Indole-3-carbinol (I3C) analogues are potent small molecule inhibitors of NEDD4-1 ubiquitin ligase activity that disrupt proliferation of human melanoma cells

Developing Small‐Molecule Inhibitors of HECT‐Type Ubiquitin Ligases for Therapeutic Applications: Challenges and Opportunities

“…To overcome the disadvantages of I3C, such as its low affinity for NEDD4‐1 and chemical instability, as caused by the formation of various condensation products, especially at low pH, a small library of N ‐benzyl or N ‐phenyl I3C analogues with different substitution patterns at the phenyl ring was prepared . The binding of these derivatives to NEDD4‐1 was confirmed by differential scanning fluorimetry, in which four out of six compounds caused significant shifts in melting temperature.…”

“…Guided by the crystal structure of the HECT domain of NEDD4‐1 in complex with the irreversible Cys‐reactive inhibitor 1 identified by Statsyuk and co‐workers (Figure A, see Section 2.1), the I3C derivatives were docked onto the hydrophobic exosite region flanking Cys627, although this binding mode has not been validated experimentally, yet.…”

Discovery of Small Molecule WWP2 Ubiquitin Ligase Inhibitors

“…The NMR screening provided clear-cutr esults for six of the hit compounds,a ss hown by positive binding resultsi nb oth the STD and CPMG NMR experiments (1,2,16,17,18 and 20, see Ta ble 1, Supporting Information Table S1 and Figure S4). Compounds 12, 19 and 21 were excludeda sthey showed very weak signals both in the binding experimentsa nd in the control spectra, probablya scribable to solubility issues;w hile compound 7 showedn oS TD signals.…”

“…Using a covalent tethering method, a new class of Nedd4 ligase inhibitors were identified and shown to react with a non‐catalytic cysteine to hinder ubiquitin binding to this exosite and subsequent enzymatic processivity . Additional attempts to discover Nedd4 inhibitors have utilised in silico modelling approaches supported by thermal shift binding experiments and revealed that derivatives of indole‐3‐carbinol may also bind to the processivity exosite in the Nedd4 catalytic HECT domain and inhibit substrate‐specific ubiquitination linked to oncogenic signalling events …”

“…[14] Additional attempts to discover Nedd4 inhibitors have utilisedi ns ilico modelling approaches supported by thermal shift binding experiments and revealed that derivatives of indole-3-carbinol may also bind to the processivity exosite in the Nedd4 catalytic HECT domain and inhibit substrate-specific ubiquitination linkedt ooncogenic signalling events. [15,16] Here, we focused our efforts on developing small molecule inhibitors of the WWP2 ubiquitin ligase, an enzymet hat can cause ubiquitin-dependent degradation of specific tumour suppressor proteins that are commonly lost in many types of cancer,n amely Smad transcriptionf actors, PTEN, and Oct4. To that aim, we have used high-throughput screening to identify hits from the NCI DiversityS et V and NCI Approved Oncology Set V smallm olecule libraries, we have confirmed binding to WWP2b yN MR spectroscopy,a nd used ac ombination of STD NMR,D iffErential EPitope mapping (DEEP)-STD NMR and docking calculations to generate for the first time an NMR-validated 3D molecular model of aW WP2-inhibitor complex.…”