Identification and Characterization of Separase Inhibitors (Sepins) for Cancer Therapy

Abstract

Separase is an endopeptidase that cleaves cohesin subunit Rad21, facilitating the repair of DNA damage during interphase and the resolution of sister chromatid cohesion at anaphase. Separase activity is negatively regulated by securin and Cdk1-cyclin B in vivo. Separase overexpression is reported in a broad range of human tumors, and its overexpression in mouse models results in tumorigenesis. To elucidate further the mechanism of separase function and to test if inhibition of overexpressed separase can be used as a strategy to inhibit tumor-cell proliferation, small-molecule inhibitors of separase enzyme are essential. Here, we report a high-throughput screening for separase inhibitors (Sepins). We developed a fluorogenic separase assay using rhodamine 110-conjugated Rad21 peptide as substrate and screened a small-molecule compound library. We identified a noncompetitive inhibitor of separase called Sepin-1 that inhibits separase enzymatic activity with a half maximal inhibitory concentration (IC 50 ) of 14.8 µM. Sepin-1 can inhibit the growth of human cancer cell lines and breast cancer xenograft tumors in mice by inhibiting cell proliferation and inducing apoptosis. The sensitivity to Sepin-1 in most cases is positively correlated to the level of separase in both cancer cell lines and tumors.

Molecular mechanism for the regulation of yeast separase by securin

“…Separase is over-expressed in human tumors, making it a potential target for drug discovery 8 . The protease activity of separase is strictly regulated by the inhibitor securin, which forms a tight complex with separase and may also stabilize this enzyme 9–16 .…”

Clonal Evolution and Blast Crisis Correlate with Enhanced Proteolytic Activity of Separase in BCR-ABL b3a2 Fusion Type CML under Imatinib Therapy

“…Prospective studies on the Separase regulatory network in CML may give rise to new concepts in carcinogenesis and leukemia therapy using selective Separase inhibitors. [55]…”

Measurement of Separase Proteolytic Activity in Single Living Cells by a Fluorogenic Flow Cytometry Assay

“…The design is based on the previously described work of the Debananda Pati group who employed a 7-amido-4-methyl coumaric acid (AMC)- or a rhodamine 110-conjugated Rad21 peptide (D-R-E-I-M-R) that contains a Separase-cleaving consensus motif (E-X-X-R) as specific Separase substrate in whole cell extract-based activity assays. [ 26 , 30 ] Since the Rh110 fluorophore (Ex max = 488 nm, Em max = 535 nm) displays a much higher extinction coefficient, higher quantum yield and better photostability than AMC, we have used the latter ([Ac-D-R-E-I-M-R] 2 -Rh110; Ac = acetyl group) to further develop a dual fluorochrome, so-called “smart” probe suitable for use in flow cytometry. [ 30 ] As shown in Fig 1 the acetyl group was replaced by the fluorophore Cy5 (Ex max = 635 nm, Em max = 650 nm) resulting in the final dual fluorophore substrate [Cy5-D-R-E-I-M-R] 2 -Rh110 where two Cy5-conjugated peptides were linked to one Rh110 molecule.…”

“…[ 26 , 30 ] Since the Rh110 fluorophore (Ex max = 488 nm, Em max = 535 nm) displays a much higher extinction coefficient, higher quantum yield and better photostability than AMC, we have used the latter ([Ac-D-R-E-I-M-R] 2 -Rh110; Ac = acetyl group) to further develop a dual fluorochrome, so-called “smart” probe suitable for use in flow cytometry. [ 30 ] As shown in Fig 1 the acetyl group was replaced by the fluorophore Cy5 (Ex max = 635 nm, Em max = 650 nm) resulting in the final dual fluorophore substrate [Cy5-D-R-E-I-M-R] 2 -Rh110 where two Cy5-conjugated peptides were linked to one Rh110 molecule. In this bi-conjugated form, Rh110 has very low autofluorescence due to the lactone state of the fluorophore.…”

“…Current assays employ fluorogenic peptides that contain the Rad21 mitotic cleavage site as substrates and measure Separase activity in whole cell extracts after cell lysis. [ 26 , 30 ] In our hands, these assays worked quite well but were difficult to standardize due to vague assay loading controls. Moreover, depending on sample composition i.e.…”