Synthetic derivatives of the SUMO consensus sequence provide a basis for improved substrate recognition

Abstract

Protein sumoylation is a dynamic posttranslational modification that regulates a diverse subset of the proteome. The mechanism by which sumoylation enzymes recognize their cognate substrates is unclear, and the consequences of sumoylation remain difficult to predict. While small molecule probes of the sumoylation process could be valuable for understanding SUMO biology, few small molecules that modulate this process exist. Here, we report the synthesis and evaluation of over 600 oxime-containing peptide sumoylation substrates. Our work demonstrates that higher modification efficiency can be achieved with non-natural side chains that deviate substantially from the consensus site requirement of a hydrophobic substituent. Furthermore, docking studies suggest that these improved substrates mimic binding interactions that are used by other endogenous protein sequences through tertiary interactions. The development of these high efficiency substrates provides key mechanistic insights toward specific recognition of low molecular weight species in the sumoylation pathway.

Macrophilones from the Marine Hydroid Macrorhynchia philippina Can Inhibit ERK Cascade Signaling

“…Treatment with 1 leads to an increase in reactive oxygen species (ROS) in cells, but it also demonstrated potent cytotoxic activity that was not related to ROS or general oxidative stress. Compounds 2 – 7 were also evaluated for their ability to block SUMOylation of a substrate peptide in an in vitro electrophoretic mobility shift assay. , They showed relatively modest inhibitory activity in the SUMO assay with IC 50 values that ranged from 11.9 μM for 2 to >100 μM for 6 (Supporting Information). To further explore their cytotoxic properties, macrophilones A ( 1 ), C ( 3 ), and D ( 4 ) were tested in the NCI-60 cell line screening assay.…”

“…Inhibition of the SUMO conjugation enzymatic cascade was assessed using a fluorescently tagged model substrate peptide following the procedures previously reported. 29,30 DMSO solutions of macrophilones A ( 1 ), C ( 3 ), and D ( 4 ) were tested for cytotoxicity against 60 human tumor cell lines in the NCI-60 cell screening assay, and the results were analyzed using bioinformatics tools on the publicly accessible CellMiner Web site (https://discover.nci.nih.gov/cellminer/). 31 Western blot analysis used antibodies to B-Raf, C-Raf, and ERK2 from Santa Cruz Biotechnology, antibodies to pS217/221-MEK and pT202/Y204-ERK from Cell Signaling Technologies, antibodies to pERK from Sigma, and antibodies to MEK1 from BD Biosciences.…”

MS‐based strategies for identification of protein SUMOylation modification

“…SUMO interacts with the substrate proteins which possess the ε‐amino group of certain lysine (K) residues. The SUMO‐modified K residues often reside in the consensus motif composed of ψKxE or ψKxD (“ψ” represents a hydrophobic residue and “x” means any sort of amino acid residue) or inverted consensus motif . Of course, the SUMO‐modified sites of non‐consensus K residues have been also reported .…”

Macrophilone A: Structure Elucidation, Total Synthesis, and Functional Evaluation of a Biologically Active Iminoquinone from the Marine Hydroid Macrorhynchia philippina

“…21 A microfluidic electrophoretic mobility shift assay was utilized to measure the ability of 1 and methoxy analogue 4 to inhibit sumoylation of a fluorescently tagged model substrate peptide. 8,28 Macrophilone A ( 1 ) exhibited an IC 50 of 8.0 μ M, while synthetic analogue 4 was more potent with an IC 50 of 2.5 μM (Figure S3). Quinones and related redox active molecules can generate reactive oxygen species (ROS), an imbalance of which causes oxidative stress in cells that results in damage to proteins, lipids, and DNA.…”