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A non-natural nucleotide uses a specific pocket to selectively inhibit telomerase activity.

ABSTRACT: Telomerase, a unique reverse transcriptase that specifically extends the ends of linear chromosomes, is up-regulated in the vast majority of cancer cells. Here, we show that an indole nucleotide analog, 5-methylcarboxyl-indolyl-2'-deoxyriboside 5'-triphosphate (5-MeCITP), functions as an inhibitor of telomerase activity. The crystal structure of 5-MeCITP bound to the Tribolium castaneum telomerase reverse transcriptase reveals an atypical interaction, in which the nucleobase is flipped in the active site. In this orientation, the methoxy group of 5-MeCITP extends out of the canonical active site to interact with a telomerase-specific hydrophobic pocket formed by motifs 1 and 2 in the fingers domain and T-motif in the RNA-binding domain of the telomerase reverse transcriptase. In vitro data show that 5-MeCITP inhibits telomerase with a similar potency as the clinically administered nucleoside analog reverse transcriptase inhibitor azidothymidine (AZT). In addition, cell-based studies show that treatment with the cell-permeable nucleoside counterpart of 5-MeCITP leads to telomere shortening in telomerase-positive cancer cells, while resulting in significantly lower cytotoxic effects in telomerase-negative cell lines when compared with AZT treatment.

SUBMITTER: Hernandez-Sanchez W

PROVIDER: S-EPMC6469803 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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A non-natural nucleotide uses a specific pocket to selectively inhibit telomerase activity.

Hernandez-Sanchez Wilnelly W Huang Wei W Plucinsky Brian B Garcia-Vazquez Nelson N Robinson Nathaniel J NJ Schiemann William P WP Berdis Anthony J AJ Skordalakes Emmanuel E Taylor Derek J DJ

PLoS biology 20190405 4

Telomerase, a unique reverse transcriptase that specifically extends the ends of linear chromosomes, is up-regulated in the vast majority of cancer cells. Here, we show that an indole nucleotide analog, 5-methylcarboxyl-indolyl-2'-deoxyriboside 5'-triphosphate (5-MeCITP), functions as an inhibitor of telomerase activity. The crystal structure of 5-MeCITP bound to the Tribolium castaneum telomerase reverse transcriptase reveals an atypical interaction, in which the nucleobase is flipped in the ac ...[more]

PMID: 30951520

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Project description:BackgroundAlthough implicated in the pathogenesis of several chronic inflammatory disorders and hematologic malignancies, telomerase mutations have not been thoroughly characterized in human cancers. The present study was performed to examine the frequency and potential clinical relevance of telomerase mutations in esophageal carcinomas.MethodsSequencing techniques were used to evaluate mutational status of telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) in neoplastic and adjacent normal mucosa from 143 esophageal cancer (EsC) patients. MTS, flow cytometry, time lapse microscopy, and murine xenograft techniques were used to assess proliferation, apoptosis, chemotaxis, and tumorigenicity of EsC cells expressing either wtTERT or TERT variants. Immunoprecipitation, immunoblot, immunofluorescence, promoter-reporter and qRT-PCR techniques were used to evaluate interactions of TERT and several TERT variants with BRG-1 and β-catenin, and to assess expression of cytoskeletal proteins, and cell signaling. Fluorescence in-situ hybridization and spectral karyotyping techniques were used to examine telomere length and chromosomal stability.ResultsSequencing analysis revealed one deletion involving TERC (TERC del 341-360), and two non-synonymous TERT variants [A279T (2 homozygous, 9 heterozygous); A1062T (4 heterozygous)]. The minor allele frequency of the A279T variant was five-fold higher in EsC patients compared to healthy blood donors (p<0.01). Relative to wtTERT, A279T decreased telomere length, destabilized TERT-BRG-1-β-catenin complex, markedly depleted β-catenin, and down-regulated canonical Wnt signaling in cancer cells; these phenomena coincided with decreased proliferation, depletion of additional cytoskeletal proteins, impaired chemotaxis, increased chemosensitivity, and significantly decreased tumorigenicity of EsC cells. A279T expression significantly increased chromosomal aberrations in mouse embryonic fibroblasts (MEFs) following Zeocin™ exposure, as well as Li Fraumeni fibroblasts in the absence of pharmacologically-induced DNA damage.ConclusionsA279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal cancer cells. These findings warrant further analysis of A279T expression in esophageal cancers and premalignant esophageal lesions.

Project description:Telomerase is expressed in the majority (>85%) of tumors, but has restricted expression in normal tissues. Long-term telomerase inhibition in malignant cells results in progressive telomere shortening and reduction in cell proliferation. Here we report the synthesis and characterization of radiolabeled oligonucleotides that target the RNA subunit of telomerase, hTR, simultaneously inhibiting enzymatic activity and delivering radiation intracellularly. Oligonucleotides complementary (Match) and noncomplementary (Scramble or Mismatch) to hTR were conjugated to diethylenetriaminepentaacetic dianhydride (DTPA), allowing radiolabeling with the Auger electron-emitting radionuclide indium-111 (111In). Match oligonucleotides inhibited telomerase activity with high potency, which was not observed with Scramble or Mismatch oligonucleotides. DTPA-conjugation and 111In-labeling did not change telomerase inhibition. In telomerase-positive cancer cells, unlabeled Match oligonucleotides had no effect on survival, however, 111In-labeled Match oligonucleotides significantly reduced clonogenic survival and upregulated the DNA damage marker γH2AX. Minimal radiotoxicity and DNA damage was observed in telomerase-negative cells exposed to 111In-Match oligonucleotides. Match oligonucleotides localized in close proximity to nuclear Cajal bodies in telomerase-positive cells. In comparison with Match oligonucleotides, 111In-Scramble or 111In-Mismatch oligonucleotides demonstrated reduced retention and negligible impact on cell survival. This study indicates the therapeutic activity of radiolabeled oligonucleotides that specifically target hTR through potent telomerase inhibition and DNA damage induction in telomerase-expressing cancer cells and paves the way for the development of novel oligonucleotide radiotherapeutics targeting telomerase-positive cancers. SIGNIFICANCE: These findings present a novel radiolabeled oligonucleotide for targeting telomerase-positive cancer cells that exhibits dual activity by simultaneously inhibiting telomerase and promoting radiation-induced genomic DNA damage.

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A non-natural nucleotide uses a specific pocket to selectively inhibit telomerase activity.

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A non-natural nucleotide uses a specific pocket to selectively inhibit telomerase activity.

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