2.https://clinicaltrials.gov/ct2/show/”type”:”clinical-trial”,”attrs”:”text”:”NCT03451084″,”term_id”:”NCT03451084″NCT03451084. 3.https://clinicaltrials.gov/ct2/show/”type”:”clinical-trial”,”attrs”:”text”:”NCT03404726″,”term_id”:”NCT03404726″NCT03404726. 4.IMU-838 derived from Vidofludimus calcium salt originally from 4SC AG (https://www.immunic-therapeutics.com/imu-838/). 5.https://clinicaltrials.gov/ct2/show/”type”:”clinical-trial”,”attrs”:”text”:”NCT02509052″,”term_id”:”NCT02509052″NCT02509052. 6.http://www.panoptes-pharma.com/pipeline/technology-and-product/. 7.http://ir.ptcbio.com/static-files/2057bc70-bce7-468e-bd67-abc1417ba1c4.. with hematologic malignancies. Historically, the use of small molecule inhibitors of DNA and RNA synthesis is common in cancer chemotherapy, and we have decades of experience with effective molecules such as 5-Fluorouracil, cytarabine, and methotrexate. Given the essential role of uridine monophosphate (UMP) in DNA and RNA synthesis, it is no surprise that multiple inhibitors of pyrimidine synthesis have been identified as hits during cancer cell line screening Amadacycline methanesulfonate efforts through the 1970s and 1980s. Indeed, inhibitors at every step of pyrimidine synthesis have been studied in clinical trials; N-(phosphonacetyl)-L-aspartate (PALA) as an inhibitor of the aspartate-transcarbamylase function of CAD, brequinar sodium as an inhibitor of DHODH, and pyrazofurin as an inhibitor of the orotate-phosphoribosyltransferase function of UMPS (Figure 1). Open in a separate window Figure 1. pyrimidine synthesis feeds myriad downstream metabolic pathways.pyrimidine synthesis begins with the conversion of glutamine to dihydroorotate (DHO) through the action of the trifunctional Amadacycline methanesulfonate enzyme CAD, which acts as a carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase. DHODH resides in the inner mitochondrial membrane and catalyzes the ubiquinone-dependent fourth step in the production of orotate. UMPS (uridine monophosphate Rabbit Polyclonal to CDCA7 synthetase) encodes the bifunctional enzyme to catalyze the final two steps; first as an orotate phosphoribosyltransferase which generates orotidine-5-monophosphate (OMP) and then as an OMP decarboxylase which converts OMP to UMP. Extracellular uridine can be salvaged by the SLC28A and SLC29A family of membrane transporters. Once intracellular, uridine is then phosphorylated to UMP by the action of UCK (uridine-cytidine kinase 1 or 2 2).UMP is the initial building block in the production of ribonucleotides and deoxyribonucleotides and is therefore critical for the synthesis of RNA and DNA. However, UMP also feeds the production of phosphatidylcholine, phospatidylserine, phosphatidylinositol, glycogen, hyaluronic acid, proteoglycans, glucuronidation, and the post-translational modification of proteins by O-linked N-acetylglucosamine (GlcNAc). 1.1. What is DHODH? Dihydroorotate dehydrogenase (DHODH) is a ubiquitous enzyme located within the Amadacycline methanesulfonate inner membrane of the mitochondria. Using the cofactor ubiquinone, DHODH catalyzes the fourth step of pyrimidine synthesis: the conversion of dihydroorotate to orotate. DHODH is the only enzyme capable of performing this conversion and it is therefore essential for the cells ability to produce uridine monophosphate (Figure 1). UMP is the first building block in the production of pyrimidine ribonucleosides and deoxyribonuclesides for RNA and DNA synthesis, respectively. Inhibitors of DHODH function by blocking this proximal step leading to the rapid depletion of UMP, UDP, and UTP. Beyond RNA and DNA synthesis, UDP acts as a shuttle for myriad other metabolic pathways in the form of UDP-glucose, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine (UDP-GlcNAc) (Figure 1). Cells are capable of scavenging uridine from the extracellular environment via nucleoside transporters [1]. However, while the concentrations of extracellular uridine are as high as 10 M, this is insufficient to sustain a dividing cell, and therefore the lack of DHODH activity is not compatible with life. Indeed, there are no DHODH-deficient animals. Even in the very rare Miller syndrome in humans, these severely affected individuals have hypomorphic alleles rather that complete loss of DHODH activity [2]. Of interest, leukemia cell lines (mouse and human) can be rendered DHODH-deficient by CRISPR/Cas9 gene editing; these knockout lines are dependent on supraphysiologic concentrations (~ 100 M) of extracellular uridine (D.B. Sykes, unpublished data). However, the Amadacycline methanesulfonate observation that these DHODH-null cells are viable suggests that the electron-transport function of DHODH is dispensable and is not the mechanistic target of the anti-leukemia effect of small molecule DHODH inhibitors. 1.2. DHODH inhibitors: initial clinical trial experience The DHODH inhibitor brequinar (NSC 368,390, DuP-785) demonstrated potent and anti-tumor activity across multiple tumor models [3]. Brequinar was quickly advanced to phase I clinical trials in patients with advanced solid tumor malignancies [4C6]. While brequinar was generally well-tolerated, and demonstrated occasional durable responses, the clinical experience was disappointing across more than a dozen trials encompassing more than 500 patients [7C11]. 1.3. Renewed interest in DHODH Further clinical development of brequinar was halted as the patent life neared an end. However, the DHODH inhibitor leflunomide was FDA-approved in 1998 as an immune-suppressive medication for the treatment of rheumatoid arthritis. In the ensuing years, investigators suggested.