Identification of the Antineoplastic Agent 6-Mercaptopurine as an Activator of the Orphan Nuclear Hormone Receptor Nurr1

Abstract

The purine anti-metabolite 6-mercaptopurine is one of the most widely used drugs for the treatment of acute childhood leukemia and chronic myelocytic leukemia. Developed in the 1950s, the drug is also being used as a treatment for inflammatory diseases such as Crohn's disease. The antiproliferative mechanism of action of this drug and other purine anti-metabolites has been demonstrated to be through inhibition of de novo purine synthesis and incorporation into nucleic acids. Despite the extensive clinical use and study of 6-mercaptopurine and other purine analogues, the cellular effects of these compounds remain relatively unknown. More recently, purine anti-metabolites have been shown to function as protein kinase inhibitors and to regulate gene expression. In an attempt to find small molecule regulators of the orphan nuclear receptor Nurr1, interestingly, we identified 6-mercaptopurine as a specific activator of this receptor. A detailed analysis of 6-mercaptopurine regulation of Nurr1 demonstrates that 6-mercaptopurine regulates Nurr1 through a region in the amino terminus. This activity can be inhibited by components of the purine biosynthesis pathway. These findings indicate that Nurr1 may play a role in mediating some of the antiproliferative effects of 6-mercaptopurine and potentially implicate Nurr1 as a molecular target for treatment of leukemias.The Nobel prize-winning work of Elion et al.(1) demonstrated that differences in nucleic acid metabolism between cancerous cells and normal cells or between cells from different organisms led to the design and development of nucleic acid analogs that would effectively and selectively block nucleic acid synthesis in the desired target cells. Among the drugs that emanated from this work are 6-mercaptopurine (6-MP), 1 6-thioguanine (6-TG), azathioprine, allopurinol, and acyclovir (2, 3). These drugs are still in use for the treatment of leukemias (6-MP and 6-TG) and autoimmune disorders and the prevention of organ transplant rejection (azathioprine), gout (allopurinol), and herpes virus infections (acyclovir). Additional nucleic acid anti-metabolites that were developed are effective in bacterial infections and malaria.The clinical efficacy of 6-MP is due in part to antiproliferative and cytotoxic effects resulting primarily from the inhibition of purine biosynthesis at multiple steps and incorporation into nucleic acids as thioguanine nucleotides (2-7). More recent work has expanded the function of purine anti-metabolites by demonstrating that compounds such as 6-thioguanine or 6-mercaptopurine can target biological activities outside of the purine biosynthesis pathway including telomerase (5), protein kinase N (6, 7), axon growth and regeneration (8, 9), and apoptosis in B cells through the regulation of the Bcl-2/Bax ratio (10).In this study, we have identified, from a high throughput screen, 6-mercaptopurine as a regulator of the transcriptional activity of the orphan nuclear hormone receptor Nurr1. There are three members of the NGFI-B group, including Nurr1 ...

Nuclear Receptor Nr4a2 Promotes Alternative Polarization of Macrophages and Confers Protection in Sepsis

“…Nr4a2 Imparts Protection in Septic Animals-To ascertain the pathological relevance of Nr4a2 in vivo, we evaluated systemic inflammation induced by an intraperitoneal injection of a lethal dose of LPS with or without 6-mercaptopurine. 6-Mercaptopurine was used because it induces the transcriptional activity of Nr4a2 (40). Within 60 h of LPS administration, 90% of the control mice died, whereas mice that received prior treatment of 6-mercaptopurine had better survival during the same period (Fig.…”

“…In the absence of ligands, the N-terminal AF1 domain of Nr4a2, which is involved in ligand-independent transcriptional activity and is the target for various post-translational modifications, is crucial for the regulation of the receptor (52). Ligands including 6-mercaptopurine and methylene-substituted diindolylmethanes (C-DIM), such as 1, 1-bis (3Ј-indolyl)-1-(p-chlorophenyl) methane (DIM-C-pPHCI), transactivate Nr4a2, pos- sibly by targeting the post-translational modifications in the AF1 domain (40,53). In this study, we observed that 6-mercaptopurine prevented mortality in animals challenged with a lethal dose of LPS (Fig.…”

The Orphan Nuclear Receptor Nur77 Suppresses Endothelial Cell Activation Through Induction of IκBα Expression

“…22 Indeed, overexpression of Nur77 in macrophages has been shown to inhibit inflammatory cytokine expression and reduce oxidized LDL uptake in these cells. 22 Moreover, 6-mercaptopurine, which is used clinically in the treatment of leukemia and inflammatory bowel disease, has been shown to enhance the transcriptional activity of Nur77, 23,24 attenuate smooth muscle cell proliferation, and prevent cuff injury-induced neointimal formation in a mouse model in a Nur77-dependent manner. 25 In ECs, Nur77 overexpression has been shown to increase cell survival and angiogenesis.…”

Orphan nuclear receptors in drug discovery

“…In addition to ligands that bind directly to the conserved ligand binding pocket, compounds that target regions outside of the LBD in the receptor may also regulate receptorcofactor interactions and receptor functions, thus serve as potential drugs for related diseases. One such example is Nurr1 agonist, 6-mercaptopurine (6-MP) that activates Nurr1 through its AF1 domain [4].…”

“…The identification of potent and selective agonists of Nurr1 will allow the development of new therapeutic interventions for CNS disorders. Recently, 6-MP was reported as a modest agonist of Nurr1, which activates Nurr1 through its amino-terminal AF1 instead of the classic ligand binding domain [4]. More Nurr1 agonists with high potency (EC50: 8-70 nM) have been identified, which can activate Nurr1 transcription activity with excellent bioavailability and can easily cross the blood-brain barrier [47].…”