| Chromosomes in vertebrate cells are protected at both ends by telomere DNA. Telomere DNA shortening during each round of cell division, due to the end replication problem and as a result, limit the proliferative potential of normal somatic cells. In cancer cells, the erosion of telomere DNA is compensated by telomerase or the alternative lengthening of telomere (ALT) mechanism to maintain telomere length homeostasis so that these cells can undergo unlimited division.Telomere DNA consists of tandem TTAGGG repeats. In the presence of metal ions, such as K+ and Na+, four TTAGGG tracks can fold into G-quadruplex structure. This structure is incompatible with the telomere maintenance mechanisms. On one hand, telomere quadruplex is not a substrate for telomerase and G-quadruplex formation may inhibit the binding of telomerase to and promote dissociation of telomerase from telomere substrate. On the other hand, G-quadruplex has been reported to preferentially form at the very 3'end of the G-rich telomere DNA strand. This property should inhibit addition of telomere repeats to the 3'end of telomere DNA by either telomerase or the ALT mechanism where base-pairing of the 3'end with the RNA template on the telomerase complex or a telomere C-rich strand is required.Currently there is intense interest in exploring small chemical ligands as new anti-cancer drugs that can disrupt telomere maintenance by stabilizing telomere G-quadruplex. Stabilization of quadruplex by G-quadruplex ligands has been shown to inhibit telomerase activity and induce growth arrest, senescence and apoptosis in cancer cells. Over the past years large number of compounds have been synthesized and screened for their ability to stabilized G-quadruplexes formed by telomere DNA or other target sequences. So far the analysis and evaluation of G-quadruplex ligands are exclusively performed in dilute solution. However, the cytoplasmic environment in living cells where the ligands are delivered to interact with G-quadruplexes is highly crowded with various biomolecules, which can be as high as 300-400 gram per liter. In general, molecular crowding has fundamental impact on the rates, equilibria, and mechanisms of biomolecular reactions. In particular, molecular crowding has been shown to affect several properties of G-quadruplex, such as its formation, stability, conformational transition and competition with formation duplex DNA. Therefore, it can be anticipated that molecular crowding may influence G-quadruplex-ligand interaction by alterations in environment and G-quadruplex properties.To evaluate how G-quadruplex ligands may perform under molecular crowding environment, we studied three ligands, i.e. TMPyP4, BMVC and Hoechst 33258, for their ability to interact with and stabilize telomere G-quadruplex, and inhibit telomerase under both dilute and molecular crowding condition. All these ligands showed significant stabilization of telomere G-quadruplex and inhibition on telomerase activity in dilute solution. However, the effects of TMPyP4 and BMVC were dramatically reduced and Hoechst 33258 even completely lost its ability to stabilize G-quadruplex and inhibit telomerase under molecular crowding condition. The cause of these reduced or diminished events can be attributed to the decreasing binding affinity between ligands and G-quadruplex which result from the decrease in water activity and increase in viscosity under molecular'crowding condition. These examples illustrate the possibility that certain ligands, although effective in dilute solution, may not be so or be totally ineffective when supplied in vivo. Therefore it is important that G-quadruplex ligands should be evaluated under a more physiologically relevant assay condition and drug designing should take into account the effect of molecular crowding. |
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