Screening process for a library of 27,520 compounds. The schema shows the steps involved in recovering inhibitors of ResT. The fluorescent screen was performed on a library of 27,520 small molecules at a 10 μM final concentration. Compounds that exhibited less than 70% inhibition of the ResT reaction were not studied further. Molecules that lost their abilities to inhibit upon the addition of BSA or DTT were eliminated as nonspecific inhibitors. Compounds were also tested for the ability to act as fluorescent signal quenchers, resulting in the removal of 21 compounds. The remaining 25 molecules were tested by the gel-based telomere resolution assay used for biochemical analysis of ResT. Low-efficiency compounds were eliminated, leaving 16 ResT inhibitors. Compounds were reordered, and the IC₅₀ of each was determined by the gel-based assay. Six compounds exhibiting IC₅₀ values of <10 μM were recovered.

Summary of small-molecule inhibitors of ResT. The top panel displays the type of information provided for each inhibitor at each corner.

Summary of compound properties. Compounds were organized into three classes based on the data displayed in this figure. The graph for each compound shows the percentages of ResT_164-449 sequence-specific DNA binding, telomere cleavage, and telomere resolution, normalized to the values obtained with the positive control.

Fluorescence-based high-throughput screen for inhibitors of the telomere resolvase, ResT. (A) Schematic of the high-throughput assay, which is described in detail in Materials and Methods. The linearized substrate plasmid, pYT1, is shown with a dashed line indicating the axis of 180° rotational symmetry in the replicated telomere. The dots indicate the positions of the scissile phosphates. ResT catalyzes telomere resolution, resulting in two DNA fragments, each with covalently closed hairpin ends. Addition of Na₃PO₄ at pH 12 to a final concentration of 42 mM results in denaturation of the substrate DNA; however, the hairpin products can snap back to form double-stranded DNA, which is detected and stabilized by SYBR green dye. This dye binds specifically to double-stranded DNA, producing a fluorescent signal in the presence of a product but not in the presence of an unreacted substrate. (B) Positive-control (red) and negative-control (blue) data for the screening of a library of 27,520 compounds using the assay in 384-well plates. Positive samples were treated with ResT, whereas negative samples were not treated.

IC₅₀ curves for ResT inhibitors. (A) IC₅₀ determination using a gel-based telomere resolution assay. The schematized structures to the left of the gel represent the substrate and product structures corresponding to the migration of the gel bands visible on an ethidium bromide-stained 0.8% agarose gel. The upper band is PstI-linearized pYT1, as shown in Fig. 1. The two lower bands represent the hairpin products resulting from telomere resolution by ResT. Decreasing concentrations of inhibitor were incubated at 30°C with 1.1 nM linearized plasmid substrate in the presence of 110 nM ResT for 30 min. (Results for compound 5 are shown in this example.) (B) IC₅₀ values of the six most effective inhibitors. Each gel assay was performed in duplicate (except for compound 11, due to a limiting amount of the compound), and the mean IC₅₀ values and standard errors were plotted as the percentage of inhibition of in vitro telomere resolution versus inhibitor concentration. The IC₅₀ values of each compound were obtained by nonlinear regression curve fits (sigmoidal dose-response curves) for the curves shown.

Effects of inhibitors on the two chemical steps of telomere resolution and on sequence-specific DNA binding. (A) Telomere cleavage, the first transesterification step in the telomere resolution reaction, was monitored using a substrate (S) containing an OPS at each of the cleavage sites, represented as dots within square brackets and expanded at the bottom left. As described in the text, and as shown at the top left, cleavage of an OPS substrate results in the irreversible formation of a CPD between ResT and the cleavage site. An example of the gel assay, showing the migration of the substrate and CPD, appears on the right (see Materials and Methods). (B) Cleavage of replicated telomeres by ResT in the presence of inhibitors. The conditions for the cleavage reactions were the same as those for telomere resolution reactions except that a 5′-³²P-labeled OPS oligonucleotide substrate was used. The average percentage of substrate cleavage by the positive control (+) was 14.4%. Reactions were performed in triplicate, and the percentages of CPD formation for reaction mixtures containing inhibitors were normalized to that for the positive control (taken as 100%) and plotted with standard errors. *, >95% significance by a two-tailed Student t test. (C) Telomere resolution reactions were performed in the presence of a 5′-labeled 62-nucleotide substrate at 5.4 nM, 165 nM ResT, and 10 μM inhibitor at 30°C for 30 min. Reactions were stopped with 0.1% (final concentration) SDS, and products were run on a 5% SDS-polyacrylamide gel. The average percentage of resolution for the positive control (+) was 35.05%. Reactions were performed in triplicate, and the percentages of resolution for reaction mixtures containing inhibitors were normalized to that for the positive control (taken as 100%) and plotted with standard errors. (D) Binding of ResT_164-449 to a replicated telomere substrate in the presence of each inhibitor. ResT (245 nM) was incubated with 5.4 nM 5′-³²P-labeled 140-bp replicated telomere substrate in the presence of 10 μM inhibitor at 0°C for 20 min. Samples were run on a 5% acrylamide gel and were analyzed for mobility shifts of the substrate band by ResT as described previously (32). The average percentage of shift for the positive control (+) was 17.15%. The results for reactions with inhibitor treatment were normalized to those of positive-control samples (taken as 100%). Reactions were performed in triplicate.