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Identification of Inhibitors of the dNTP Triphosphohydrolase SAMHD1 Using a Novel and Direct High-Throughput Assay.

ABSTRACT: The dNTP triphosphohydrolase SAMHD1 is a regulator of cellular dNTP pools. Given its central role in nucleotide metabolism, SAMHD1 performs important functions in cellular homeostasis, cell cycle regulation, and innate immunity. It therefore represents a high-profile target for small molecule drug design. SAMHD1 has a complex mechanism of catalytic activation that makes the design of an activating compound challenging. However, an inhibitor of SAMHD1 could serve multiple therapeutic roles, including the potentiation of antiviral and anticancer drug regimens. The lack of high-throughput screens that directly measure SAMHD1 catalytic activity has impeded efforts to identify inhibitors of SAMHD1. Here we describe a novel high-throughput screen that directly measures SAMHD1 catalytic activity. This assay results in a colorimetric end point that can be read spectrophotometrically and utilizes bis(4-nitrophenyl) phosphate as the substrate and Mn2+ as the activating cation that facilitates catalysis. When used to screen a library of Food and Drug Administration-approved drugs, this HTS identified multiple novel compounds that inhibited SAMHD1 dNTPase activity at micromolar concentrations.

SUBMITTER: Mauney CH

PROVIDER: S-EPMC6322912 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Identification of Inhibitors of the dNTP Triphosphohydrolase SAMHD1 Using a Novel and Direct High-Throughput Assay.

Mauney Christopher H CH Perrino Fred W FW Hollis Thomas T

Biochemistry 20181113 47

The dNTP triphosphohydrolase SAMHD1 is a regulator of cellular dNTP pools. Given its central role in nucleotide metabolism, SAMHD1 performs important functions in cellular homeostasis, cell cycle regulation, and innate immunity. It therefore represents a high-profile target for small molecule drug design. SAMHD1 has a complex mechanism of catalytic activation that makes the design of an activating compound challenging. However, an inhibitor of SAMHD1 could serve multiple therapeutic roles, inclu ...[more]

PMID: 30380297

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Project description:Anti-cancer topoisomerase I (Top1) inhibitors (camptothecin and its derivatives irinotecan and topotecan, and indenoisoquinolines) induce lethal DNA lesions by stabilizing Top1-DNA cleavage complex (Top1cc). These lesions are repaired by parallel repair pathways including the tyrosyl-DNA phosphodiesterase 1 (TDP1)-related pathway and homologous recombination. As TDP1-deficient cells in vertebrates are hypersensitive to Top1 inhibitors, small molecules inhibiting TDP1 should augment the cytotoxicity of Top1 inhibitors. We developed a cell-based high-throughput screening assay for the discovery of inhibitors for human TDP1 using a TDP1-deficient chicken DT40 cell line (TDP1-/-) complemented with human TDP1 (hTDP1). Any compounds showing a synergistic effect with the Top1 inhibitor camptothecin (CPT) in hTDP1 cells should either be a TDP1-related pathway inhibitor or an inhibitor of alternate repair pathways for Top1cc. We screened the 400,000-compound Small Molecule Library Repository (SMLR, NIH Molecular Libraries) against hTDP1 cells in the absence or presence of CPT. After confirmation in a secondary screen using both hTDP1 and TDP1-/- cells in the absence or presence of CPT, five compounds were confirmed as potential TDP1 pathway inhibitors. All five compounds showed synergistic effect with CPT in hTDP1 cells, but not in TDP1-/- cells, indicating that the compounds inhibited a TDP1-related repair pathway. Yet, in vitro gel-based assay revealed that the five compounds did not inhibit TDP1 catalytic activity directly. We tested the compounds for their ability to inhibit poly(ADP-ribose)polymerase (PARP) because PARP inhibitors are known to potentiate the cytotoxicity of CPT by inhibiting the recruitment of TDP1 to Top1cc. Accordingly, we found that the five compounds inhibit catalytic activity of PARP by ELISA and Western blotting. We identified the most potent compound (Cpd1) that offers characteristic close to veliparib, a leading clinical PARP inhibitor. Cpd1 may represent a new scaffold for the development of PARP inhibitors.

Dataset Information

Identification of Inhibitors of the dNTP Triphosphohydrolase SAMHD1 Using a Novel and Direct High-Throughput Assay.

Publications

Identification of Inhibitors of the dNTP Triphosphohydrolase SAMHD1 Using a Novel and Direct High-Throughput Assay.

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