Stabilizers of the Max Homodimer Identified in Virtual Ligand Screening Inhibit Myc Function

  1. Hao Jiang,
  2. Kristen E. Bower1,
  3. Albert E. Beuscher IV2,
  4. Bin Zhou,
  5. Andrey A. Bobkov,
  6. Arthur J. Olson and
  7. Peter K. Vogt
  1. Departments of Molecular and Experimental Medicine (H.J., K.E.B., P.K.V.), Molecular Biology (A.E.B., A.J.O.), and Chemistry (B.Z.), The Scripps Research Institute, La Jolla, California; and Protein Production and Analysis Facility, Burnham Institute for Medical Research, La Jolla, California (A.A.B.)
  1. Address correspondence to:
    Dr. Hao Jiang, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail: hjiang{at}scripps.edu

Abstract

Many human cancers show constitutive or amplified expression of the transcriptional regulator and oncoprotein Myc, making Myc a potential target for therapeutic intervention. Here we report the down-regulation of Myc activity by reducing the availability of Max, the essential dimerization partner of Myc. Max is expressed constitutively and can form unstable homodimers. We have isolated stabilizers of the Max homodimer by applying virtual ligand screening (VLS) to identify specific binding pockets for small molecule interactors. Candidate compounds found by VLS were screened by fluorescence resonance energy transfer, and from these screens emerged a potent, specific stabilizer of the Max homodimer. In vitro binding assays demonstrated that the stabilizer enhances the formation of the Max-Max homodimer and interferes with the heterodimerization of Myc and Max in a dose-dependent manner. Furthermore, this compound interferes with Myc-induced oncogenic transformation, Myc-dependent cell growth, and Myc-mediated transcriptional activation. The Max-Max stabilizer can be considered a lead compound for the development of inhibitors of the Myc network.

Footnotes

  • This work was supported by the National Institutes of Health National Cancer Institute [Grant CA078045].

  • H.J., K.E.B., and A.E.B. contributed equally to this work.

  • ABBREVIATIONS: 10058-F4, (Z,E)-5-(4-ethylbenzylidine)-2-thioxothiazolidin-4-one; bHLHLZ, basic helix-loop-helix leucine zipper; CEF, chicken embryonic fibroblast; CFP, cyan fluorescence protein; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; FRET, fluorescence resonance energy transfer; GFP, green fluorescence protein; HEK, human embryonic kidney; HLH, helix-loop-helix; ICI 182,780, fulvestrant; NCI, National Cancer Institute; NSC13728, 4-methyl-6-[(4-methyl-2-piperidin-1-ylquinolin-6-yl)methyl]-2-piperidin-1-ylquinoline; NSC292213, 4-[2-(3-carboxy-4-hydroxynaphthalen-1-yl)-2-oxoacetyl]-1-hydroxynaphthalene-2-carboxylic acid; NSC2979, 7,8,8a,9-tetrachloro-1,4a-dimethyl-7-propan-2-yl-2,3,4,4b,5,6,8,9,10,10a-decahydrophenanthrene-1-carboxylic acid; NSC299137, N-(9,10-dioxoanthracen-1-yl)-7-oxobenzo[e]perimidine-4-carboxamide; NSC30188, 4-[(3-carboxy-2-hydroxynaphthalen-1-yl)-methyl]-3-hydroxynaphthalene-2-carboxylic acid; NSC38777, 2-(2-nitrophenyl)butanedioic acid; NSC39863, 1,11a,13a-trimethyl-8-phenyl-2,3,3a,3b,4,5,5a,6,11,11a,11b,12,13,13a-tetradecahydro-1H-cyclopenta[5,6]naphtho[1,2-g]quinazolin-1-ol; NSC40837, 2-phosphonoethylphosphonic acid; NSC41309, furan-2,3,4,5-tetracarboxylic acid; NSC42258, 3-[[4-(1H-indol-3-ylmethyl)piperazin-1-yl]methyl]-1H-indole; NSC601364, 5-[3-pyridin-2-yl-6-(5-sulfofuran-2-yl)-1,2,4-triazin-5-yl]furan-2-sulfonic acid; NSC610938, 4-[(3-carboxy-4-oxonaphthalen-1-ylidene)-phenylmethyl]-1-hydroxynaphthalene-2-carboxylic acid; NSC66207, naphthalene-1,4,5,8-tetracarboxylic acid; NSC683770, (4R)-4-[(3R,5R, 8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoic acid; NSC73100, 2-(9H-fluoren-2-ylcarbamoyl)terephthalic acid; NSC7616, achilleic acid; NSC93354, (8S,9S,10R,11S,13S,14S,17S)-11-hydroxy-10,13-dimethyl-17-(2-phenyl-1,3-thiazol-4-yl)-1,2,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-3-one; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; QEF, quail embryonic fibroblast; RCAS, replication-competent Avian sarcoma-leukosis virus long terminal repeat with a splice acceptor; SPR, surface plasmon resonance; Src, Rous sarcoma virus oncogene cellular homolog; VLS, virtual ligand screening; YFP, yellow fluorescent protein.

  • ↵Graphic The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • ↵1 Current affiliation: CovX Research LLC, San Diego, California.

  • ↵2 Current affiliation: Institute of Oral Health Research, School of Dentistry, University of Alabama at Birmingham, Birmingham, Alabama.

    • Accepted June 4, 2009.
    • Received January 15, 2009.
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  1. Published online before print June 4, 2009, doi: 10.1124/mol.109.054858 Molecular Pharmacology September 2009 vol. 76 no. 3 491-502
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