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  • Here we describe a novel series of arylazoderivatives develo


    Here, we describe a novel series of arylazoderivatives developed from CAN508, one of the first known selective CDK9 inhibitors. We focused on modification of both ends of parental molecule, CAN508, in order to improve cytotoxicity and CDK activity. We therefore analyzed how changes in molecules will affect the affinity toward CDK2 or CDK9 and antiproliferative properties. By employing several changes at different positions of the parental molecule, we succeeded in synthesizing novel derivatives that differed in their CDK-selectivity profile. Besides biochemical assays, we attempted to confirm the selectivity of one prepared compound in MCF-7 cells synchronized by various methods and compared with CDK4 inhibitor PD-0332991, CDK2 inhibitor CGP74514A and CDK1 inhibitor RO-3306.
    Results and discussion
    Experimental part
    Introduction The white shrimp Litopenaeus vannamei is a native species from the Pacific Ocean; it is found from the coasts of Sonora, Mexico, to Northern Peru. The adult animals live in the open sea, while the postlarvae migrate to the coasts and stay until the pre-adult stage in estuaries. The white shrimp is the major crustacean species cultivated around the world. Low oxygen environments or hypoxia, affects the growth of shrimp. Levels lower than 2 mg of dissolved oxygen per liter (DO L−1) are considered hypoxic (Diaz and Rosenberg, 2008). Hypoxia occurs both in marine ecosystems due to high salinity, eutrophication and high temperature (Diaz and Breitburg, 2009), and in farming conditions (Parrilla-Taylor and Zenteno-Savín, 2011). Exposure to hypoxia can affect normal silibinin by arresting the transition of the G1 to S phase resulting in cell cycle arrest (Åmellem and Pettersen, 1991; Gardner et al., 2001). Cell cycle comprises a series of events necessary for cell growth and ends with cell division. These events include DNA replication and chromosome segregation that must occur properly and without errors (Nigg, 1995; Russo et al., 1996; Behl and Ziegler, 2014). The proteins responsible for cell cycle regulation are the cyclin-dependent kinases (Cdk) (Morgan, 1997; Li et al., 2002); in mammals 20 Cdks are known (Cdk 1–20) (Malumbres, 2014). All Cdks are structurally related to each other and these kinases contain a serine/threonine catalytic domain. Cdks by themselves are inactive and are activated upon binding to cyclins, a regulatory subunit (Kim, 1998; Peyressatre et al., 2015; Lim and Kaldis, 2013). In addition, the activity of Cdks can be affected by the presence of Cdk inhibitor proteins (Hengst et al., 1994; Behl and Ziegler, 2014). In mammals, Cdk-2 is necessary in the G1/S transition and progress through the S phase (Ortega et al., 2003). The Cdk-2/Cyclin-E complex controls DNA replication during the G1/S phase while the Cdk-2/Cyclin A complex allows S phase progression (Nigg, 1995; Moreau et al., 1998; Lacey et al., 1999). Some studies have shown that cell cycle arrest in the G1/S phase is associated with the decrease in the activity of the Cdk-2/Cyclin-E complex, which causes hypo-phosphorylation of the retinoblastoma protein (Rb), a tumor suppressor protein (Krtolica et al., 1998). The role of Cdks in mammalian cells has been extensively studied, but there is little information about them in invertebrates, specifically in crustaceans. In the prawn Macrobrachium rosenbergii Cdk-2 is an important regulator involved in meiotic maturation (Chen et al., 2013) and in the shrimp Penaeus monodon the complex Cdk-2/Cyclin-E is involved in ovarian development (Zhao et al., 2016). However, Cdk-2 from the white shrimp Litopenaeus vannamei has not been described. In this study, we report the molecular characterization of Cdk-2 from the white shrimp L. vannamei and its expression during hypoxia and reoxygenation.
    Materials and methods
    Discussion In eukaryotic cells, progression through cell cycle is dependent on the activity of Cdks (Pavletich, 1999; Malumbres and Barbacid, 2005; Li et al., 2015), a group of proline directed proteins that are inactive as monomers (Peyressatre et al., 2015; Li et al., 2015), and are activated upon association to a cyclin, a regulatory subunit through the serine/threonine catalytic domain (Nigg, 1995; Honda et al., 2005; Shapiro, 2006). All kinases that have been characterized to date present a conserved catalytic core, known as S_TKc domain that contains an ATP binding site, a PSTAIRE cyclin binding domain and an activating T-loop motif. The predicted structure of the shrimp Cdk-2 herein reported shows the characteristic bi-lobed structure of all kinases (Malumbres, 2014). This structure presents the N-terminal lobe composed of several β-sheet segments, the C-helix also known as the PSTAIRE helix, a glycine rich region that contains the Thr16 and Tyr17 and its phosphorylation causes the inhibition of the kinase. The C-terminal lobe of the Cdk-2 contains the T-loop motif which includes the activation segment that spans from the DFG to the APE motives where the Thr161 is present, a sensitive to phosphorylation residue. Phosphorylation of this residue results in activation of the Cdk/Cyclin complex (Brown et al., 1999; Li et al., 2015). In addition, the ATP binding site was found between the two lobes (Fig. 4). Activation of Cdks occurs in two ways, first Cdks binds to the cyclin subunit gaining partial kinase activity; then, the formed Cdk-Cyclin complex is phosphorylated (Pavletich, 1999; Malumbres, 2014). This activation processes result in conformational modifications of the Cdk upon cyclin binding (Harper and Adams, 2001). After the binding occurs, the C-helix located in the N-terminal lobe packs against a helix located in the cyclin through hydrophobic interactions and adjusts the ATP binding site (Peyressatre et al., 2015). Also, the cyclin removes the activation segment of the C-terminal lobe out of the catalytic site, thus, the threonine residue is prone to phosphorylation and stabilization of the activated form of the Cdk-Cyclin complex (Honda et al., 2005; Malumbres, 2014). The predicted structure of the active conformation of the Cdk-2 showed changes in the T-loop and C-helix, in which both elements were separated, exposed the active site and presented the conserved phospho-threonine (Thr161). Activation of Cdk proteins is regulated by the Cdk-activating kinases (CAKs). These proteins phosphorylate the conserved phospho-threonine residue present in the T-loop, this improves the stability of the complex as well as substrate binding, thus causing Cdk activation (Harper and Adams, 2001; Lim and Kaldis, 2013). Cdks are grouped into two distinctive groups that are based on their functions, one related to cell cycle progression and one related to transcriptional regulation, Cdk-2 is grouped in the first category which is thought to be essential in cell cycle progression (Ortega et al., 2003; Shapiro, 2006). The Cdk-2/Cyclin-E complex is necessary in the transition of the G1/S phase and later the formation of the Cdk-2/Cyclin-A complex permits DNA replication at the beginning of the S phase (Matsumoto et al., 1999; Shapiro, 2006). Some cancer cells exposed to severe hypoxia suffer cell cycle arrest in the G1 phase or at the beginning of the S phase, while cells that are in the late S phase, G2 phase or M phase will finish division and arrest occurs in the G1 phase (Åmellem and Pettersen, 1991; Box and Demetrick, 2004; Muz et al., 2015). Under moderate hypoxia (2.5–5% oxygen), the cell cycle arrest in the G1 phase is associated with the decrease in the activity of the Cdk-cyclin complex (Krtolica et al., 1998).