Urea Cycle Dysregulation Generates Clinically Relevant Genomic and Biochemical Signatures.

The urea cycle (UC) is the main pathway by which mammals dispose of waste nitrogen. We find that specific alterations in the expression of most UC enzymes occur in many tumors, leading to a general metabolic hallmark termed "UC dysregulation" (UCD). UCD elicits nitrogen diversion toward carbamoyl-phosphate synthetase2, aspartate transcarbamylase, and dihydrooratase (CAD) activation and enhances pyrimidine synthesis, resulting in detectable changes in nitrogen metabolites in both patient tumors and their bio-fluids. The accompanying excess of pyrimidine versus purine nucleotides results in a genomic signature consisting of transversion mutations at the DNA, RNA, and protein levels. This mutational bias is associated with increased numbers of hydrophobic tumor antigens and a better response to immune checkpoint inhibitors independent of mutational load. Taken together, our findings demonstrate that UCD is a common feature of tumors that profoundly affects carcinogenesis, mutagenesis, and immunotherapy response.

Physiologic Medium Rewires Cellular Metabolism and Reveals Uric Acid as an Endogenous Inhibitor of UMP Synthase.

A complex interplay of environmental factors impacts the metabolism of human cells, but neither traditional culture media nor mouse plasma mimic the metabolite composition of human plasma. Here, we developed a culture medium with polar metabolite concentrations comparable to those of human plasma (human plasma-like medium [HPLM]). Culture in HPLM, relative to that in traditional media, had widespread effects on cellular metabolism, including on the metabolome, redox state, and glucose utilization. Among the most prominent was an inhibition of de novo pyrimidine synthesis-an effect traced to uric acid, which is 10-fold higher in the blood of humans than of mice and other non-primates. We find that uric acid directly inhibits uridine monophosphate synthase (UMPS) and consequently reduces the sensitivity of cancer cells to the chemotherapeutic agent 5-fluorouracil. Thus, media that better recapitulates the composition of human plasma reveals unforeseen metabolic wiring and regulation, suggesting that HPLM should be of broad utility.

ppGpp Coordinates Nucleotide and Amino-Acid Synthesis in E. coli During Starvation.

(p)ppGpp is a nucleotide messenger universally produced in bacteria following nutrient starvation. In E. coli, ppGpp inhibits purine nucleotide synthesis by targeting several different enzymes, but the physiological significance of their inhibition is unknown. Here, we report the structural basis of inhibition for one target, Gsk, the inosine-guanosine kinase. Gsk creates an unprecedented, allosteric binding pocket for ppGpp by restructuring terminal sequences, which restrains conformational dynamics necessary for catalysis. Guided by this structure, we generated a chromosomal mutation that abolishes Gsk regulation by ppGpp. This mutant strain accumulates abnormally high levels of purine nucleotides following amino-acid starvation, compromising cellular fitness. We demonstrate that this unrestricted increase in purine nucleotides is detrimental because it severely depletes pRpp and essential, pRpp-derived metabolites, including UTP, histidine, and tryptophan. Thus, our results reveal the significance of ppGpp's regulation of purine nucleotide synthesis and a critical mechanism by which E. coli coordinates biosynthetic processes during starvation.

Oncogenic ß-catenin stimulation of AKT2-CAD-mediated pyrimidine synthesis is targetable vulnerability in liver cancer.

CTNNB1, encoding ß-catenin protein, is the most frequently altered proto-oncogene in hepatic neoplasms. In this study, we studied the significance and pathological mechanism of CTNNB1 gain-of-function mutations in hepatocarcinogenesis. Activated ß-catenin not only triggered hepatic tumorigenesis but also exacerbated Tp53 deletion or hepatitis B virus infection-mediated liver cancer development in mouse models. Using untargeted metabolomic profiling, we identified boosted de novo pyrimidine synthesis as the major metabolic aberration in ß-catenin mutant cell lines and livers. Oncogenic ß-catenin transcriptionally stimulated AKT2, which then phosphorylated the rate-limiting de novo pyrimidine synthesis enzyme CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotase) on S1406 and S1859 to potentiate nucleotide synthesis. Moreover, inhibition of ß-catenin/AKT2-stimulated pyrimidine synthesis axis preferentially repressed ß-catenin mutant cell proliferation and tumor formation. Therefore, ß-catenin active mutations are oncogenic in various preclinical liver cancer models. Stimulation of ß-catenin/AKT2/CAD signaling cascade on pyrimidine synthesis is an essential and druggable vulnerability for ß-catenin mutant liver cancer.

SARS-CoV-2 couples evasion of inflammatory response to activated nucleotide synthesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolves rapidly under the pressure of host immunity, as evidenced by waves of emerging variants despite effective vaccinations, highlighting the need for complementing antivirals. We report that targeting a pyrimidine synthesis enzyme restores inflammatory response and depletes the nucleotide pool to impede SARS-CoV-2 infection. SARS-CoV-2 deploys Nsp9 to activate carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase (CAD) that catalyzes the rate-limiting steps of the de novo pyrimidine synthesis. Activated CAD not only fuels de novo nucleotide synthesis but also deamidates RelA. While RelA deamidation shuts down NF-κB activation and subsequent inflammatory response, it up-regulates key glycolytic enzymes to promote aerobic glycolysis that provides metabolites for de novo nucleotide synthesis. A newly synthesized small-molecule inhibitor of CAD restores antiviral inflammatory response and depletes the pyrimidine pool, thus effectively impeding SARS-CoV-2 replication. Targeting an essential cellular metabolic enzyme thus offers an antiviral strategy that would be more refractory to SARS-CoV-2 genetic changes.

Human uridine 5'-monophosphate synthase stores metabolic potential in inactive biomolecular condensates.

Human uridine 5'-monophosphate synthase (HsUMPS) is a bifunctional enzyme that catalyzes the final two steps in de novo pyrimidine biosynthesis. The individual orotate phosphoribosyl transferase and orotidine monophosphate domains have been well characterized, but little is known about the overall structure of the protein and how the organization of domains impacts function. Using a combination of chromatography, electron microscopy, and complementary biophysical methods, we report herein that HsUMPS can be observed in two structurally distinct states, an enzymatically active dimeric form and a nonactive multimeric form. These two states readily interconvert to reach an equilibrium that is sensitive to perturbations of the active site and the presence of substrate. We determined that the smaller molecular weight form of HsUMPS is an S-shaped dimer that can self-assemble into relatively well-ordered globular condensates. Our analysis suggests that the transition between dimer and multimer is driven primarily by oligomerization of the orotate phosphoribosyl transferase domain. While the cellular distribution of HsUMPS is unaffected, quantification by mass spectrometry revealed that de novo pyrimidine biosynthesis is dysregulated when this protein is unable to assemble into inactive condensates. Taken together, our data suggest that HsUMPS self-assembles into biomolecular condensates as a means to store metabolic potential for the regulation of metabolic rates.

Cyclin D1 extensively reprograms metabolism to support biosynthetic pathways in hepatocytes.

Cell proliferation requires metabolic reprogramming to accommodate biosynthesis of new cell components, and similar alterations occur in cancer cells. However, the mechanisms linking the cell cycle machinery to metabolism are not well defined. Cyclin D1, along with its main partner cyclin-dependent kinase 4 (Cdk4), is a pivotal cell cycle regulator and driver oncogene that is overexpressed in many cancers. Here, we examine hepatocyte proliferation to define novel effects of cyclin D1 on biosynthetic metabolism. Metabolomic studies reveal that cyclin D1 broadly promotes biosynthetic pathways including glycolysis, the pentose phosphate pathway, and the purine and pyrimidine nucleotide synthesis in hepatocytes. Proteomic analyses demonstrate that overexpressed cyclin D1 binds to numerous metabolic enzymes including those involved in glycolysis and pyrimidine synthesis. In the glycolysis pathway, cyclin D1 activates aldolase and GAPDH, and these proteins are phosphorylated by cyclin D1/Cdk4 in vitro. De novo pyrimidine synthesis is particularly dependent on cyclin D1. Cyclin D1/Cdk4 phosphorylates the initial enzyme of this pathway, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and metabolomic analysis indicates that cyclin D1 depletion markedly reduces the activity of this enzyme. Pharmacologic inhibition of Cdk4 along with the downstream pyrimidine synthesis enzyme dihydroorotate dehydrogenase synergistically inhibits proliferation and survival of hepatocellular carcinoma cells. These studies demonstrate that cyclin D1 promotes a broad network of biosynthetic pathways in hepatocytes, and this model may provide insights into potential metabolic vulnerabilities in cancer cells.

SGLT2 inhibition leads to a restoration of hepatic and circulating metabolites involved in the folate cycle and pyrimidine biosynthesis.

Inhibition of sodium-glucose cotransporter 2 (SGLT2) by empagliflozin (EMPA) and other "flozins" can improve glycemic control under conditions of diabetes and kidney disease. Though they act on the kidney, they also offer cardiovascular and liver protection. Previously, we found that EMPA decreased circulating triglycerides and hepatic lipid and cholesterol esters in male TallyHo mice fed a high-milk-fat diet (HMFD). The goal of this study was to determine whether the liver protection is associated with a change in metabolic function by characterizing the hepatic and circulating metabolic and lipidomic profiles using targeted LC-MS. In both male and female mice, HMFD feeding significantly altered the circulating and hepatic metabolome compared with low-fat diet (LFD). Addition of EMPA resulted in the restoration of circulating orotate (intermediate in pyrimidine biosynthesis) and hepatic dihydrofolate (intermediate in the folate and methionine cycles) levels in males and acylcarnitines in females. These changes were partially explained by altered expression of rate-limiting enzymes in these pathways. This metabolic signature was not detected when EMPA was incorporated into an LFD, suggesting that the restoration requires the metabolic shift that accompanies the HMFD. Notably, the HMFD increased expression of 18 of 20 circulating amino acids in males and 11 of 20 in females, and this pattern was reversed by EMPA. Finally, we confirmed that SGLT2 inhibition upregulates ketone bodies including ß-hydroxybutyrate. Collectively, this study highlights the metabolic changes that occur with EMPA treatment, and sheds light on the possible mechanisms by which this drug offers liver and systemic protection.NEW & NOTEWORTHY Sodium-glucose cotransporter 2 (SGLT2) inhibitors, including empagliflozin, have emerged as a new treatment option for individuals with type 2 diabetes that have positive impacts on kidney and cardiovascular disease. However, less is known about their impact on other tissues, including the liver. Here, we report that empagliflozin reduces hepatic steatosis that is associated with restoring metabolic intermediates in the folate and pyrimidine biosynthesis pathways. These changes may lead to new approaches to treat nonalcoholic fatty liver disease.

TOR coordinates nucleotide availability with ribosome biogenesis in plants.

TARGET OF RAPAMYCIN (TOR) is a conserved eukaryotic Ser/Thr protein kinase that coordinates growth and metabolism with nutrient availability. We conducted a medium-throughput functional genetic screen to discover essential genes that promote TOR activity in plants, and identified a critical regulatory enzyme, cytosolic phosphoribosyl pyrophosphate (PRPP) synthetase (PRS4). PRS4 synthesizes cytosolic PRPP, a key upstream metabolite in nucleotide synthesis and salvage pathways. We found that prs4 knockouts are embryo-lethal in Arabidopsis thaliana, and that silencing PRS4 expression in Nicotiana benthamiana causes pleiotropic developmental phenotypes, including dwarfism, aberrant leaf shape, and delayed flowering. Transcriptomic analysis revealed that ribosome biogenesis is among the most strongly repressed processes in prs4 knockdowns. Building on these results, we discovered that TOR activity is inhibited by chemical or genetic disruption of nucleotide biosynthesis, but that this effect can be reversed by supplying plants with nucleobases. Finally, we show that TOR transcriptionally promotes nucleotide biosynthesis to support the demands of ribosomal RNA synthesis. We propose that TOR coordinates ribosome biogenesis with nucleotide availability in plants to maintain metabolic homeostasis and support growth.

Biocatalytic Synthesis of Ruxolitinib Intermediate via Engineered Imine Reductase.

An enzyme catalyzed strategy for the synthesis of a chiral hydrazine from 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 is presented. An imine reductase (IRED) from Streptosporangium roseum was identified to catalyze the reaction between 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 to produce trace amounts of (R)-3-cyclopentyl-3-hydrazineylpropanenitrile 4. We employed a 2-fold approach to optimize the catalytic performance of this enzyme. First, a transition state analogue (TSA) model was constructed to illuminate the enzyme-substrate interactions. Subsequently, the Enzyme_design and Funclib methods were utilized to predict mutants for experimental evaluation. Through three rounds of site-directed mutagenesis, site saturation mutagenesis, and combinatorial mutagenesis, we obtained mutant M6 with a yield of 98% and an enantiomeric excess (ee) of 99%. This study presents an effective method for constructing a hydrazine derivative via IRED-catalyzed reductive amination of ketone and hydrazine. Furthermore, it provides a general approach for constructing suitable enzymes, starting from nonreactive enzymes and gradually enhancing their catalytic activity through active site modifications.

Miro proteins connect mitochondrial function and intercellular transport.

Mitochondria are organelles present in most eukaryotic cells, where they play major and multifaceted roles. The classical notion of the main mitochondrial function as the powerhouse of the cell per se has been complemented by recent discoveries pointing to mitochondria as organelles affecting a number of other auxiliary processes. They go beyond the classical energy provision via acting as a relay point of many catabolic and anabolic processes, to signaling pathways critically affecting cell growth by their implication in de novo pyrimidine synthesis. These additional roles further underscore the importance of mitochondrial homeostasis in various tissues, where its deregulation promotes a number of pathologies. While it has long been known that mitochondria can move within a cell to sites where they are needed, recent research has uncovered that mitochondria can also move between cells. While this intriguing field of research is only emerging, it is clear that mobilization of mitochondria requires a complex apparatus that critically involves mitochondrial proteins of the Miro family, whose role goes beyond the mitochondrial transfer, as will be covered in this review.

Inhibition of the de novo pyrimidine biosynthesis pathway limits ribosomal RNA transcription causing nucleolar stress in glioblastoma cells.

Glioblastoma is the most common and aggressive type of cancer in the brain; its poor prognosis is often marked by reoccurrence due to resistance to the chemotherapeutic agent temozolomide, which is triggered by an increase in the expression of DNA repair enzymes such as MGMT. The poor prognosis and limited therapeutic options led to studies targeted at understanding specific vulnerabilities of glioblastoma cells. Metabolic adaptations leading to increased synthesis of nucleotides by de novo biosynthesis pathways are emerging as key alterations driving glioblastoma growth. In this study, we show that enzymes necessary for the de novo biosynthesis of pyrimidines, DHODH and UMPS, are elevated in high grade gliomas and in glioblastoma cell lines. We demonstrate that DHODH's activity is necessary to maintain ribosomal DNA transcription (rDNA). Pharmacological inhibition of DHODH with the specific inhibitors brequinar or ML390 effectively depleted the pool of pyrimidines in glioblastoma cells grown in vitro and in vivo and impaired rDNA transcription, leading to nucleolar stress. Nucleolar stress was visualized by the aberrant redistribution of the transcription factor UBF and the nucleolar organizer nucleophosmin 1 (NPM1), as well as the stabilization of the transcription factor p53. Moreover, DHODH inhibition decreased the proliferation of glioblastoma cells, including temozolomide-resistant cells. Importantly, the addition of exogenous uridine, which reconstitutes the cellular pool of pyrimidine by the salvage pathway, to the culture media recovered the impaired rDNA transcription, nucleolar morphology, p53 levels, and proliferation of glioblastoma cells caused by the DHODH inhibitors. Our in vivo data indicate that while inhibition of DHODH caused a dramatic reduction in pyrimidines in tumor cells, it did not affect the overall pyrimidine levels in normal brain and liver tissues, suggesting that pyrimidine production by the salvage pathway may play an important role in maintaining these nucleotides in normal cells. Our study demonstrates that glioblastoma cells heavily rely on the de novo pyrimidine biosynthesis pathway to generate ribosomal RNA (rRNA) and thus, we identified an approach to inhibit ribosome production and consequently the proliferation of glioblastoma cells through the specific inhibition of the de novo pyrimidine biosynthesis pathway.

Functional characterization of the HMP-P synthase of Legionella pneumophila (Lpg1565).

The production of the pyrimidine moiety in thiamine synthesis, 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate (HMP-P), has been described to proceed through the Thi5-dependent pathway in Saccharomyces cerevisiae and other yeast. Previous work found that ScThi5 functioned poorly in a heterologous context. Here we report a bacterial ortholog to the yeast HMP-P synthase (Thi5) was necessary for HMP synthesis in Legionella pneumophila. Unlike ScThi5, LpThi5 functioned in vivo in Salmonella enterica under multiple growth conditions. The protein LpThi5 is a dimer that binds pyridoxal-5'-phosphate (PLP), apparently without a solvent-exposed Schiff base. A small percentage of LpThi5 protein co-purifies with a bound molecule that can be converted to HMP. Analysis of variant proteins both in vivo and in vitro confirmed that residues in sequence motifs conserved across bacterial and eukaryotic orthologs modulate the function of LpThi5. IMPORTANCE: Thiamine is an essential vitamin for the vast majority of organisms. There are multiple strategies to synthesize and salvage this vitamin. The predominant pathway for synthesis of the pyrimidine moiety of thiamine involves the Fe-S cluster protein ThiC. An alternative pathway utilizes Thi5, a novel enzyme that uses PLP as a substrate. The Thi5-dependent pathway is poorly characterized in yeast and has not been characterized in Bacteria. Here we demonstrate that a Thi5-dependent pathway is necessary for thiamine biosynthesis in Legionella pneumophila and provide biochemical data to extend knowledge of the Thi5 enzyme, the corresponding biosynthetic pathway, and the role of metabolic network architecture in optimizing its function.

Diversion of aspartate in ASS1-deficient tumours fosters de novo pyrimidine synthesis.

Cancer cells hijack and remodel existing metabolic pathways for their benefit. Argininosuccinate synthase (ASS1) is a urea cycle enzyme that is essential in the conversion of nitrogen from ammonia and aspartate to urea. A decrease in nitrogen flux through ASS1 in the liver causes the urea cycle disorder citrullinaemia. In contrast to the well-studied consequences of loss of ASS1 activity on ureagenesis, the purpose of its somatic silencing in multiple cancers is largely unknown. Here we show that decreased activity of ASS1 in cancers supports proliferation by facilitating pyrimidine synthesis via CAD (carbamoyl-phosphate synthase 2, aspartate transcarbamylase, and dihydroorotase complex) activation. Our studies were initiated by delineating the consequences of loss of ASS1 activity in humans with two types of citrullinaemia. We find that in citrullinaemia type I (CTLN I), which is caused by deficiency of ASS1, there is increased pyrimidine synthesis and proliferation compared with citrullinaemia type II (CTLN II), in which there is decreased substrate availability for ASS1 caused by deficiency of the aspartate transporter citrin. Building on these results, we demonstrate that ASS1 deficiency in cancer increases cytosolic aspartate levels, which increases CAD activation by upregulating its substrate availability and by increasing its phosphorylation by S6K1 through the mammalian target of rapamycin (mTOR) pathway. Decreasing CAD activity by blocking citrin, the mTOR signalling, or pyrimidine synthesis decreases proliferation and thus may serve as a therapeutic strategy in multiple cancers where ASS1 is downregulated. Our results demonstrate that ASS1 downregulation is a novel mechanism supporting cancerous proliferation, and they provide a metabolic link between the urea cycle enzymes and pyrimidine synthesis.

Pyrimidine Biosynthetic Enzyme CAD: Its Function, Regulation, and Diagnostic Potential.

CAD (Carbamoyl-phosphate synthetase 2, Aspartate transcarbamoylase, and Dihydroorotase) is a multifunctional protein that participates in the initial three speed-limiting steps of pyrimidine nucleotide synthesis. Over the past two decades, extensive investigations have been conducted to unmask CAD as a central player for the synthesis of nucleic acids, active intermediates, and cell membranes. Meanwhile, the important role of CAD in various physiopathological processes has also been emphasized. Deregulation of CAD-related pathways or CAD mutations cause cancer, neurological disorders, and inherited metabolic diseases. Here, we review the structure, function, and regulation of CAD in mammalian physiology as well as human diseases, and provide insights into the potential to target CAD in future clinical applications.

Plumbagin, a Natural Product with Potent Anticancer Activities, Binds to and Inhibits Dihydroorotase, a Key Enzyme in Pyrimidine Biosynthesis.

Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway for pyrimidine nucleotides, and an attractive target for potential anticancer chemotherapy. By screening plant extracts and performing GC-MS analysis, we identified and characterized that the potent anticancer drug plumbagin (PLU), isolated from the carnivorous plant Nepenthes miranda, was a competitive inhibitor of DHOase. We also solved the complexed crystal structure of yeast DHOase with PLU (PDB entry 7CA1), to determine the binding interactions and investigate the binding modes. Mutational and structural analyses indicated the binding of PLU to DHOase through loop-in mode, and this dynamic loop may serve as a drug target. PLU exhibited cytotoxicity on the survival, migration, and proliferation of 4T1 cells and induced apoptosis. These results provide structural insights that may facilitate the development of new inhibitors targeting DHOase, for further clinical anticancer chemotherapies.

Combined small molecule and loss-of-function screen uncovers estrogen receptor alpha and CAD as host factors for HDV infection and antiviral targets.

OBJECTIVE: Hepatitis D virus (HDV) is a circular RNA virus coinfecting hepatocytes with hepatitis B virus. Chronic hepatitis D results in severe liver disease and an increased risk of liver cancer. Efficient therapeutic approaches against HDV are absent. DESIGN: Here, we combined an RNAi loss-of-function and small molecule screen to uncover host-dependency factors for HDV infection. RESULTS: Functional screening unravelled the hypoxia-inducible factor (HIF)-signalling and insulin-resistance pathways, RNA polymerase II, glycosaminoglycan biosynthesis and the pyrimidine metabolism as virus-hepatocyte dependency networks. Validation studies in primary human hepatocytes identified the carbamoyl-phosphatesynthetase 2, aspartate transcarbamylase and dihydroorotase (CAD) enzyme and estrogen receptor alpha (encoded by ESR1) as key host factors for HDV life cycle. Mechanistic studies revealed that the two host factors are required for viral replication. Inhibition studies using N-(phosphonoacetyl)-L-aspartic acid and fulvestrant, specific CAD and ESR1 inhibitors, respectively, uncovered their impact as antiviral targets. CONCLUSION: The discovery of HDV host-dependency factors elucidates the pathogenesis of viral disease biology and opens therapeutic strategies for HDV cure.

Atovaquone Inhibits Arbovirus Replication through the Depletion of Intracellular Nucleotides.

Arthropod-borne viruses represent a significant public health threat worldwide, yet there are few antiviral therapies or prophylaxes targeting these pathogens. In particular, the development of novel antivirals for high-risk populations such as pregnant women is essential to prevent devastating disease such as that which was experienced with the recent outbreak of Zika virus (ZIKV) in the Americas. One potential avenue to identify new and pregnancy-acceptable antiviral compounds is to repurpose well-known and widely used FDA-approved drugs. In this study, we addressed the antiviral role of atovaquone, an FDA Pregnancy Category C drug and pyrimidine biosynthesis inhibitor used for the prevention and treatment of parasitic infections. We found that atovaquone was able to inhibit ZIKV and chikungunya virus virion production in human cells and that this antiviral effect occurred early during infection at the initial steps of viral RNA replication. Moreover, we were able to complement viral replication and virion production with the addition of exogenous pyrimidine nucleosides, indicating that atovaquone functions through the inhibition of the pyrimidine biosynthesis pathway to inhibit viral replication. Finally, using an ex vivo human placental tissue model, we found that atovaquone could limit ZIKV infection in a dose-dependent manner, providing evidence that atovaquone may function as an antiviral in humans. Taken together, these studies suggest that atovaquone could be a broad-spectrum antiviral drug and a potential attractive candidate for the prophylaxis or treatment of arbovirus infection in vulnerable populations, such as pregnant women and children.IMPORTANCE The ability to protect vulnerable populations such as pregnant women and children from Zika virus and other arbovirus infections is essential to preventing the devastating complications induced by these viruses. One class of antiviral therapies may lie in known pregnancy-acceptable drugs that have the potential to mitigate arbovirus infections and disease, yet this has not been explored in detail. In this study, we show that the common antiparasitic drug atovaquone inhibits arbovirus replication through intracellular nucleotide depletion and can impair ZIKV infection in an ex vivo human placental explant model. Our study provides a novel function for atovaquone and highlights that the rediscovery of pregnancy-acceptable drugs with potential antiviral effects can be the key to better addressing the immediate need for treating viral infections and preventing potential birth complications and future disease.

CAD, A Multienzymatic Protein at the Head of de Novo Pyrimidine Biosynthesis.

CAD is a 1.5 MDa particle formed by hexameric association of a 250 kDa protein that carries the enzymatic activities for the first three steps in the de novo biosynthesis of pyrimidine nucleotides: glutamine-dependent Carbamoyl phosphate synthetase, Aspartate transcarbamoylase and Dihydroorotase. This metabolic pathway is essential for cell growth and proliferation and is conserved in all living organisms. However, the fusion of the first three enzymatic activities of the pathway into a single multienzymatic protein only occurs in animals. In prokaryotes, by contrast, these activities are encoded as distinct monofunctional enzymes that function independently or by forming more or less transient complexes. Whereas the structural information about these enzymes in bacteria is abundant, the large size and instability of CAD has only allowed a fragmented characterization of its structure. Here we retrace some of the most significant efforts to decipher the architecture of CAD and to understand its catalytic and regulatory mechanisms.

Global metabolic reprogramming of colorectal cancer occurs at adenoma stage and is induced by MYC.

Cancer cells alter their metabolism for the production of precursors of macromolecules. However, the control mechanisms underlying this reprogramming are poorly understood. Here we show that metabolic reprogramming of colorectal cancer is caused chiefly by aberrant MYC expression. Multiomics-based analyses of paired normal and tumor tissues from 275 patients with colorectal cancer revealed that metabolic alterations occur at the adenoma stage of carcinogenesis, in a manner not associated with specific gene mutations involved in colorectal carcinogenesis. MYC expression induced at least 215 metabolic reactions by changing the expression levels of 121 metabolic genes and 39 transporter genes. Further, MYC negatively regulated the expression of genes involved in mitochondrial biogenesis and maintenance but positively regulated genes involved in DNA and histone methylation. Knockdown of MYC in colorectal cancer cells reset the altered metabolism and suppressed cell growth. Moreover, inhibition of MYC target pyrimidine synthesis genes such as CAD, UMPS, and CTPS blocked cell growth, and thus are potential targets for colorectal cancer therapy.