Fig 1.
Nucleotide 5-MeCITP is an inhibitor of telomerase activity.
(A) Chemical structures and electron density surface potentials of indolyl nucleotide analogs used in this study. For clarity, only nucleobase structures are shown. Abbreviations at the bottom of each nucleobase correspond to the full name of the nucleotide as described in S1 Table. (B) Screening of nucleotide analogs via the direct telomerase activity assay reveals that 5-MeCITP reduces telomerase activity by 80% compared with untreated control. Numbers at the bottom of the gel correspond to the analog number indicated in panel A. Control refers to untreated reaction. Values to the left of the gel depict the number of deoxynucleotides added by telomerase to the primer d(GGGTTA)3. (C) Quantitation of G (dGTP) incorporation relative to control for samples analyzed in panel B. Error bars indicate the standard deviation calculated from three replicates. A two-tailed Student t test was used to determine p-values. *p < 0.05, ***p < 0.0001. Data associated with this figure can be found in the supplemental data file (S1 Data). dATP, deoxyadenosine triphosphate; dGTP, deoxyguanosine triphosphate; LC, loading control; 5-MeITP, 5-methylindolyl-2′-deoxyriboside 5′-triphosphate; 4-NITP, 4-nitroindolyl-2′-deoxynucleoside 5′-triphosphate; 6-NITP, 6-nitroindolyl-2′-deoxynucleoside 5′-triphosphate; 5-AITP, 5-aminoindolyl-2′-deoxyriboside 5′-triphosphate; 5-CITP, 5-carboxylindolyl-2′-deoxyriboside 5′-triphosphate; 5-EyITP, 5-ethyleneindolyl-2′-deoxyriboside 5′-triphosphate; 5-MeCITP, 5-methylcarboxyl-indolyl-2′-deoxyriboside 5′-triphosphate.
Fig 2.
Nucleotides 5-MeCITP and AZT-TP inhibit telomerase in a dose-dependent manner.
Direct telomerase extension assay with increasing concentrations of (A) 5-MeCITP (0–2 mM) and (B) AZT-TP (0–2 mM) reveal inhibition of telomerase activity in a dose-dependent manner. Activity (normalized G incorporation) was quantified and plotted against inhibitor concentration to determine relative K’1/2 values for each compound (n = 4). Data associated with this figure can be found in the supplemental data file (S1 Data). AZT-TP, azidothymidine triphosphate; G, deoxyguanosine triphosphate; LC, loading control; 5-MeCITP, 5-methylcarboxyl-indolyl-2′-deoxyriboside 5′-triphosphate.
Fig 3.
Nucleotide 5-MeCITP occupies the telomerase deoxynucleotide binding site and displaces the RNA template base.
(A) Structure of T. castaneum TERT in complex with a hybrid RNA-DNA and 5-MeCITP. The RNA-binding domain is shown in blue, the finger domain in orange, and the palm domain in yellow. The RNA template strand is shown in purple and the DNA strand in orange. (B) Panel (A) rotated 90°. (C) Two-dimensional diagram of the molecular interactions of 5-MeCITP with T. castaneum TERT. (D) Sequence alignment reveals sequence comparison of residues in the T-motif, and motifs 1 and 2 of Homo sapiens and Tribolium castaneum TERT. TERT, telomerase reverse transcriptase; 5-MeCITP, 5-methylcarboxyl-indolyl-2′-deoxyriboside 5′-triphosphate.
Fig 4.
Nucleoside 5-MeCIdR leads to telomere shortening in telomerase-positive cells selectively.
Trypan exclusion assay for telomerase-negative (A) and telomerase-positive cells (B) shows that 5-MeCIdR (abbreviated as 5-M, green scale bars) treatment is less toxic than AZT (gray scale bars) to all treated cells. *p < 0.05, ***p < 0.001. Error bars indicate the standard deviation values from three replicate experiments. (C) Treatment of telomerase-negative U2OS cells with 100 μM 5-MeCIdR does not alter telomere length, while telomeres in telomerase-positive A549 cells (D) get shorter with increasing population doublings (PDs). Numbers at the bottom of the Southern blots indicate the average telomere lengths. Data associated with this figure can be found in the supplemental data file (S1 Data). AZT, azidothymidine; PD, population doubling; 5-M/5-MeCIdR, cell-permeable nucleoside form of 5-MeCITP.
Fig 5.
Proposed mechanism of action of 5-MeCITP for inhibiting telomerase activity.
(A) Pre-polymerization state of a natural telomerase substrate (dNTP). The two magnesium ions help to coordinate the nucleophilic attack of the 3′-OH of the terminal dNTP of the telomeric DNA strand on the α-phosphate group of the dNTP in the TERT active site. (B) Post-polymerization state of a natural telomerase dNTP substrate. The dNTP forms a Watson-Crick base pair with the RNA template to aid in nucleophilic attack of the phosphate backbone for incorporation into the synthesized DNA strand. (C) 5-MeCITP does not base pair appropriately with the RNA template, thereby occluding the RNA from the active site. As a consequence, 5-MeCITP is not incorporated into the DNA chain. dNTP, deoxynucleotide triphosphate; TERT, telomerase reverse transcriptase; 5-MeCITP, 5-methylcarboxyl-indolyl-2′-deoxyriboside 5′-triphosphate.