Summary assay for small molecule inhibitors of Artemis endonuclease activity
Nonhomologous DNA end joining (NHEJ) is the primary DNA repair pathway in human cells for the repair of double-strand DNA breaks. Like most DNA repair pathways, NHEJ relies on three enzyme activities: a nuclease to remove damaged DNA, polymerases to fill-in new DNA, and a ligase to restore DNA strand integrity. The primary nuclease for NHEJ is Artemis, which is activated when a protein kinase more ..
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1 R03 MH095489-01A1
Assay Provider: Michael Lieber, M.D., Ph.D., University of Southern California, Los Angeles, CA
Nonhomologous DNA end joining (NHEJ) is the primary DNA repair pathway in human cells for the repair of double-strand DNA breaks. Like most DNA repair pathways, NHEJ relies on three enzyme activities: a nuclease to remove damaged DNA, polymerases to fill-in new DNA, and a ligase to restore DNA strand integrity. The primary nuclease for NHEJ is Artemis, which is activated when a protein kinase called DNA-PKcs makes contact with a double-stranded DNA end at a chromosomal break site. Artemis is an endonuclease which nicks the DNA at transitions between single- and double-stranded DNA. It is very important for repairing a critical subset of double-strand breaks caused by X-rays, as found in therapeutic radiation. Artemis is even more critical for the repair of DNA damage caused by Type II topoisomerase inhibitors, such as the etoposide, VP-16, which is used in many human cancer chemotherapy regimens. It has been shown that human pre-B acute lymphoblastic lymphoma (ALL) cells that lack Artemis are much more susceptible to the cytotoxic effects of VP-16 at concentrations well below those used clinically. Therefore, inhibitors of Artemis might be synergistic with topoisomerase II inhibitors. Hence, complete inhibition of Artemis activity by a small molecule would be expected to recapitulate this topoisomerase II inhibitor hypersensitivity.
In addition to being critical in repairing etoposide and a subset of X-ray induced DNA damage, the most essential role of Artemis is one that is specific to B and T cell precursors, and the ALL malignancies derived from those precursors. The endonuclease activity of Artemis includes not only its ability to cut 5' and 3' overhangs (which are its role in etoposide and X-ray repair), but also the opening of DNA hairpins which are involved in the immunoglobin and T-cell receptor gene rearrangement process called V(D)J recombination. Without Artemis, the hairpins remain unopened, the DNA ends are not joined efficiently or at all, and the cell cycle is markedly delayed while the cells attempt a variety of less efficient pathways for their repair.
For the reasons stated above, it has been hypothesized that a small molecule that strongly inhibits Artemis might have strong potential therapeutic use for human ALL and other human cancers. The purpose of the assay described herein, is to identify inhibitors of Artemis endonuclease activity. This is accomplished via the development of an assay that utilizes a quenched, labeled substrate that fluoresces upon endonucleolytic cleavage.
Sequence: DCLRE1C DNA cross-link repair 1C [ Homo sapiens (human) ]
Probe molecules are defined as the positives of this assay and assigned a score of 100. Testing has not progressed to the point where a probe molecule has been identified.