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. 2015 Jun 25;58(12):5075-87.
doi: 10.1021/acs.jmedchem.5b00468. Epub 2015 Jun 5.

Identification of a Potent Inhibitor of CREB-Mediated Gene Transcription with Efficacious in Vivo Anticancer Activity

Fuchun Xie, Bingbing X Li, Alina Kassenbrock, Changhui Xue, Xiaoyan Wang, David Z Qian, Rosalie C Sears, Xiangshu Xiao

Identification of a Potent Inhibitor of CREB-Mediated Gene Transcription with Efficacious in Vivo Anticancer Activity

Fuchun Xie et al. J Med Chem. .

Abstract

Recent studies have shown that nuclear transcription factor cyclic adenosine monophosphate response element binding protein (CREB) is overexpressed in many different types of cancers. Therefore, CREB has been pursued as a novel cancer therapeutic target. Naphthol AS-E and its closely related derivatives have been shown to inhibit CREB-mediated gene transcription and cancer cell growth. Previously, we identified naphthamide 3a as a different chemotype to inhibit CREB's transcription activity. In a continuing effort to discover more potent CREB inhibitors, a series of structural congeners of 3a was designed and synthesized. Biological evaluations of these compounds uncovered compound 3i (666-15) as a potent and selective inhibitor of CREB-mediated gene transcription (IC50 = 0.081 ± 0.04 μM). 666-15 also potently inhibited cancer cell growth without harming normal cells. In an in vivo MDA-MB-468 xenograft model, 666-15 completely suppressed the tumor growth without overt toxicity. These results further support the potential of CREB as a valuable cancer drug target.

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Figures

Figure 1
Figure 1
Chemical structures of previously reported CREB inhibitors: naphthol AS-E (1) and compounds 2 and 3a. Compound 2 is rapidly transformed into 3a through an O,N-acyl transfer reaction at pH 7.4.
Figure 2
Figure 2
Chemical structures of newly designed structural congeners (3b–j) of 3a. The key structural difference between 3b–d and 3a is highlighted in red.
Scheme 1
Scheme 1. Synthesis of Compound 3b
Scheme 2
Scheme 2. Synthesis of Compound 3c
Scheme 3
Scheme 3. Synthesis of Compound 3d
Scheme 4
Scheme 4. Synthesis of Compounds 3e–g
Scheme 5
Scheme 5. Synthesis of Compound 3h
Scheme 6
Scheme 6. Synthesis of Compound 3i
Scheme 7
Scheme 7. Synthesis of Compound 3j
Figure 3
Figure 3
Conformation of the global energy minimum of 3a (A), 3j (B), 3h (C), and 3i (D). The distance between the amide oxygen and ammonium nitrogen was labeled in each conformation to indicate formation of an intramolecular hydrogen bond.
Figure 4
Figure 4
Compound 3i decreased endogenous CREB target gene expression. HEK 293T cells were treated with different concentrations of 3i followed by treatment with forskolin. Then the relative mRNA level of Nurr1/NR4A2 was determined by qRT-PCR analysis.
Figure 5
Figure 5
Compound 3i selectively inhibited CREB-mediated gene transcription. HEK 293T cells were transfected with indicated combinations of plasmids. Then the cells were treated with different concentrations of 3i before RLuc activity measurement. Forskolin (Fsk, 10 μM) was added to CRE-Rluc only transfected cells at 30 min after drug treatment to stimulate CREB’s activity. The RLuc activity was normalized to the protein concentration and presented as relative luciferase unit (RLU)/μg protein.
Figure 6
Figure 6
Compound 3i selectively inhibited tumor cell growth. Shown are antiproliferative dose–response curves of 3i in breast cancer MDA-MB-468 (A) and MDA-MB-231 (B) cells as well as normal HMEC (C) and HFF (D) cells. The cells were incubated with 3i for 72 h, and then the remaining live cells were quantified by the MTT assay.
Figure 7
Figure 7
Compound 3i suppressed MDA-MB-468 tumor growth in vivo. MDA-MB-468 tumor-bearing mice were treated either with vehicle or with 3i at 10 mg/kg once a day for 5 days a week. The duration of treatment was 5 weeks. The relative tumor volume (A) and body weight (B) of the treated mice are shown: (∗) P < 0.05 by Student’s t test.
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