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  • In this review we present


    In this review, we present compelling evidence in support of DHODH as an essential enzyme for the survival of cancer cells. DHODH, and its relationship to de novo pyrimidine metabolism, will be discussed along with factors that influence its regulation and expression. We will show evidence of DHODH's potential clinical relevance and co-expression network using data from The Cancer Genome Atlas (TCGA) and additional published datasets in the context of glioma. Additionally, results using previous DHODH inhibitors in cancer clinical trials and potential improvements for DHODH-targeted therapy will be discussed.
    DHODH is a vital enzyme in the de novo pyrimidine biosynthesis pathway. Through this pathway, cancer phosphodiesterase inhibitors generate the required substrates for continual DNA replication and protein synthesis without the limitations of nucleotide salvage pathways (discussed below). Pharmacological inhibition of this pathway provides a selective approach to targeting cells undergoing rapid growth. When cells are not preparing for growth, nucleotide demand is primarily maintained via nucleotide salvage pathways (Evans & Guy, 2004; Fairbanks, Bofill, Ruckemann, & Simmonds, 1995). Alternatively, cells preparing for proliferation depend on de novo nucleotide biosynthesis to fuel nucleotide demands. As a result, enzymes that are a part of the de novo nucleotide biosynthesis pathways are frequently overexpressed in cancer to sustain growth and are attractive targets to suppress cancer cell proliferation (Weber, 2001). In the de novo pyrimidine biosynthetic pathway, DHODH catalyzes a committed step and thus presents as a desirable target for halting pathway flux. Overall, the de novo pyrimidine pathway generates UMP from glutamine (Fig. 1). Flux through this pathway begins with a large enzymatic complex that catalyzes the first three steps. This complex, known as CAD (an acronym for its domains), is made up of carbamoyl phosphate synthetase, aspartate carbamoyltransferase, and dihydroorotase. The carbamoyl phosphate synthetase domain catalyzes the first reaction and generates carbamoyl phosphate from bicarbonate, ATP, and glutamine or ammonia (Fig. 1, step 1) (Evans & Guy, 2004). The second step is catalyzed by the aspartate carbamoyltransferase domain, converting carbamoyl phosphate into carbamoyl aspartate (Fig. 1, step 2) (Evans & Guy, 2004). The dihydroorotase domain hydrolyzes carbamoyl aspartate into dihydroorotate and generates the substrate for DHODH (Fig. 1, step 3). DHODH oxidizes dihydroorotate into orotate (Fig. 1, step 4) and is the only pathway enzyme located in the mitochondria. The final two steps in the pathway are catalyzed by another large enzyme complex known as uridine monophosphate synthetase. This enzyme is comprised of two domains: orotate phosphoribosyltransferase and orotidine 5′-monophosphate decarboxylase (OMP decarboxylase). The orotate phosphoribosyltransferase domain catalyzes the transfer of a phosphoribosyl group to orotate (Fig. 1, step 5). A final decarboxylation by the OMP decarboxylase domain generates the uridine monophosphate nucleotide (Fig. 1, step 6). Two enzymes, carbamoyl phosphate synthetase and DHODH that catalyze committed steps, primarily control flux through this pathway (Baumgartner et al., 2006; Evans & Guy, 2004; Lane & Fan, 2015). Inhibition of either enzyme halts flux through the de novo pyrimidine pathway, but carbamoyl phosphate synthetase is not expressed in all cancer cells. In fact, carbamoyl phosphate synthetase has been observed to have low expression or is completely downregulated in most liver carcinomas (Liu, Dong, Robertson, & Liu, 2011; Siddiqui, Saboorian, Gokaslan, & Ashfaq, 2002). Therefore, inhibition of DHODH's catalytic activity presents as a more pharmacologically relevant approach to treat cancer (Reis et al., 2017). DHODH catalyzes two redox reactions: the oxidation of dihydroorotate to orotate and subsequent flavin mononucleotide (FMN) regeneration (Fig. 2). The oxidation of dihydroorotate is carried out via a stepwise mechanism with highly conserved residues. This first enzymatic reaction follows a deprotonation of dihydroorotate at the C5 position and a subsequent hydride transfer (from dihydororotate's C6 position) to the FMN cofactor (Fig. 2) (Reis et al., 2017). The catalytic base for the initial C5 deprotonation is likely S215 (Fig. 3), which has been reported as the catalytic base for DHODH in other organisms (Bjornberg, Gruner, Roepstorff, & Jensen, 1999; Liu, Neidhardt, Grossman, Ocain, & Clardy, 2000; Reis et al., 2017). Adjacent residues T218 and F149 are highly conserved as well and may increase the basicity of S215 (Fig. 3C) (Liu et al., 2000). However, their precise roles have not been determined. Additional non-catalytic conserved residues contributing to dihydroorotate oxidation are N212, S214, P216, L221, R222, and Q225. These residues are located on a loop region that may be responsible for substrate/product exchange (Liu et al., 2000). The second redox reaction, resulting in regeneration of FMN, requires ubiquinone from the mitochondrial electron transport chain (ETC) (Fig. 2) (Reis et al., 2017). FMN is thought to bind with DHODH's G119 and V282, however the catalytic mechanism of FMN regeneration from ubiquinone is not well understood (Liu et al., 2000). FMNH2 is perceived to undergo multiple single-electron transfers but the exact residues that facilitate this oxidation are not known (Liu et al., 2000; Palfey, Bjornberg, & Jensen, 2001; Reis et al., 2017). Nonetheless, FMN regeneration is necessary for continued DHODH catalysis. Two of the most well-known DHODH inhibitors, leflunomide and brequinar (discussed below), are proposed to act as competitive inhibitors of ubiquinone (Liu et al., 2000). The necessity to regenerate FMN via ubiquinone may correspond to DHODH localization within the inner mitochondrial membrane. This localization increases exposure to ubiquinone from the mitochondrial ETC.