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.

Membrane permeable lipophilic cations as mitochondrial directing groups.

The mitochondrion's negatively charged membrane potential has been well documented to drive the accumulation of membrane permeable delocalized lipophilic cations (DLC). DLC attachments to known bioactive compounds can direct organelle localization and improve drug exposure to targets within the mitochondria. Due to the mitochondria's essential function and its regulation of cell death, DLC targeted therapies are the focus of drug discovery projects altering cellular fate via mitochondrial targets. This review provides an update on recent developments for the two main applications of DLCs: cytoprotective therapies aimed at reducing oxidative stress and cytotoxic therapies aimed at initiating cell death for the treatment of various cancers. Both approaches have produced significant improvements using DLC conjugated compounds that include improved potency, pharmacokinetic properties, and the potential to overcome resistance mechanisms.