Structural basis of human separase regulation by securin and CDK1-cyclin B1.

In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex1. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD212-4). Separase is activated by degradation of its inhibitors, securin5 and cyclin B6, but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalytic site and at nearby docking sites. As in Caenorhabditis elegans7 and yeast8, human securin contains its own pseudosubstrate motifs. By contrast, CDK1-cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1-cyclin B1 to block the catalytic sites of both separase and CDK19,10. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine6 that promotes complex assembly by binding to a conserved phosphate-binding pocket in cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation.

Structure and Function of the Separase-Securin Complex.

Separase is a large cysteine protease in eukaryotes and has crucial roles in many cellular processes, especially chromosome segregation during mitosis and meiosis, apoptosis, DNA damage repair, centrosome disengagement and duplication, spindle stabilization and elongation. It dissolves the cohesion between sister chromatids by cleaving one of the subunits of the cohesin ring for chromosome segregation. The activity of separase is tightly controlled at many levels, through direct binding of inhibitory proteins as well as posttranslational modification. Dysregulation of separase activity is linked to cancer and genome instability, making it a target for drug discovery. One of the best-known inhibitors of separase is securin, which has been identified in yeast, plants, and animals. Securin forms a tight complex with separase and potently inhibits its catalytic activity. Recent structures of the separase-securin complex have revealed the molecular mechanism for the inhibitory activity of securin. A segment of securin is bound in the active site of separase, thereby blocking substrate binding. Securin itself is not cleaved by separase as its binding mode is not compatible with catalysis. Securin also has extensive interactions with separase outside the active site, consistent with its function as a chaperone to stabilize this enzyme.

Centrosome reduction in newly-generated tetraploid cancer cells obtained by separase depletion.

Tetraploidy, a common feature in cancer, results in the presence of extra centrosomes, which has been associated with chromosome instability (CIN) and aneuploidy. Deregulation in the number of centrosomes triggers tumorigenesis. However, how supernumerary centrosomes evolve during the emergence of tetraploid cells remains yet to be elucidated. Here, generating tetraploid isogenic clones in colorectal cancer and in non-transformed cells, we show that near-tetraploid clones exhibit a significant increase in the number of centrosomes. Moreover, we find that centrosome area in near-tetraploids is twice as large as in near-diploids. To evaluate whether centrosome clustering was occurring, we next analysed the number of centrioles revealing centriole amplification. Notwithstanding, more than half of the near-tetraploids maintained in culture do not present centrosome aberrations. To test whether cells progressively lost centrioles after becoming near-tetraploid, we transiently transfected diploid cells with siRNA against ESPL1/Separase, a protease responsible for triggering anaphase, to generate newly near-tetraploid cells. Finally, using this model, we assessed the number of centrioles at different time-points after tetraploidization finding that near-tetraploids rapidly lose centrosomes over time. Taken together, these data demonstrate that although most cells reduce supernumerary centrosomes after tetraploidization, a small fraction retains extra centrioles, potentially resulting in CIN.

Securin-independent regulation of separase by checkpoint-induced shugoshin-MAD2.

Separation of eukaryotic sister chromatids during the cell cycle is timed by the spindle assembly checkpoint (SAC) and ultimately triggered when separase cleaves cohesion-mediating cohesin1-3. Silencing of the SAC during metaphase activates the ubiquitin ligase APC/C (anaphase-promoting complex, also known as the cyclosome) and results in the proteasomal destruction of the separase inhibitor securin1. In the absence of securin, mammalian chromosomes still segregate on schedule, but it is unclear how separase is regulated under these conditions4,5. Here we show that human shugoshin 2 (SGO2), an essential protector of meiotic cohesin with unknown functions in the soma6,7, is turned into a separase inhibitor upon association with SAC-activated MAD2. SGO2-MAD2 can functionally replace securin and sequesters most separase in securin-knockout cells. Acute loss of securin and SGO2, but not of either protein individually, resulted in separase deregulation associated with premature cohesin cleavage and cytotoxicity. Similar to securin8,9, SGO2 is a competitive inhibitor that uses a pseudo-substrate sequence to block the active site of separase. APC/C-dependent ubiquitylation and action of the AAA-ATPase TRIP13 in conjunction with the MAD2-specific adaptor p31comet liberate separase from SGO2-MAD2 in vitro. The latter mechanism facilitates a considerable degree of sister chromatid separation in securin-knockout cells that lack APC/C activity. Thus, our results identify an unexpected function of SGO2 in mitotically dividing cells and a mechanism of separase regulation that is independent of securin but still supervised by the SAC.

Stability and pharmacokinetics of separase inhibitor-Sepin-1 in Sprague-Dawley rats.

Separase, a sister chromatid cohesion-resolving enzyme, is an oncogene and overexpressed in many human cancers. Sepin-1 (2,2-dimethyl-5-nitro-2H-benzimidazole-1,3-dioxide) is a potent separase inhibitor that impedes cancer cell growth, cell migration, and wound healing, suggesting that Sepin-1 possesses a great potential to target separase-overexpressing tumors. As a part of the IND-enabling studies to bring Sepin-1 to clinic, herein we report the results from a 28-day repeat-dose pharmacokinetic study of Sepin-1 in rats. Sepin-1 was intravenously administered to Sprague-Dawley rats once daily for 28 days at three different (5, 10, and 20 mg/kg) doses. Blood samples were collected after administration of doses on days 1 and 28. Sepin-1 is unstable and isomerizes in basic solutions, but it is stable in acidic buffer such as citrate-buffered saline (pH 4.0). UHPLC-MS analysis indicated Sepin-1 was rapidly metabolized in vivo. One of the major metabolites was an amine adduct of 2,2-dimethyl-5-nitro-2H-benzimidazole (named Sepin-1.55). The concentration of Sepin-1.55 in blood samples was Sepin-1 dose-dependent and used for pharmacokinetic analysis of Sepin-1. Tmax was approximately 5-15 min. The data suggest that no Sepin-1 accumulation occurred from daily repeat dosing and similar exposures on the first and final day of dosing. Data also suggest a gender difference, namely that female rats have more exposure and slower clearance than male rats. The data support that Sepin-1 is a potential drug candidate that can be further developed to treat Separase-overexpressing human tumors.

Identification of Bioactive Small Molecule Inhibitors of Separase.

Separase, a cysteine protease of the CD clan, triggers chromosome segregation during mitosis by cleaving the cohesin ring entrapping the two sister chromatids. Deregulated separase activity is associated with aneuploidy, a hallmark of most human cancers. In fact, separase is highly overexpressed in many solid cancers, making it an attractive chemotherapeutic target. To identify small molecules capable of inhibiting separase in its complex cellular environment, we established a highly sensitive assay to quantify separase activity in cells and screened a 51 009-member library for separase inhibitors. In vitro assays confirmed that the identified compounds efficiently inhibited separase, while not affecting caspase-1, another CD-clan protease structurally related to separase. Importantly, HeLa cells with compromised separase activity displayed severe chromosome segregation defects upon compound treatment, confirming that the identified inhibitors are bioactive in tumor tissue culture cells. Structure-activity relationship studies succeeded in the optimization of the most promising inhibitor. Overall, this study demonstrates the feasibility of identifying separase-specific inhibitors, which serve as promising lead compounds for the development of clinically relevant separase inhibiting drugs.

Molecular mechanism for the regulation of yeast separase by securin.

Separase is a cysteine protease with a crucial role in the dissolution of cohesion among sister chromatids during chromosome segregation. In human tumours separase is overexpressed, making it a potential target for drug discovery. 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. Separases are large, 140-250-kilodalton enzymes, with an amino-terminal α-helical region and a carboxy-terminal caspase-like catalytic domain. Although crystal structures of the C-terminal two domains of separase and low-resolution electron microscopy reconstructions of the separase-securin complex have been reported, the atomic structures of full-length separase and especially the complex with securin are unknown. Here we report crystal structures at up to 2.6 Å resolution of the yeast Saccharomyces cerevisiae separase-securin complex. The α-helical region of separase (also known as Esp1) contains four domains (I-IV), and a substrate-binding domain immediately precedes the catalytic domain and has tight associations with it. The separase-securin complex assumes a highly elongated structure. Residues 258-373 of securin (Pds1), named the separase interaction segment, are primarily in an extended conformation and traverse the entire length of separase, interacting with all of its domains. Most importantly, residues 258-269 of securin are located in the separase active site, illuminating the mechanism of inhibition. Biochemical studies confirm the structural observations and indicate that contacts outside the separase active site are crucial for stabilizing the complex, thereby defining an important function for the helical region of separase.

Synthesis and activity of benzimidazole-1,3-dioxide inhibitors of separase.

Due to the oncogenic activity of cohesin protease, separase in human cancer cells, modulation of separase enzymatic activity could constitute a new therapeutic strategy for targeting resistant, separase-overexpressing aneuploid tumors. Herein, we report the synthesis, structural information, and structure-activity relationship (SAR) of separase inhibitors based on modification of the lead molecule 2,2-dimethyl-5-nitro-2H-benzimidazole-1,3-dioxide, named Sepin-1, (1) identified from a high-throughput-screen. Replacement of -NO2 at C5 with other functional groups reduce the inhibitory activity in separase enzymatic assay. Substitution of the two methyl groups with other alkyl chains at the C2 moderately improves the effects on the inhibitory activity of those compounds. Modifications on 2H-benzimidazole-1,3-dioxide or the skeleton have variable effect on inhibition of separase enzymatic activity. Density-functional theory (DFT) calculations suggest there may be a correlation between the charges on the oxide moieties on these compounds and their activity in inhibiting separase enzyme.

Structural basis of cohesin cleavage by separase.

Accurate chromosome segregation requires timely dissolution of chromosome cohesion after chromosomes are properly attached to the mitotic spindle. Separase is absolutely essential for cohesion dissolution in organisms from yeast to man. It cleaves the kleisin subunit of cohesin and opens the cohesin ring to allow chromosome segregation. Cohesin cleavage is spatiotemporally controlled by separase-associated regulatory proteins, including the inhibitory chaperone securin, and by phosphorylation of both the enzyme and substrates. Dysregulation of this process causes chromosome missegregation and aneuploidy, contributing to cancer and birth defects. Despite its essential functions, atomic structures of separase have not been determined. Here we report crystal structures of the separase protease domain from the thermophilic fungus Chaetomium thermophilum, alone or covalently bound to unphosphorylated and phosphorylated inhibitory peptides derived from a cohesin cleavage site. These structures reveal how separase recognizes cohesin and how cohesin phosphorylation by polo-like kinase 1 (Plk1) enhances cleavage. Consistent with a previous cellular study, mutating two securin residues in a conserved motif that partly matches the separase cleavage consensus converts securin from a separase inhibitor to a substrate. Our study establishes atomic mechanisms of substrate cleavage by separase and suggests competitive inhibition by securin.

Separase Cleaves the N-Tail of the CENP-A Related Protein CPAR-1 at the Meiosis I Metaphase-Anaphase Transition in C. elegans.

Centromeres are defined epigenetically in the majority of eukaryotes by the presence of chromatin containing the centromeric histone H3 variant CENP-A. Most species have a single gene encoding a centromeric histone variant whereas C. elegans has two: HCP-3 (also known as CeCENP-A) and CPAR-1. Prior RNAi replacement experiments showed that HCP-3 is the functionally dominant isoform, consistent with CPAR-1 not being detectable in embryos. GFP::CPAR-1 is loaded onto meiotic chromosomes in diakinesis and is enriched on bivalents until meiosis I. Here we show that GFP::CPAR-1 signal loss from chromosomes precisely coincides with homolog segregation during anaphase I. This loss of GFP::CPAR-1 signal reflects proteolytic cleavage between GFP and the histone fold of CPAR-1, as CPAR-1::GFP, in which GFP is fused to the C-terminus of CPAR-1, does not exhibit any loss of GFP signal. A focused candidate screen implicated separase, the protease that initiates anaphase by cleaving the kleisin subunit of cohesin, in this cleavage reaction. Examination of the N-terminal tail sequence of CPAR-1 revealed a putative separase cleavage site and mutation of the signature residues in this site eliminated the cleavage reaction, as visualized by retention of GFP::CPAR-1 signal on separating homologous chromosomes at the metaphase-anaphase transition of meiosis I. Neither cleaved nor uncleavable CPAR-1 were centromere-localized in mitosis and instead localized throughout chromatin, indicating that centromere activity has not been retained in CPAR-1. Although the functions of CPAR-1 and of its separase-dependent cleavage remain to be elucidated, this effort reveals a new substrate of separase and provides an in vivo biosensor to monitor separase activity at the onset of meiosis I anaphase.

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

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 (IC50) 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.