A Review of Small-Molecule Inhibitors of One-Carbon Enzymes: SHMT2 and MTHFD2 in the Spotlight.

Metabolic reprogramming is a key hallmark of cancer and shifts cellular metabolism to meet the demands of biomass production necessary for abnormal cell reproduction. One-carbon metabolism (1CM) contributes to many biosynthetic pathways that fuel growth and is comprised of a complex network of enzymes. Methotrexate and 5-fluorouracil were pioneering drugs in this field and are still widely used today as anticancer agents as well as for other diseases such as arthritis. Besides dihydrofolate reductase and thymidylate synthase, two other enzymes of the folate cycle arm of 1CM have not been targeted clinically: serine hydroxymethyltransferase (SHMT) and methylenetetrahydrofolate dehydrogenase (MTHFD). An increasing body of literature suggests that the mitochondrial isoforms of these enzymes (SHMT2 and MTHFD2) are clinically relevant in the context of cancer. In this review, we focused on the 1CM pathway as a target for cancer therapy and, in particular, SHMT2 and MTHFD2. The function, regulation, and clinical relevance of SHMT2 and MTHFD2 are all discussed. We expand on previous clinical studies and evaluate the prognostic significance of these critical enzymes by performing a pan-cancer analysis of patient data from the The Cancer Genome Atlas and a transcriptional coexpression network enrichment analysis. We also provide an overview of preclinical and clinical inhibitors targeting the folate pathway, the methionine cycle, and folate-dependent purine biosynthesis enzymes.

The Dihydroorotate Dehydrogenase Inhibitor Brequinar Is Synergistic with ENT1/2 Inhibitors.

The dihydroorotate dehydrogenase (DHODH) inhibitor brequinar failed all clinical trials for solid tumors. To investigate mechanisms to increase brequinar's efficacy, we employed a combination strategy to simultaneously inhibit the nucleotide salvage pathways. Brequinar is synergistic with the equilibrative nucleoside transporter (ENT) inhibitor dipyridamole, but not the concentrative nucleoside transporter inhibitor phlorizin. This synergy carries over to ENT1/2 inhibition, but not ENT4. Our previously described brequinar analogue 41 was also synergistic with dipyridamole as were the FDA-approved DHODH inhibitors leflunomide and teriflunomide but the latter required much higher concentrations than brequinar. Therefore, a combination of brequinar and ENT inhibitors presents a potential anti-cancer strategy in select tumors.

Revisiting the role of dihydroorotate dehydrogenase as a therapeutic target for cancer.

Identified as a hallmark of cancer, metabolic reprogramming allows cancer cells to rapidly proliferate, resist chemotherapies, invade, metastasize, and survive a nutrient-deprived microenvironment. Rapidly growing cells depend on sufficient concentrations of nucleotides to sustain proliferation. One enzyme essential for the de novo biosynthesis of pyrimidine-based nucleotides is dihydroorotate dehydrogenase (DHODH), a known therapeutic target for multiple diseases. Brequinar, leflunomide, and teriflunomide, all of which are potent DHODH inhibitors, have been clinically evaluated but failed to receive FDA approval for the treatment of cancer. Inhibition of DHODH depletes intracellular pyrimidine nucleotide pools and results in cell cycle arrest in S-phase, sensitization to current chemotherapies, and differentiation in neural crest cells and acute myeloid leukemia (AML). Furthermore, DHODH is a synthetic lethal susceptibility in several oncogenic backgrounds. Therefore, DHODH-targeted therapy has potential value as part of a combination therapy for the treatment of cancer. In this review, we focus on the de novo pyrimidine biosynthesis pathway as a target for cancer therapy, and in particular, DHODH. In the first part, we provide a comprehensive overview of this pathway and its regulation in cancer. We further describe the relevance of DHODH as a target for cancer therapy using bioinformatic analyses. We then explore the preclinical and clinical results of pharmacological strategies to target the de novo pyrimidine biosynthesis pathway, with an emphasis on DHODH. Finally, we discuss potential strategies to harness DHODH as a target for the treatment of cancer.

Design, Synthesis, and Biological Evaluation of 4-Quinoline Carboxylic Acids as Inhibitors of Dihydroorotate Dehydrogenase.

We pursued a structure-guided approach toward the development of improved dihydroorotate dehydrogenase (DHODH) inhibitors with the goal of forming new interactions between DHODH and the brequinar class of inhibitors. Two potential residues, T63 and Y356, suitable for novel H-bonding interactions, were identified in the brequinar-binding pocket. Analogues were designed to maintain the essential pharmacophore and form new electrostatic interactions through strategically positioned H-bond accepting groups. This effort led to the discovery of potent quinoline-based analogues 41 (DHODH IC50 = 9.71 ± 1.4 nM) and 43 (DHODH IC50 = 26.2 ± 1.8 nM). A cocrystal structure between 43 and DHODH depicts a novel water mediated H-bond interaction with T63. Additional optimization led to the 1,7-naphthyridine 46 (DHODH IC50 = 28.3 ± 3.3 nM) that forms a novel H-bond with Y356. Importantly, compound 41 possesses significant oral bioavailability ( F = 56%) and an elimination t1/2 = 2.78 h (PO dosing). In conclusion, the data supports further preclinical studies of our lead compounds toward selection of a candidate for early-stage clinical development.

Design, Synthesis, and Characterization of Brequinar Conjugates as Probes to Study DHODH Inhibition.

Brequinar, a potent dihydroorotate dehydrogenase (DHODH) inhibitor, has been evaluated in multiple clinical trials as a potential treatment for cancer. To further understand brequinar-based DHODH inhibition and DHODH's therapeutic relevance in cancer, we have developed novel brequinar-based probes. We disclose a 16-step convergent synthesis of the first brequinar-PROTAC and a four-step approach towards the first mitochondrial-directed brequinar probe. A PROTAC and mitochondria-directed probe of brequinar both possess cytotoxicity that is superior to brequinar in a colony formation assay.