Rational design of human DNA ligase inhibitors that target cellular DNA replication and repair

Abstract

Based on the crystal structure of human DNA ligase I complexed with nicked DNA, computer-aided drug design was used to identify compounds in a database of 1.5 million commercially available low molecular weight chemicals that were predicted to bind to a DNA-binding pocket within the DNA-binding domain of DNA ligase I, thereby inhibiting DNA joining. Ten of 192 candidates specifically inhibited purified human DNA ligase I. Notably, a subset of these compounds was also active against the other human DNA ligases. Three compounds that differed in their specificity for the three human DNA ligases were analyzed further. L82 inhibited DNA ligase I, L67 inhibited DNA ligases I and III, and L189 inhibited DNA ligases I, III, and IV in DNA joining assays with purified proteins and in cell extract assays of DNA replication, base excision repair, and nonhomologous end-joining. L67 and L189 are simple competitive inhibitors with respect to nicked DNA, whereas L82 is an uncompetitive inhibitor that stabilized complex formation between DNA ligase I and nicked DNA. In cell culture assays, L82 was cytostatic whereas L67 and L189 were cytotoxic. Concordant with their ability to inhibit DNA repair in vitro, subtoxic concentrations of L67 and L189 significantly increased the cytotoxicity of DNA-damaging agents. Interestingly, the ligase inhibitors specifically sensitized cancer cells to DNA damage. Thus, these novel human DNA ligase inhibitors will not only provide insights into the cellular function of these enzymes but also serve as lead compounds for the development of anticancer agents.

Figure 1. Small molecule inhibitors of human DNA ligases identified by CADD

A Key residues in the DNA binding pocket, Gly448 (green) Arg451 (orange) and Ala455 (blue), within the hLigI DBD (aqua ribbon format) are shown in VDW representation with the nicked DNA in cartoon format. The sphere set used to direct the docking of small molecules is indicated by red transparent spheres. Docked orientations of the three characterized compounds, L67 (purple), L82 (red), and L189 (green). B. Chemical structures of L67, L82 and L189. C. Representative gels of DNA ligation assays. The results of three independent experiments are shown graphically. For clarity, the data for T4 DNA ligase, which was not significantly inhibited, has been omitted (hLigI, □; hLigIIIβ, ○; hLigIV/XRCC4, ▽).

Figure 2. Effect of the ligase inhibitors on the second and third steps of the ligation reaction

A Formation of the DNA adenylate reaction intermediate. Labeled ligase-adenylate forms of hLigI (I), hLigIIIβ (III), hLigIV (IV) and T4 (T4) DNA ligase were incubated with a linear DNA substrate containing a single non-ligatable nick in the absence or presence of L67, L82 and L189. The positions of the labeled 19 nucleotide DNA-adenylate (A*ppDNA) and a 5′ end–labeled 18 mer oligonucleotide (*pDNA) are indicated (left panel). The results of 2 independent experiments are shown graphically (right panel). Formation of DNA-adenylate is expressed as a percentage of DNA-adenylate formed by the DNA ligase in the absence of inhibitor. B. Phosphodiester bond formation. hLigI (I), hLigIIIβ (III), hLigIV (IV) and T4 (T4) DNA ligase were incubated with labeled linear DNA molecule containing a single ligatable nick with 3′ hydroxyl and 5′ adenylate termini in the absence (Mock) or presence of L67, L82 and L189. The postions of the labeled 19 nucleotide DNA-adenylate (Ap*pDNA) and labeled ligated product (43 mer) are indciated (left panel). The results of 2 independent experiments are shown graphically (right panel). Ligation is expressed as a percentage of DNA joining by the DNA ligase in the absence of inhibitor.

Figure 3. Michaelis-Menten analysis of ligase inhibitors: Effect of ligase inhibitors on DNA-protein complex formation by hLigI

A hLig1 (0.05 pmol) was incubated in the absence (○) and presence of L189 (left panel), L67 (middle panel) and L82 (right panel) at 25 (△) and 50 μM (□) with increasing amounts of a linear nicked DNA substrate. Lineweaver-Burk double reciprocal plots of initial reaction velocity (1/V) versus substrate concentration (1/S) are shown. B. A labeled linear substrate with a single non-ligatable nick (1 pmol) was incubated with; lane 1, no addition; lanes 2 and 3, 0.25 pmol hLigI; lanes 4 and 5, 0.5 pmol hLigI; lanes 6 and 7, 1 pmol hLigI in the absence (−) or presence (+) of 100 μM L189. C. A labeled linear substrate with a single non-ligatable nick (1 pmol) and hLigI (3 pmol) were incubated with either no additon (lane 2) or L82 at; 100 μM (lane 3); 60 μM (lane 4); 50 μM (lane 5); 30 μM (lane 6); 20 μM (lane 7); 10μM (lane 8). Lane 1, 1 pmol of DNA substrate alone. The positions of the labeled DNA substrate (DNA) and DNA-protein complexes (Lig-DNA) are indicated.

Figure 4. Effect of ligase inhibitors on replication and repair reactions catalyzed by human cell extracts

A The flap substrate (0.1 pmol) was incubated with cell extract (20 μg) in the absence (lane 2, Mock) or presence of 100 μM of; lane 3, L189; lane 4, L67; lane 5, L82. hLigI (lane 6, I-dp) and hLigIIIα (lane 7, III-dp) were immunodepeleted from the cell extracts prior to incubation with the DNA substrate. Lane 1, DNA substrate alone (Sub). The positions of the DNA substrate (24 mer), cleaved product (18 mer) and fully repaired product (43 mer) are shown. B. The linear DNA substrate with an incised AP site (0.3 pmol) was incubated with a cell extract (20 μg) and [α³²P]dTTP in the absence (lane l, Mock) or presence of 100 μM of; lane 2, L189; lane 3, L67; lane 4, L82. hLigI (lane 5, I-dp) and hLigIIIα (lane 6, III-dp) were immunodepleted from the cell extracts prior to incubation with the DNA substrate. The positions of the single nucleotide insertion reaction intermediate (31 mer, Incorporated) and the ligated product (73 mer, Repaired) are indicated. C. A 1 kb fragment with cohesive ends (0.1 pmol) was incubated with cell extract (20 μg) in the absence (lane 3, Mock) or presence of 100 μM of; lane 4, L189; lane 5, L67; lane 6, L82. hLigI (lane 7, I-dp) hLigIIIα (lane 8, III-dp) and hLigIV (lane 9, IV-dp) were immunodepleted from the cell extracts prior to incubation with the DNA substrate. Lane 1, molecular mass standard (M). Lane 2, DNA substrate alone (Sub). The positions of the DNA substrate and dimers and multimers of the substrate are indicated.

Figure 5. Characterization of the cytostatic effect of L82

A MCF10A (■), MCF7 (◇), HCT116 (▲) and HeLa (▼;) cells were plated in the absence or presence of L82 (left panel), L67 (middle panel) and L189 (right panel). After 6 days, cell viability was measured and is expressed as a percentage of the value obtained with untreated cells. B. MCF7 cells were plated out in the absence or presence of L82 (upper panel) and L67 (lower panel) at the indicated concentrations. After two weeks, colonies were stained with crystal violet. C. After serum starvation for 4 days, MCF 7 cells were returned to serum-containing media either without (□) or with 50 μM L82 (●). The cell cycle distribution at various time intervals was determined by FACS. D. Asynchronous populations of MCF cells were either untreated (upper panel) or treated with L82 at 10 μM (middle panel) and 50 μM (lower panel). After 3 days, tubulin and DNA were visualized by fluorescence microscopy (Scale bars, 0.5 mm).

Figure 6. L67 and L189 are cytotoxic and potentiate the cytotoxic effects of DNA damaging agents; altered levels of DNA ligase in cancer cells

A. Effect of L67 (left panel) and L189 (right panel) on the survival of MCF7 (△), HCT116 (□) and HeLa (◇) and MCF10A (▽) cells. B. Normal breast epithelial MCF10A cells (open symbols) and breast cancer MCF7 cells (filled symbols) in the absence (circles) or presence of 3 μM L67 (squares) were exposed to increasing concentrations of MMS (left panel). Normal breast epithelial MCF10A cells (open symbols) and colon cancer HCT116 cells (filled symbols) in the absence (circles) or presence of 20 μM L189 (squares) were exposed to increasing doses of ionizing radiation (right panel). C. hLigI (I), hLigIIIα (III) and hLigIV (IV) were detected in extracts (400 μg) of the indicated cell lines by immunoblotting. To control for extract loading, β-actin was also detected by immunoblotting.