title: Inhibition of telomerase activity by preventing proper assemblage.
abstract: Telomerase is a ribonucleoprotein complex that acts as a reverse transcriptase in the maintenance of chromosome ends. Because the vast majority of cancer cells require telomerase activity, telomerase has become a target for anticancer drug discovery. Here, we describe a new approach for targeting telomerase by blocking the association between the telomerase catalytic subunit, hTERT, and key elements of the human telomerase RNA subunit, hTR. By examining the effects of oligonucleotides that hybridize to various regions of hTR, we identified two regions of the RNA subunit that are sensitive to molecular interactions leading to telomerase inhibition. Oligonucleotides that hybridize to either the P3/P1 pairing region or to the CR4-CR5 domain of hTR, hTRas009, and hTRas010, respectively, inhibit telomerase activity when added to recombinant hTERT and hTR prior to assemblage. However, addition of hTRas009 or hTRas010 to preassembled telomerase resulted in little or no inhibition. We also examined the ability of hTRas009 and hTRas010 to inhibit binding of hTR and hTR fragments to hTERT. We found that hTRas009 inhibited approximately 50% of the maximum binding between the pseudoknot fragment of hTR (nucleotides 46-209) and hTERT, whereas hTRas010 inhibited over 90% of the maximum binding between the CR4-CR5 fragment of hTR (nucleotides 243-328) and hTERT. In addition, neither oligonucleotide was able to appreciably inhibit the binding of full-length hTR to hTERT, although both oligonucleotides used in conjunction decreased binding by approximately 50%. We propose that the P3/P1 pairing region and CR4-CR5 domain represent viable targets to inhibit telomerase by perturbing proper assemblage of the active complex.
authors.affiliation: School of Pharmacy, Division of Medicinal Chemistry and Natural Products, University of North Carolina, Chapel Hill, North Carolina 27599-7360, USA.
authors.forename: Brian R
authors.initials: BR
authors.lastname: Keppler
authors.forename: Michael B
authors.initials: MB
authors.lastname: Jarstfer
mesh_terms: D017124:Conserved Sequence; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems; D006801:Humans; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000037:antagonists & inhibitors / Q000737:chemistry; D018075:RNA, Complementary / Q000737:chemistry; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry; D019098:Telomerase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D009841:Oligonucleotides; D021122:Protein Subunits; D018075:RNA, Complementary; D018894:Reverse Transcriptase Inhibitors; D012313:RNA; D019098:Telomerase