.2088.
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