A small molecule inhibitor of Pot1 binding to telomeric DNA

Abstract

Chromosome ends are complex structures, consisting of repetitive DNA sequence terminating in an ssDNA overhang with many associated proteins. Because alteration of the regulation of these ends is a hallmark of cancer, telomeres and telomere maintenance have been prime drug targets. The universally conserved ssDNA overhang is sequence-specifically bound and regulated by Pot1 (protection of telomeres 1), and perturbation of Pot1 function has deleterious effects for proliferating cells. The specificity of the Pot1/ssDNA interaction and the key involvement of this protein in telomere maintenance have suggested directed inhibition of Pot1/ssDNA binding as an efficient means of disrupting telomere function. To explore this idea, we developed a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) screen for inhibitors of Pot1/ssDNA interaction. We conducted this screen with the DNA-binding subdomain of Schizosaccharomyces pombe Pot1 (Pot1pN), which confers the vast majority of Pot1 sequence-specificity and is highly similar to the first domain of human Pot1 (hPOT1). Screening a library of ∼20 000 compounds yielded a single inhibitor, which we found interacted tightly with sub-micromolar affinity. Furthermore, this compound, subsequently identified as the bis-azo dye Congo red (CR), was able to competitively inhibit hPOT1 binding to telomeric DNA. Isothermal titration calorimetry and NMR chemical shift analysis suggest that CR interacts specifically with the ssDNA-binding cleft of Pot1, and that alteration of this surface disrupts CR binding. The identification of a specific inhibitor of ssDNA interaction establishes a new pathway for targeted telomere disruption.

Figure 1

High-throughput screen and secondary validation of compounds identify as single inhibitor. (A) Pot1pN (wheat) superposed with OB1 of hPOT1 (white) shows structural similarity; the N- and C-terminal portions of each protein have been removed for clarity. Conserved residues on the ssDNA-binding surface are colored according to chemical character: aromatic (green), hydrophobic (yellow), polar (orange), acidic (red), and basic (blue). Pot1pN F88 (and corresponding hPOT1 F62) is shown as green sticks. Superposition was performed using LSQMAN (91) and the image was generated using MacPymol version 1.3 (92). (B) Schematic of the TR-FRET assay used to identify inhibitors of Pot1pN interaction with 6mer ssDNA. 6xHis-tagged Pot1pN is bound by the Eu-chelate anti-6xHis antibody (donor), which transfers energy to ULight-Streptavidin (acceptor) bound to biotinylated ssDNA. Excitation and FRET emission wavelengths are indicated. (C) Plot of the FRET-based dose-dependence of the activity of small molecules identified by the TR-FRET screen. Percent FRET signal is plotted as a function of compound concentration. (D) Plot of the dose-dependent inhibitory activity of the compounds from (C) by secondary filter-binding assay revealing only one compound (Congo red) that directly inhibits the Pot1pN/6mer interaction. Percent 6mer ssDNA bound is plotted as a function of compound concentration.

Figure 2

Pot1pN binds CR but not Thioflavin T. The left panels show baseline corrected raw ITC data (upper) for 1.2 mM CR titrated into buffer (blue) or 100 μM Pot1pN and the reference-subtracted integrated heat of binding (lower). The right panels show baseline corrected raw ITC data (upper) for 2 mM Thioflavin T titrated into buffer (blue) or 100 μM Pot1pN and the reference-subtracted integrated heat of binding (lower). K_D and n values for fitting triplicate Pot1pN/CR experiments to a one-site binding model are reported; errors are the standard error of the mean.

Figure 3

Particle size distribution obtained by DLS shows that CR-bound Pot1pN is a trimer. (A) A monomeric species of calculated radius and MW of 1.3 nm and 25 kDa, respectively, accounts for 100% of sample mass for free Pot1pN. (B) The calculated radius and MW for the Pot1pN/CR sample are 3.8 nm and 77 kDa, respectively, and this species accounts for 99% of the total sample mass.

Figure 4

Comparison of CR-bound (red) and free (blue) Pot1pN ¹H-¹⁵N HSQC spectra obtained at 900 MHz. reveals differences upon CR binding, but no global changes or aggregation of Pot1pN. Boxed region of CR-bound spectrum in top panel is expanded below and superposed with free Pot1pN.

Figure 5

Mapping of amide backbone chemical shift changes onto the Pot1pN crystal structure demonstrates that CR specifically binds the Pot1pN ssDNA-binding surface. Pot1pN residues definitively unperturbed by CR binding (magenta), residues that undergo significant chemical shift changes in the presence of CR (teal), and unassigned residues (gray) are shown. Pot1pN N-terminus is indicated (N) and 6mer (blue sticks) is shown for reference in the ssDNA-binding cleft. Images were generated using MacPymol version 1.3 (92).

Figure 6

Plotting of the global fits from triplicate filter-binding experiments show that CR equivalently inhibits SpPot1-DBD (black) and hPOT1-DBD (blue) binding cognate telomeric ssDNA. Apparent IC_50rel values for triplicate experiments are reported; errors are the standard error of the mean.

Figure 7

Mutation of Pot1pN and alteration of CR structure alter the characteristics of binding. (A) Baseline corrected raw ITC data (upper) for 1.2 mM CR titrated into buffer (blue) or 100 μM Pot1pN_F88A and the reference-subtracted integrated heat of binding (lower). (B) Baseline corrected raw ITC data (upper) for 1 mM TB titrated into buffer (blue) or 103 μM Pot1pN and the reference-subtracted integrated heat of binding (lower). The structures of CR and TB are shown below for comparison Titrations were performed at 20 °C with 21 injections. Integrated heat data were fit to a one-site binding model.