title: Indole-3-carbinol (I3C) analogues are potent small molecule inhibitors of NEDD4-1 ubiquitin ligase activity that disrupt proliferation of human melanoma cells.
abstract: The HECT domain-containing E3 ubiquitin ligase NEDD4-1 (Neural precursor cell Expressed Developmentally Down regulated gene 4-1) is frequently overexpressed in human cancers and displays oncogenic-like properties through the ubiquitin-dependent regulation of multiple protein substrates. However, little is known about small molecule enzymatic inhibitors of HECT domain-containing ubiquitin ligases. We now demonstrate that indole-3-carbinol (I3C), a natural anti-cancer phytochemical derived from cruciferous vegetables such as cabbage and broccoli, represents a new chemical scaffold of small molecule enzymatic inhibitors of NEDD4-1. Using in vitro ubiquitination assays, I3C, its stable synthetic derivative 1-benzyl-I3C and five novel synthetic analogues were shown to directly inhibit NEDD4-1 ubiquitination activity. Compared to I3C, which has an IC50 of 284μM, 1-benzyl-I3C was a significantly more potent NEDD4-1 enzymatic inhibitor with an IC50 of 12.3μM. Compounds 2242 and 2243, the two indolecarbinol analogues with added methyl groups that results in a more nucleophilic benzene ring π system, further enhanced potency with IC50s of 2.71μM and 7.59μM, respectively. Protein thermal shift assays that assess small ligand binding, in combination with in silico binding simulations with the crystallographic structure of NEDD4-1, showed that each of the indolecarbinol compounds bind to the purified catalytic HECT domain of NEDD4-1. The indolecarbinol compounds inhibited human melanoma cell proliferation in a manner that generally correlated with their effectiveness as NEDD4-1 enzymatic inhibitors. Taken together, we propose that I3C analogues represent a novel set of anti-cancer compounds for treatment of human melanomas and other cancers that express indolecarbinol-sensitive target enzymes.
authors.affiliation: Dept. of Molecular and Cell Biology and The Cancer Research Laboratory, University of California at Berkeley, Berkeley, CA, USA. Electronic address: jgquirit@berkeley.edu.
authors.forename: Jeanne G
authors.initials: JG
authors.lastname: Quirit
authors.affiliation: Gause Institute of New Antibiotics, B. Pirogovskaya 11, Moscow 119021, Russia. Electronic address: satory@mail.ru.
authors.forename: Sergey N
authors.initials: SN
authors.lastname: Lavrenov
authors.affiliation: Dept. of Molecular and Cell Biology and The Cancer Research Laboratory, University of California at Berkeley, Berkeley, CA, USA. Electronic address: kpoindexter25@gmail.com.
authors.forename: Kevin
authors.initials: K
authors.lastname: Poindexter
authors.affiliation: Dept. of Molecular and Cell Biology and The Cancer Research Laboratory, University of California at Berkeley, Berkeley, CA, USA. Electronic address: jamicexu@scripps.edu.
authors.forename: Janice
authors.initials: J
authors.lastname: Xu
authors.affiliation: Dept. of Molecular and Cell Biology and The Cancer Research Laboratory, University of California at Berkeley, Berkeley, CA, USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Kyauk
authors.affiliation: Molecular Graphics and Computational Facility, College of Chemistry, University of California, Berkeley, CA, USA. Electronic address: durkin@berkeley.edu.
authors.forename: Kathleen A
authors.initials: KA
authors.lastname: Durkin
authors.affiliation: Dept. of Molecular and Cell Biology and The Cancer Research Laboratory, University of California at Berkeley, Berkeley, CA, USA. Electronic address: idaaronchik@gmail.com.
authors.forename: Ida
authors.initials: I
authors.lastname: Aronchik
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA. Electronic address: tomasiak@msg.ucsf.edu.
authors.forename: Thomas
authors.initials: T
authors.lastname: Tomasiak
authors.affiliation: Gause Institute of New Antibiotics, B. Pirogovskaya 11, Moscow 119021, Russia. Electronic address: hercules44@yandex.ru.
authors.forename: Yaroslav A
authors.initials: YA
authors.lastname: Solomatin
authors.affiliation: Gause Institute of New Antibiotics, B. Pirogovskaya 11, Moscow 119021, Russia. Electronic address: satory@mail.ru.
authors.forename: Maria N
authors.initials: MN
authors.lastname: Preobrazhenskaya
authors.affiliation: Dept. of Molecular and Cell Biology and The Cancer Research Laboratory, University of California at Berkeley, Berkeley, CA, USA. Electronic address: glfire@berkeley.edu.
authors.forename: Gary L
authors.initials: GL
authors.lastname: Firestone
mesh_terms: D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D001943:Breast Neoplasms; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003198:Computer Simulation; D004354:Drug Screening Assays, Antitumor; D056827:Endosomal Sorting Complexes Required for Transport / Q000037:antagonists & inhibitors*; D005260:Female; D006801:Humans; D007211:Indoles / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D008545:Melanoma; D062105:Molecular Docking Simulation; D000075702:Nedd4 Ubiquitin Protein Ligases; D013329:Structure-Activity Relationship; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors*
chemical_list: D000970:Antineoplastic Agents; D056827:Endosomal Sorting Complexes Required for Transport; D007211:Indoles; C016517:indole-3-carbinol; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; D044767:Ubiquitin-Protein Ligases
keywords: Anti-cancer compounds; In silico computational modeling; Indole-3-carbinol analogues; Ligand-target protein interactions; Melanoma cells; NEDD4-1 ubiquitin ligase