Cells were allowed to attach overnight and treated with vehicle (DMSO) or 10 M of indicated compounds. (LIG I). Natamycin significantly inhibited proliferation of PCa cells in an androgen depleted environment at 1 M concentration, however, growth inhibition did not occur with nonmalignant prostate cell lines, suggesting that BER inhibition may improve efficacy of the castration therapies. Exo III was from New England BioLabs (Ipswitch, MA). All chemical reagents were from Sigma-Aldrich (St Louis, MO) and Thermo Fisher Scientific Inc (Weston, FL). 2.2. High throughput screening assay for inhibitors of the BER pathway The high throughput screening assay was described in US Patent No. 9809843 B1. Briefly, a fluorescence-tagged oligonucleotide substrate that contains a synthesized abasic site, i.e., tetrahydrofuran (THF) was designed to determine the total capacity of BER in prostate cancer whole cell extracts. The sequence of the oligonucleotides for constructing the substrate is: 5-CTGGA[FluorT]ACACGAACTTTAAGCATHFAGTCAATGAAGGACGCATATCAGTG-3 (upper strand) and 5-CACTGATATGCGTCCTTCATTGACTCTGCTTAAAGTTCG TG[T(BHQ-1)]ATCCAG-3 (bottom strand). A 6-carboxyfluorescein (6-FAM)-tagged-T is inserted upstream of the abasic site in the damaged strand and close to a black hole quencher (BHQ) tagged-T, which was inserted in the template strand (Figure 1). The substrate was constructed by annealing the damaged strand with the template strand at 1:1 ratio. The substrate (25 nM) was precut with 25 nM purified human AP endonuclease 1 (APE1) at 37 C for 30 min. Subsequently, the substrate was incubated with 25 g prostate cancer cell extracts (total volume of 10 L) at 37 C for 30 min allowing repair of the abasic site by BER. Unrepaired substrates were then subject to digestion by the 3-5 exonuclease activity of Exo III (0.5U) (New England BioLabs, Ipswitch, MA) at 37 C for 10 min. This cleaved the upstream strand in the unrepaired substrates releasing the 6-FAM-tagged T and allowing the emission of fluorescence detected by a fluorescence plate reader at 52820 nm (Biotek Instruments, Winoski, VT). Inhibition of BER enzymatic activity and/or the coordination among BER enzymes and their cofactors reduced the amount of repaired products and led to the accumulation of unrepaired substrates thereby significantly increasing the intensity of fluorescence signal. The approach was used with a 384-well platform in high throughput screening for inhibitors of the BER pathway. Open in a separate window Figure 1. The schematic diagram of the fluorescence-based high throughput screening of BER capacity inhibitors.A fluorescence-tagged oligonucleotide substrate that contains the analog of an abasic lesion, tetrahydrofuran (THF) was employed to determine the inhibitory effects of 774 compounds from The Screen-Well? FDA Approved Drug Library V2 on the Endothelin-2, human BER capacity of prostate cancer whole cell extracts. The procedure of the screening was conducted as descried in the Materials and Methods. 2.2.1. High Throughput Screening for BER inhibitors The Screen-Well? FDA Approved Drug Library V2 with 774 compounds were purchased from Enzo. The 10 mM stock solutions in DMSO were diluted to 2 mM before 0.5 L was added to 10 L of each assay reaction mixture of 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 0.1 mM EDTA, 0.1 mg/ml bovine serum albumin, 0.01% Nonidet P-40, 25 nM APE1 pre-cut substrate and cancer cell extract (72 g of LNCaP cell lysate per assay) in 384-well black plates (Corning 3821), for a final compound concentration of 100 M. The control reaction also has 5% DMSO added. After mixing for 2 min and spinning at 200 g for 1 min, the plates were incubated at 37C for 30 min. Freshly diluted Exo III (0.5 U, New England BioLabs) was then added for.After mixing for 2 min and spinning at 200 g for 1 min, the plates were incubated at 37C for 30 min. screen. Five compounds were then selected for further testing in the independently derived, androgen-dependent prostate cancer cell lines, LNCaP and LAPC4, and in the nonmalignant prostate derived cell lines PNT1A and RWPE1. Further analysis led to the identification of a lead compound, natamycin, as an effective inhibitor of key BER enzymes DNA polymerase (pol ) and DNA Ligase I (LIG I). Natamycin significantly inhibited proliferation of PCa cells in an androgen depleted environment at 1 M concentration, however, growth inhibition did not occur with nonmalignant prostate cell lines, suggesting that BER inhibition may improve efficacy of the castration therapies. Exo III was from New England BioLabs (Ipswitch, MA). All chemical reagents were from Sigma-Aldrich (St Louis, MO) and Thermo Fisher Scientific Inc (Weston, FL). 2.2. High throughput screening assay for inhibitors of the BER pathway The high throughput screening assay was described in US Patent No. 9809843 B1. Briefly, a fluorescence-tagged oligonucleotide substrate that contains a synthesized abasic site, i.e., tetrahydrofuran (THF) was designed to determine the total capacity of BER in prostate cancer whole cell extracts. The sequence of the oligonucleotides for constructing the substrate is: 5-CTGGA[FluorT]ACACGAACTTTAAGCATHFAGTCAATGAAGGACGCATATCAGTG-3 (upper strand) and 5-CACTGATATGCGTCCTTCATTGACTCTGCTTAAAGTTCG TG[T(BHQ-1)]ATCCAG-3 (bottom strand). A 6-carboxyfluorescein (6-FAM)-tagged-T is inserted upstream of the abasic site in the damaged strand and close to a black hole quencher (BHQ) tagged-T, which was inserted in the template strand (Figure 1). The substrate was constructed by annealing the damaged strand with the template strand at 1:1 ratio. The substrate (25 nM) was precut with 25 nM purified human AP endonuclease 1 (APE1) at 37 C for 30 min. Subsequently, the substrate was incubated with 25 g prostate cancer cell extracts (total volume of 10 L) at 37 C for 30 min allowing repair of the abasic site by BER. Unrepaired substrates were then subject to digestion by the 3-5 exonuclease activity of Exo III (0.5U) (New England BioLabs, Ipswitch, MA) at 37 C for 10 min. This cleaved the upstream strand in the unrepaired substrates releasing the 6-FAM-tagged T and allowing the emission of fluorescence detected by a fluorescence plate reader at 52820 nm (Biotek Instruments, Winoski, VT). Inhibition of BER enzymatic activity and/or the coordination among BER enzymes and their cofactors reduced the amount of repaired products and led to the accumulation of unrepaired substrates thereby significantly increasing the intensity of fluorescence signal. The approach was used with a 384-well platform Endothelin-2, human in high throughput screening for inhibitors of the BER pathway. Open in a separate window Figure 1. The schematic diagram of the fluorescence-based high throughput screening of BER capacity inhibitors.A fluorescence-tagged oligonucleotide substrate that contains the analog of an abasic lesion, tetrahydrofuran (THF) was employed to determine the inhibitory effects of 774 compounds from The Screen-Well? FDA Approved Drug Library ABCG2 V2 on the BER capacity of prostate cancer whole cell extracts. The procedure of the screening was conducted as descried in the Materials and Methods. 2.2.1. High Throughput Screening for BER inhibitors The Screen-Well? FDA Approved Drug Library V2 with 774 compounds were purchased from Enzo. The 10 mM stock solutions in DMSO were diluted to 2 mM before 0.5 L was added to 10 L of each assay reaction mixture of 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 0.1 mM EDTA, 0.1 mg/ml bovine serum albumin, 0.01% Nonidet P-40, 25 nM APE1 pre-cut substrate and cancer cell extract (72 g of LNCaP cell lysate per assay) in 384-well black plates (Corning 3821), for a final compound concentration of 100 M. The control reaction also has 5% DMSO added. After mixing for 2 min and spinning at 200 g for 1 min, the plates were incubated at 37C for 30 min. Freshly diluted Exo III (0.5 U, New England BioLabs) was then added for an additional incubation at 37C for 10 min, followed by 30 min at 50C. The reactions were terminated by adding 1 L of 500 mM EDTA. Fluorescence signal (excitation wavelength of 48520 nm and emission wavelength of 52820 nm) were recorded with the Biotek Synergy HT Plate Reader. Compounds that showed a signal greater than DMSO control + 3 S.D for each plate were chosen as hits. Twenty-six hits were selected from.Primary screening of compounds that inhibit the BER pathway To identify FDA approved compounds that can directly inhibit the activities of BER enzymes and co-factors we invented a high throughput, BER pathway C specific screening approach (Figure 1). inhibitor of important BER enzymes DNA polymerase (pol ) and DNA Ligase I (LIG I). Natamycin significantly inhibited proliferation of PCa cells in an androgen depleted environment at 1 M concentration, however, growth inhibition did not occur with nonmalignant prostate cell lines, suggesting that BER inhibition may improve effectiveness of the castration therapies. Exo III was from New England BioLabs (Ipswitch, MA). All chemical reagents were from Sigma-Aldrich (St Louis, MO) and Thermo Fisher Scientific Inc (Weston, FL). 2.2. Large throughput screening assay for inhibitors of the BER pathway The high throughput screening assay was explained in US Patent No. 9809843 B1. Briefly, a fluorescence-tagged oligonucleotide substrate that contains a synthesized abasic site, i.e., tetrahydrofuran (THF) was designed to determine the total capacity of BER in prostate malignancy whole cell components. The sequence of the oligonucleotides for building the substrate is definitely: 5-CTGGA[FluorT]ACACGAACTTTAAGCATHFAGTCAATGAAGGACGCATATCAGTG-3 (top strand) and 5-CACTGATATGCGTCCTTCATTGACTCTGCTTAAAGTTCG TG[T(BHQ-1)]ATCCAG-3 (bottom strand). A 6-carboxyfluorescein (6-FAM)-tagged-T is definitely put upstream of the abasic site in the damaged strand and close to a black opening quencher (BHQ) tagged-T, which was put in the template strand (Number 1). The substrate was constructed by annealing the damaged strand with the template strand at 1:1 percentage. The substrate (25 nM) was precut with 25 nM purified human being AP endonuclease 1 (APE1) at 37 C for 30 min. Subsequently, the substrate was incubated with 25 g prostate malignancy cell components (total volume of 10 L) at 37 C for 30 min permitting repair of the abasic site by BER. Unrepaired substrates were then subject to digestion from the 3-5 exonuclease activity of Exo III (0.5U) (New England BioLabs, Ipswitch, MA) at 37 C for 10 min. This cleaved the upstream strand in the unrepaired substrates liberating the 6-FAM-tagged T and permitting the emission of fluorescence recognized by a fluorescence plate reader at 52820 nm (Biotek Tools, Winoski, VT). Inhibition of BER enzymatic activity and/or the coordination among BER enzymes and their cofactors reduced the amount of repaired products and led to the build up of unrepaired substrates therefore significantly increasing the intensity of fluorescence transmission. The approach was used with a 384-well platform in high throughput screening for inhibitors of the BER pathway. Open in a separate window Number 1. The schematic diagram of the fluorescence-based high throughput screening of BER capacity inhibitors.A fluorescence-tagged oligonucleotide substrate that contains the analog of an abasic lesion, tetrahydrofuran (THF) was employed to determine the inhibitory effects of 774 compounds from your Screen-Well? FDA Authorized Drug Library V2 within the BER capacity of prostate malignancy whole cell components. The procedure of the screening was carried out as descried in the Materials and Methods. 2.2.1. Large Throughput Screening for BER inhibitors The Screen-Well? FDA Authorized Drug Library V2 with 774 compounds were purchased from Enzo. The 10 mM stock solutions in DMSO were diluted to 2 mM before 0.5 L was added to 10 L of each assay reaction mixture of 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 0.1 mM EDTA, 0.1 mg/ml bovine serum albumin, 0.01% Nonidet P-40, 25 nM APE1 pre-cut substrate and cancer cell extract (72 g of LNCaP cell lysate per assay) in 384-well black plates (Corning 3821), for a final compound concentration of 100 M. The control reaction also has 5% DMSO added. After combining for 2 min and spinning at 200 g for 1 min, the plates were incubated at 37C for 30 min. Freshly diluted Exo III (0.5 U, New England BioLabs) was then added for an additional incubation at 37C for 10 min, followed by 30 min at 50C. The reactions were terminated by adding 1 L of 500 mM EDTA. Fluorescence transmission (excitation wavelength of 48520 nm and emission wavelength of 52820 nm) were recorded with the Biotek Synergy HT Plate Reader. Compounds that showed a signal greater than DMSO control + 3 S.D for each plate were chosen while hits. Twenty-six hits were selected from 774 compounds (3.4%). 2.2.2. Secondary Assays of BER inhibitors The hit compounds were subject to secondary Endothelin-2, human assays to determine their ability to reduce the BER capacity when reconstituted with purified core BER enzymes, as well as their inhibitory effects on individual BER enzymes including pol , FEN1 and LIG I. The effect of hit compounds.