Identification of Natural Product Sulfuretin Derivatives as Inhibitors for the Endoplasmic Reticulum Redox Protein ERO1α.

The flavin adenine dinucleotide containing Endoplasmic Reticulum Oxidoreductase-1 α (ERO1α) catalyzes the formation of de novo disulfide bond formation of secretory and transmembrane proteins and contributes towards proper protein folding. Recently, increased ERO1α expression has been shown to contribute to increased tumor growth and metastasis in multiple cancer types. In this report we sought to define novel chemical space for targeting ERO1α function. Using the previously reported ERO1α inhibitor compound, EN-460, as a benchmark pharmacological tool we were able to identify a sulfuretin derivative, T151742 which was approximately two-fold more potent using a recombinant enzyme assay system (IC50 = 8.27 ± 2.33 µM) compared to EN-460 (IC50= 16.46 ± 3.47 µM). Additionally, T151742 (IC50 = 16.04 µM) was slightly more sensitive than EN-460 (IC50= 19.35µM) using an MTT assay as an endpoint. Utilizing a cellular thermal shift assay (CETSA), we determined that the sulfuretin derivative T151742 demonstrated isozyme specificity for ERO1α as compared to ERO1ß and showed no detectable binding to the FAD containing enzyme LSD-1. T151742 retained activity in PC-9 cells in a clonogenicity assay while EN-460 was devoid of activity. Furthermore, the activity of T151742 inhibition of clonogenicity was dependent on ERO1α expression as CRISPR edited PC-9 cells were resistant to treatment with T151742. In summary we identified a new scaffold that shows specificity for ERO1α compared to the closely related paralog ERO1ß or the FAD containing enzyme LSD-1 that can be used as a tool compound for inhibition of ERO1α to allow for pharmacological validation of the role of ERO1α in cancer.

Bioactivity improvement via display of the hydrophobic core of HYD1 in a cyclic ß-hairpin-like scaffold, MTI-101.

HYD1 is an all D-amino acid linear 10-mer peptide that was discovered by one-bead-one-compound screening. HYD1 has five hydrophobic amino acids flanked by polar amino acids. Alanine scanning studies showed that alternating hydrophobic amino acid residues and N- and C-terminal lysine side chains were contributors to the biological activity of the linear 10-mer analogs. This observation led us to hypothesize that display of the hydrophobic pentapeptide sequence of HYD1 in a cyclic beta-hairpin-like scaffold could lead to better bioavailability and biological activity. An amphipathic pentapeptide sequence was used to form an antiparallel strand and those strands were linked via dipeptide-like sequences selected to promote ß-turns. Early cyclic analogs were more active but otherwise mimicked the biological activity of the linear HYD1 peptide. The cyclic peptidomimetics were synthesized using standard Fmoc solid phase synthesis to form linear peptides, followed by solution phase or on-resin cyclization. SAR studies were carried out with an aim to increase the potency of these drug candidates for the killing of multiple myeloma cells in vitro. The solution structures of 1, 5, and 10 were elucidated using NMR spectroscopy. 1H NMR and 2D TOCSY studies of these peptides revealed a downfield Hα proton chemical shift and 2D NOE spectral analysis consistent with a ß-hairpin-like structure.