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Small-molecule inhibitors of USP1 target ID1 degradation in leukemic cells.

ABSTRACT: Inhibitor of DNA binding 1 (ID1) transcription factor is essential for the proliferation and progression of many cancer types, including leukemia. However, the ID1 protein has not yet been therapeutically targeted in leukemia. ID1 is normally polyubiquitinated and degraded by the proteasome. Recently, it has been shown that USP1, a ubiquitin-specific protease, deubiquitinates ID1 and rescues it from proteasome degradation. Inhibition of USP1 therefore offers a new avenue to target ID1 in cancer. Here, using a ubiquitin-rhodamine-based high-throughput screening, we identified small-molecule inhibitors of USP1 and investigated their therapeutic potential for leukemia. These inhibitors blocked the deubiquitinating enzyme activity of USP1 in vitro in a dose-dependent manner with an IC50 in the high nanomolar range. USP1 inhibitors promoted the degradation of ID1 and, concurrently, inhibited the growth of leukemic cell lines in a dose-dependent manner. A known USP1 inhibitor, pimozide, also promoted ID1 degradation and inhibited growth of leukemic cells. In addition, the growth of primary acute myelogenous leukemia (AML) patient-derived leukemic cells was inhibited by a USP1 inhibitor. Collectively, these results indicate that the novel small-molecule inhibitors of USP1 promote ID1 degradation and are cytotoxic to leukemic cells. The identification of USP1 inhibitors therefore opens up a new approach for leukemia therapy.

SUBMITTER: Mistry H

PROVIDER: S-EPMC4089878 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Small-molecule inhibitors of USP1 target ID1 degradation in leukemic cells.

Mistry Helena H Hsieh Grace G Buhrlage Sara J SJ Huang Min M Park Eunmi E Cuny Gregory D GD Galinsky Ilene I Stone Richard M RM Gray Nathanael S NS D'Andrea Alan D AD Parmar Kalindi K

Molecular cancer therapeutics 20131015 12

Inhibitor of DNA binding 1 (ID1) transcription factor is essential for the proliferation and progression of many cancer types, including leukemia. However, the ID1 protein has not yet been therapeutically targeted in leukemia. ID1 is normally polyubiquitinated and degraded by the proteasome. Recently, it has been shown that USP1, a ubiquitin-specific protease, deubiquitinates ID1 and rescues it from proteasome degradation. Inhibition of USP1 therefore offers a new avenue to target ID1 in cancer. ...[more]

PMID: 24130053

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Project description:BackgroundSirtuin 2 (SIRT2) is a member of the sirtuin family, nicotinamide adenine dinucleotide+-dependent deacylases, which participates in modulation of cell cycle control, neurodegeneration, and tumorigenesis. SIRT2 expression increases in acute myeloid leukemia blasts. Downregulation of SIRT2 using siRNA causes apoptosis of HeLa cells. Therefore, selective inhibitors of SIRT2 are candidate therapeutic agents for cancer. Adult T-cell leukemia/lymphoma (ATL) is a T-cell malignancy that has a poor prognosis and develops after long-term infection with human T-cell leukemia virus (HTLV)-1. Sirtuin 1 inhibition has been shown to induce apoptosis and autophagy in HTLV-1-infected cell lines, whereas the effects of SIRT2 inhibition alone have not been elucidated.MethodsWe assessed the efficacy of our small molecule selective SIRT2 inhibitors NCO-90/141 to induce leukemic cell death. Cell viability was examined using the cell proliferation reagent Cell Count Reagent SF. Apoptotic cells were detected by annexin V-FITC and terminal deoxynucleotidyl transferase dUTP nick end labeling assays by flow cytometry. Caspase activity was detected using an APOPCYTO Intracellular Caspase Activity Detection Kit. The presence of autophagic vacuoles was assessed using a Cyto-ID Autophagy Detection Kit.ResultsOur novel small molecule SIRT2-specific inhibitors NCO-90/141 inhibited cell growth of leukemic cell lines including HTLV-1-transformed T-cells. NCO-90/141 induced apoptosis via caspase activation and mitochondrial superoxide generation in leukemic cell lines. However, a caspase inhibitor did not prevent this caspase-associated cell death. Interestingly, NCO-90/141 increased the LC3-II level together with autophagosome accumulation, indicating autophagic cell death. Thus, NCO-90/141 simultaneously caused apoptosis and autophagy.ConclusionsThese results suggest that NCO-90/141 are highly effective against leukemic cells in caspase-dependent or -independent manners via autophagy, and they may have a novel therapeutic potential for treatment of leukemias including ATL.

Project description:Target-specific scoring methods are more commonly used to identify small-molecule inhibitors among compounds docked to a target of interest. Top candidates that emerge from these methods have rarely been tested for activity and specificity across a family of proteins. In this study we docked a chemical library into CaMKII?, a member of the Ca2+ /calmodulin (CaM)-dependent protein kinase (CaMK) family, and re-scored the resulting protein-compound structures using Support Vector Machine SPecific (SVMSP), a target-specific method that we developed previously. Among the 35 selected candidates, three hits were identified, such as quinazoline compound 1 (KIN-1; N4-[7-chloro-2-[(E)-styryl]quinazolin-4-yl]-N1,N1-diethylpentane-1,4-diamine), which was found to inhibit CaMKII? kinase activity at single-digit micromolar IC50 . Activity across the kinome was assessed by profiling analogues of 1, namely 6 (KIN-236; N4-[7-chloro-2-[(E)-2-(2-chloro-4,5-dimethoxyphenyl)vinyl]quinazolin-4-yl]-N1,N1-diethylpentane-1,4-diamine), and an analogue of hit compound 2 (KIN-15; 2-[4-[(E)-[(5-bromobenzofuran-2-carbonyl)hydrazono]methyl]-2-chloro-6-methoxyphenoxy]acetic acid), namely 14 (KIN-332; N-[(E)-[4-(2-anilino-2-oxoethoxy)-3-chlorophenyl]methyleneamino]benzofuran-2-carboxamide), against 337 kinases. Interestingly, for compound 6, CaMKII? and homologue CaMKII? were among the top ten targets. Among the top 25 targets of 6, IC50 values ranged from 5 to 22??m. Compound 14 was found to be not specific toward CaMKII kinases, but it does inhibit two kinases with sub-micromolar IC50 values among the top 25. Derivatives of 1 were tested against several kinases including several members of the CaMK family. These data afforded a limited structure-activity relationship study. Molecular dynamics simulations with explicit solvent followed by end-point MM-GBSA free-energy calculations revealed strong engagement of specific residues within the ATP binding pocket, and also changes in the dynamics as a result of binding. This work suggests that target-specific scoring approaches such as SVMSP may hold promise for the identification of small-molecule kinase inhibitors that exhibit some level of specificity toward the target of interest across a large number of proteins.

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Small-molecule inhibitors of USP1 target ID1 degradation in leukemic cells.

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Small-molecule inhibitors of USP1 target ID1 degradation in leukemic cells.

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