Development of Small Molecules that Specifically Inhibit the D-loop Activity of RAD51 - PubMed (original) (raw)

Development of Small Molecules that Specifically Inhibit the D-loop Activity of RAD51

Wei Lv et al. J Med Chem. 2016.

Abstract

RAD51 is the central protein in homologous recombination (HR) DNA repair and represents a therapeutic target in oncology. Herein we report a novel class of RAD51 inhibitors that were identified by high throughput screening. In contrast to many previously reported RAD51 inhibitors, our lead compound 1 is capable of blocking RAD51-mediated D-loop formation (IC50 21.3 ± 7.8 μM) at concentrations that do not influence RAD51 binding to ssDNA. In human cells, 1 inhibits HR (IC50 13.1 ± 1.6 μM) without blocking RAD51's ability to assemble into subnuclear foci at sites of DNA damage. We determined that the active constituent of 1 is actually an oxidized derivative (termed RI(dl)-1 or 8) of the original screening compound. Our SAR campaign also yielded RI(dl)-2 (hereafter termed 9h), which effectively blocks RAD51's D-loop activity in biochemical systems (IC50 11.1 ± 1.3 μM) and inhibits HR activity in human cells (IC50 3.0 ± 1.8 μM).

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Conflict of interest statement

Notes

The authors declare no competing financial interest.

Figures

Figure 1

Figure 1

Schematic representation of the mechanism of DNA double-strand breaks repair by homologous recombination. Following a DSB, the DNA ends are resected to generate 3′ ssDNA overhangs onto which RAD51 loads. The resulting RAD51-ssDNA nucleoprotein filament is capable of invading homologous dsDNA and base-pairing with complementary DNA, thereby forming a heteroduplex and displacing a loop (D-loop) of ssDNA.

Figure 2

Figure 2

High-throughput (HT) screen for compounds that inhibit RAD51’s D-loop activity. (A) One biochemical assay monitors RAD51 binding to fluorescently labeled ssDNA, which is detected as an increase in fluorescence polarization. (B) A second parallel biochemical assay monitors fluorescence intensity, which decreases upon pairing of Black Hole Quencher 1-labeled ssDNA with a fluorescein-labeled complementary double-hairpin duplex. (C) Compounds were tested for the ability to influence the efficiency of these two RAD51-mediated processes, and the results of this HT are displayed. Blue symbols = ASDI library compounds, red symbols = LOPAC library compounds. Arrow indicates the position of compound 1.

Figure 3

Figure 3

Compound 1 inhibits RAD51’s D-loop activity with minimal interference of RAD51-ssDNA binding. (A) Electromobility shift assay showing that 1 does not inhibit interaction with RAD51 and ssDNA. (B) Gel-based D-loop formation assay showing that 1 inhibits RAD51-mediated assimilation of a radiolabeled ssDNA oligonucleotide into the homologous region of a supercoiled dsDNA plasmid by up to 74%. Error bars indicate the standard error for four replicates with a representative gel image shown.

Figure 4

Figure 4

An overview is shown for the optimization strategy of 8.

Figure 5

Figure 5

(A) Inhibition of cellular HR activity by 1 and optimized analogues of 1 using the DR-GFP assay. Data were collected at 24 h following transfection with the I-SceI expressing plasmid pCBASce. Error bars denote the standard error for three replicates. (B) Inhibition of cellular HR and SSA activity by 9h at 24 h post-transfection. Error bars denote the standard error for three replicates.

Figure 6

Figure 6

(A) Compound 9h does not inhibit the appearance of IR-induced RAD51 foci in 293-DR-GFP cells over an 8 h time course. At least 100 nuclei per condition are represented. (B) Representative micrographs showing colocalization of RAD51 foci with replication protein A (RPA) foci in DAPI-counterstained 293-DR-GFP nuclei at 8 h postirradiation in 293-DR-GFP cells treated with 9h or the DMSO vehicle control.

Figure 7

Figure 7

(A,B) Electromobility shift assay showing RAD51-ssDNA binding in the presence of 9h or RI-1, respectively. (C) 9h does not destabilize RAD51-ssDNA nucleoprotein filaments as shown by salt titration midpoint. RI-1 serves as a positive control for disruption of RAD51-ssDNA nucleoprotein filament stability. Error bars denote the standard error for three replicates. (D,E,F) Representative D-loop assay gel images showing recombinase-mediated assimilation of a radiolabeled ssDNA oligonucleotide into the homologous region of a supercoiled dsDNA plasmid in the presence of 9h by human RAD51 protein, S. cerevisiae Rad51 and Rad52 proteins, or E. coli RecA protein, respectively. Error bars denote the standard error for three replicates.

Figure 8

Figure 8

Clonogenic survival of (A) U2OS, (B) PC-3, and (C) MCF-7 tumor cell lines treated with ionizing radiation followed by outgrowth in the presence of 9h or the vehicle-only control. Error bars denote the standard error for three replicates.

Scheme 1

Scheme 1. Synthesis of 7_a_

_a_Reagents and conditions: (a) AcOH, reflux, 3 h, 90%; (b) H2, Pd/C, MeOH, 5 h, 97%; (c) 1,2,3-benzotriazole, AlCl3, THF, 4 h, 32%.

Scheme 2

Scheme 2. Synthesis of 8_a_

_a_Reagents and conditions: (a) 1,2,3-benzotriazole, AlCl3, THF, overnight; (b) MnO2, acetone, 55 °C, 24 h, 59% in two steps.

Scheme 3

Scheme 3. Synthesis of Analogues 9a–j_a_

_a_Reagents and conditions: (a) 1,2,3-benzotriazole, AlCl3, THF, overnight; (b) MnO2, acetone, 55 °C, 24 h, 19–70% in two steps; (c) H2, Pd/C, methanol, 5 h, 57–87%.

Scheme 4

Scheme 4. Synthesis of Analogues 11a–d_a_

_a_Reagents and conditions: (a) 1,2,3-benzotriazole, AlCl3, THF, overnight; (b) MnO2, acetone, 55 °C, 24 h, 35–71% in two steps.

Scheme 5

Scheme 5. Synthesis of 5b–d_a_

_a_Reagents and conditions: (a) AcOH, reflux, 3 h, 45–63%; (b) H2, Pd/C, MeOH, 5 h, 37–97%.

Scheme 6

Scheme 6. Synthesis of Analogues 12a–m_a_

_a_Reagents and conditions: (a) 1,2,3-benzotriazole, AlCl3, THF, overnight; (b) MnO2, acetone, 55 °C, 24 h, 7–77% in two steps; (c) H2, Pd/C, MeOH, 5 h, 50–93%.

Scheme 7

Scheme 7. Synthesis of Analogues 13a–b_a_

_a_Reagents and conditions: (a) 1,2,3-benzotriazole, AlCl3, THF, overnight; (b) MnO2, acetone, 55 °C, 24 h, 52–78% in two steps.

Scheme 8

Scheme 8. Synthesis of Analogues 15a–b_a_

_a_Reagents and conditions: (a) allyl bromide, Cs2CO3, DMF, microwave, 80 °C, 30 min, 58%; (b) FCH2CH2OH, DEAD, Ph3P, THF, microwave, 60 °C, 40 min; (c) H2, Pd/C, MeOH, overnight, 36–45%.

Scheme 9

Scheme 9. Synthesis of Analogues 16_a_

_a_Reagents and conditions: (a) Br(CH3)2CHCONH2, NaOH, DMA, 50 °C, 3 h, 41%.

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