Identification of phosphotyrosine mimetic inhibitors of human tyrosyl-DNA phosphodiesterase I by a novel AlphaScreen high-throughput assay

Abstract

Tyrosyl-DNA phosphodiesterase I (Tdp1) resolves topoisomerase I (Top1)-DNA adducts accumulated from natural DNA damage as well as from the action of certain anticancer drugs. Tdp1 catalyzes the hydrolysis of the phosphodiester bond between the catalytic tyrosine residue of topoisomerase I and the DNA 3'-phosphate. Only a limited number of weak inhibitors have been reported for Tdp1, and there is an unmet need to identify novel chemotypes through screening of chemical libraries. Herein, we present an easily configured, highly miniaturized, and robust Tdp1 assay using the AlphaScreen technology. Uninhibited enzyme reaction is associated with low signal, whereas inhibition leads to a gain of signal, making the present assay format especially attractive for automated large-collection high-throughput screening. We report the identification and initial characterization of four previously unreported inhibitors of Tdp1. Among them, suramin, NF449, and methyl-3,4-dephostatin are phosphotyrosine mimetics that may act as Tdp1 substrate decoys. We also report a novel biochemical assay using the SCAN1 Tdp1 mutant to study the mechanism of action of methyl-3,4-dephostatin.

Figure 1. Function of Tdp1

Topoisomerase 1 (Top1) excision by tyrosyl DNA phosphodiesterase 1 (Tdp1) requires prior proteolysis (41) or denaturation (21) of Top1 to expose the phosphotyrosyl bond to be attacked. Tdp1 generates a 3′-phosphate DNA end, which is hydrolyzed by polynucleotide kinase phosphatase (PNKP). PNKP also catalyzes the phosphorylation of the 5′ end of the DNA. Tdp1 and PNKP are part of the XRCC1 complex (shown at the bottom) (2,7).

Figure 2. Assay Design

The Tdp1-catalyzed hydrolysis is indicated by an arrow. Upon red-shifted light excitation (λ₆₈₀) of the donor bead, singlet oxygens are generated. When singlet oxygen encounters an acceptor bead within its traveling range, it triggers the emission of blue-shifted light (λ_580–620).

Figure 3. Assay Optimization

A) Signal generated by increasing concentration of AlphaScreen Tdp1 DNA substrate (squares) and Tdp1 DNA substrate lacking the 3′ FITC (triangles). B) Detection of substrate cleavage in the presence of increasing concentrations of Tdp1. C) Inhibition of Tdp1 by vanadate (circles) and neomycin (squares).

Figure 4. LOPAC¹²⁸⁰ Screens in 1536-well Plates

A) Structures of the four hits identified from the pilot screen. B) The hits were tested in dose-response under three reaction conditions: 1 nM enzyme and 5 minute reaction (condition A), 1 nM enzyme and 2 minute reaction (condition B), and 0.2 nM enzyme and 2 minute reaction (condition C). As the substrate conversion decreased, a progressive decrease in IC₅₀ values was observed for all four inhibitors. C) Dose-response curves of the hits tested under condition C. Shown from left to right are the responses for ATA (squares), methyl-3,4-dephostatin (rhombs), NF449 (inverted triangles), and suramin (triangles). D) 3-D scatter plots of qHTS data lacking (black) or showing (blue) concentration–response relationships are shown for the screens of the LOPAC¹²⁸⁰ collection performed under conditions of high (left plot, reaction condition A) and low (right plot, reaction condition C) substrate conversion. The increased sensitivity of the second screen is evident from the increase in the number of samples showing stronger inhibition (blue dots).

Figure 5. Hit confirmation and characterization in gel-based secondary assay

A) Structures of methyl-3,4-dephostatin and dephostatin. B) Representative gel image demonstrating dose-dependent inhibition by methyl-3,4-dephostatin. C) Inhibition curves for methyl-3,4 dephostatin (circles) and dephostatin (squares) derived from gels similar to the one presented in panel B (closed symbols) and from follow-up experiments using the HTS assay (open symbols). Error bars represent SEM from at least 3 independent experiments for the gel assays and SEM from duplicates for the AlphaScreen assays.

Figure 6. SCAN1 Tdp1 mutant binding experiments

The reaction catalyzed by Tdp1 is a two-step process in which the two catalytic histidine residues (H263 & H493) participate sequentially. A) The first step involves the nucleophilic attack of the phosphotyrosyl bond by the H263 residue and the donation of a proton by the H493 residue to the tyrosine-containing peptide-leaving group. B) This results in the formation of a transient covalent Tdp1-DNA intermediate. C) The H493 residue then activates a water molecule to hydrolyze the covalent intermediate. D) The presence of the SCAN1 H493R Tdp1 mutation leads to the accumulation of the covalent Tdp1-DNA intermediate. E) Representative SDS-PAGE gel showing the inhibition of Tdp1H493R-DNA complex formation by methyl-3,4-dephostatin.