(S)-(+)-Dimethindene maleate Several TERRA binding proteins

Several TERRA-binding proteins have also been discovered, especially telomeric duplex DNA binding proteins TRF1 and TRF2, pointing to a significant role of TERRA in more general chromosome biology [14], [20], [21]. TRF2 is a key component of Shelterin that protect chromosome termini [22]. Telomeres can fold into t-loops, which is facilitated by TRF2, and their formation contribute to telomere protection by masking the 3′-end overhang from being recognized as damaged DNA, preventing activation of a DNA damage response (DDR) at telomeres and inappropriate repair [23], [24]. TRF2 GAR domain has a relatively high affinity for the G-rich RNA capable of forming G-quadruplex [21], [25]. Balasubramanian and co-workers have found that TRF2 binds TERRA via interactions that necessitate the formation of a G-quadruplex structure rather than TERRA liner sequence. They also show that TRF2 simultaneously binds TERRA G-quadruplex and telomeric duplex DNA. Besides, TRF2 binds to telomeric duplex DNA more tightly than its binding to telomeric duplex DNA in the presence of TERRA G-quadruplex [26]. TRF2 helps the folding of telomere to form T-loop structure and the suppression of ATM-dependent DNA damage response activation. Telomere interacting agents are being exploited and have proven to be a new class of anticancer agents [27], [28]. It should be noted that allosteric binding inhibitors of proteins have been previously studied for inhibition of other (S)-(+)-Dimethindene maleate [29], [30]. In the present study, after screening our G-quadruplex binding ligands, we found a Quindoline derivative, CK1-14, could bind selectively with and stabilize TERRA G-quadruplex. This binding complex could interact strongly with an allosteric binding pocket of TRF2, which consequently inhibited the binding of TRF2 to telomeric duplex DNA both in vitro and in cells. The resulting delocalization of TRF2 from telomere induced DNA damage response at telomere region, which could effectively induce acute cell growth arrest in cancer cells. CK1-14 could effectively inhibit the proliferation of osteosarcoma cancer cells (U2OS cells), cause the cell G2/M phase arrest and induce apoptosis of U2OS cells. These findings illustrated that a TERRA G-quadruplex binding ligand could control the binding interaction of TRF2 with telomeric duplex DNA in ALT cell line. CK1-14 induced DNA-damage response in U2OS cells through inhibition of TRF2. TERRA G-quadruplex with bound ligand CK1-14 was found to be a novel allosteric modulator of TRF2, which possibly elicited a telomeric t-loop uncapping effect resulting in DNA damage response.
Materials and methods
Results and discussion
Discussion and conclusion TERRA G-quadruplexes are ideal therapeutic targets because they are required for telomere heterochromatin formation in all cancer cells, even in those that do not require telomerase (ALT-positive tumors). Several TERRA-binding proteins have also been discovered, especially the telomeric duplex DNA binding proteins TRF1 and TRF2, pointing to a significant role of TERRA in more general chromosome biology. TRF2 is a key component of the human telomeric protein complex Shelterin [35]. TRF2 binds to double-stranded telomeric DNA and has been implicated in the formation of the t-loop structure [40]. Inhibition of TRF2 results in immediate deprotection of chromosome ends, manifested by loss of the telomeric 3′ overhang, preventing activation of a DNA damage response (DDR) at telomeres and inappropriate repair [23], [24]. TRF2 GAR domain has a relatively high affinity for the G-rich RNA capable of forming G-quadruplex [21], [25]. Recent studies have found that TRF2 binds to TERRA via interactions that necessitate the formation of a G-quadruplex structure rather than TERRA liner sequence, and TRF2 can simultaneously bind to TERRA G-quadruplex and telomeric duplex DNA. Besides, TRF2 binds to a telomeric duplex DNA more tightly than its binding to telomeric DNA simultaneously forming a complex with TERRA G-quadruplex [26].