Inhibition Of Pyrimidine Biosynthesis Research Articles

Inhibitors of Nucleotide Biosynthesis as Candidates for a Wide Spectrum of Antiviral Chemotherapy.

Emerging and re-emerging viruses have been a challenge in public health in recent decades. Host-targeted antivirals (HTA) directed at cellular molecules or pathways involved in virus multiplication represent an interesting strategy to combat viruses presently lacking effective chemotherapy. HTA could provide a wide range of agents with inhibitory activity against current and future viruses that share similar host requirements and reduce the possible selection of antiviral-resistant variants. Nucleotide metabolism is one of the more exploited host metabolic pathways as a potential antiviral target for several human viruses. This review focuses on the antiviral properties of the inhibitors of pyrimidine and purine nucleotide biosynthesis, with an emphasis on the rate-limiting enzymes dihydroorotate dehydrogenase (DHODH) and inosine monophosphate dehydrogenase (IMPDH) for which there are old and new drugs active against a broad spectrum of pathogenic viruses.

Abstract 4037: The effects of n-acetyltransferase 1 gene knockout on the cytotoxicity of pyrimidine biosynthesis inhibitors in human breast cancer cells

Abstract Arylamine N-acetyltransferase 1 (NAT1), a polymorphic drug metabolism enzyme, is often upregulated in breast cancer cells. To develop novel hypotheses on how NAT1 contributes to breast cancer development and progression, NAT1 knockout (KO) cell lines (KO2 and KO5) were created from MDA-MB-231 using CRISPR/Cas9 technology. According to our proteomics and RNA sequencing analyses, NAT1 KO cells have increased cytidine deaminase (CDA), a player in the pyrimidine salvage pathway. Metabolomics data showed NAT1 KO cells had defects in the de novo pyrimidine pathway, which can explain the upregulation of the salvage pathway. Pyrimidine de novo synthesis and salvage pathways are essential for DNA and RNA synthesis and cell growth.We hypothesize that NAT1 plays a key role in maintaining pyrimidine biosynthesis in breast cancer cells, and hence, exhibit increased sensitivity to some of chemotherapeutic drugs that depends on pyrimidine metabolic pathways for their cytotoxicity. Parental, KO2, and KO5 MDA-MB-231 breast cancer cell lines were treated with several different classes of drugs: CDA inhibitors: Tetrahydrouridine and zebularine; pyrimidine biosynthesis inhibitors teriflunomide and leflunomide; DNA damaging agent doxorubicin; and cytidine analogues 5-hydroxymethyl-2’-deoxycytidine, 5’-formyl-2’-deoxycytidine, and 5’-deoxy-5-fluoropyridine at varying concentrations for 4 days. Cell viability was assessed by Alamarblue assay. Differences between cell lines were assessed for statistical significance by two-way ANOVA. The results were as follows Tetrahydrouridine: No statistically significant difference in cell viability between parental and NAT1 KOs except at 200 µM p= 0.0002Zebularine: Statistically significant difference between parental and NAT1 KOs at concentrations above 60 µM at p < 0.0001Teriflunomide: Statistically significant difference between parental and NAT1 KOs between 20 and 60 µM at p < 0.0001Leflunomide: No statistically significant difference between parental and NAT1 KOs except at 200 µM at p = 0.00065’- Formyl- 2'- Deoxycytidine: Statistically significant difference between parental and NAT1 KOs above 12 µM at p = 0.0042Doxorubicin: No statistically significant difference between parental and NAT1 KOs 5-Hydroxymethyl-2’-deoxycytidine: No statistically significant difference between parental and NAT15’-Deoxy-5-fluorocytidine: No statistically significant difference between parental and NAT1 KOs Whereas the results with many of the drugs were inconsistent with our initial hypothesis, it is possible that the NAT1 KO cells are more resistant to CDA or pyrimidine biosynthesis inhibitors because of the upregulation of CDA. Understanding the pharmacogenomic similarities in Tetrahydrouridine, zebularine and teriflunomide’s may help develop a new hypothesis. Citation Format: Afi Henriette Tagnedji, Kyung U. Hong, David W. Hein. The effects of n-acetyltransferase 1 gene knockout on the cytotoxicity of pyrimidine biosynthesis inhibitors in human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4037.

Abstract P5-05-01: Metabolite profiling and RNA-seq identifies novel metabolomic-genomic biomarker and therapeutic options for rapidly proliferating breast cancers

Abstract Metabolic dysregulation is one of the distinctive features of breast cancer, yet remains under-characterized in different subtypes of breast cancer. In this study, we performed full metabolome profiling and RNA-seq gene expression analyses on patient samples comprised of Triple Negative Breast Cancer (TNBC) and Estrogen Receptor (ER) positive breast cancers, as well as TNBC patient-derived xenografts (PDX). We identified two major metabolic groups using hierarchical clustering analysis of global metabolite levels: a Nucleotide/Carbohydrate-Enriched group and a Lipid/Fatty Acid-Enriched group. The Nucleotide/Carbohydrate-Enriched group is populated by the majority of TNBCs and all TNBC PDX samples, and shows high levels of energy consumption and nucleotide biosynthesis related metabolites, while the Lipid/Fatty Acid-Enriched group contained all ER+ cancers (and normal breast tissues) and showed high levels of lipids and fatty acids. Using these two metabolite-defined groups, we compared metabolic signatures to RNA-seq expression data and identified gene expression signatures that correlated with each metabolic group. This novel integrated signature identified classic proliferation-associated genes as highly expressed in the Nucleotide/Carbohydrate-Enriched metabolomics group. We next sought to therapeutically target the Nucleotide/Carbohydrate-Enriched group through targeting of nucleotide metabolism using a pyrimidine biosynthesis inhibitor (DHODH inhibitor, Brequinar), and/or a glutaminase inhibitor (CB-839), and observed tumor volume reductions and improved survival times using the TNBC PDX models. Notably,. when the DHODH inhibitor Brequinar was tested on four different immune-competent TNBC mouse models, it showed significant single agent activity, and in 3/4 cases had better activity than carboplatin/paclitaxel combination, and with less toxicity. Our study reveals a new molecularly targeted therapy for rapidly proliferating TNBCs, guided by a novel set of metabolic-genomic biomarkers, which might be translated quickly as DHODH inhibitors are already FDA approved for use in other diseases. Citation Format: Cherise R Glodowski, Chengheng Liao, Cheng Fan, Juan Liu, Kevin R Mott, Akash Kaushik, Hieu Vu, Jason W Locasale, Samuel K McBrayer, Ralph J DeBerardinis, Charles M Perou, Qing Zhang. Metabolite profiling and RNA-seq identifies novel metabolomic-genomic biomarker and therapeutic options for rapidly proliferating breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-05-01.

Pyrimidine inhibitors synergize with nucleoside analogues to block SARS-CoV-2.

The SARS-CoV-2 virus has infected more than 261million people and has led to more than 5million deaths in the past year and a half1 ( https://www.who.org/ ). Individuals with SARS-CoV-2 infection typically develop mild-to-severe flu-like symptoms, whereas infection of a subset of individuals leads to severe-to-fatal clinical outcomes2. Although vaccines have been rapidly developed to combat SARS-CoV-2, there has been a dearth of antiviral therapeutics. There is an urgent need for therapeutics, which has been amplified by the emerging threats of variants that may evade vaccines. Large-scale efforts are underway to identify antiviral drugs. Here we screened approximately 18,000 drugs for antiviral activity using live virus infection in human respiratory cells and validated 122 drugs with antiviral activity and selectivity against SARS-CoV-2. Among these candidates are 16 nucleoside analogues, the largest category of clinically used antivirals. This included the antivirals remdesivir and molnupiravir, which have been approved for use in COVID-19. RNA viruses rely on a high supply of nucleoside triphosphates from the host to efficiently replicate, and we identified a panel of host nucleoside biosynthesis inhibitors as antiviral. Moreover, we found that combining pyrimidine biosynthesis inhibitors with antiviral nucleoside analogues synergistically inhibits SARS-CoV-2 infection in vitro and in vivo against emerging strains of SARS-CoV-2, suggesting a clinical path forward.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Integrated Metabolic Profiling and Transcriptional Analysis Reveals Therapeutic Modalities for Targeting Rapidly Proliferating Breast Cancers.

A multiomics strategy that integrates metabolic and gene expression profiling in patient tumor samples and animal models identifies effective pharmacologic approaches to target rapidly proliferating breast tumor subtypes.

Inhibition of pyrimidine biosynthesis by strobilurin derivatives induces differentiation of acute myeloid leukemia cells

All-trans retinoic acid–based differentiation therapies have succeeded in the treatment of acute promyelocytic leukemia, which is a rare subtype of acute myeloid leukemia (AML). Their clinical efficacy is negligible, however, for other subtypes of AML. Here, we showed that strobilurin derivatives, a well-established class of inhibitors of mitochondrial electron transport chain (ETC) complex III, possessed differentiation-inducing activity in AML cells. Impairment of mitochondrial ETC activity was involved in the differentiation effects of strobilurin derivatives, where reactive oxygen species generation appeared unnecessary. Conversely, strobilurin derivative-mediated differentiation was triggered by pyrimidine deficiency, which resulted from the inhibition of the mitochondrial-coupled dihydroorotate dehydrogenase enzyme. Moreover, strobilurin derivative-mediated pyrimidine depletion led to the activation of the Akt/mTOR cascade, which was required for the differentiation. Our study provided evidence that strobilurin derivatives may represent a novel class of differentiation-inducing agents for the treatment of AML.

Atovaquone and Berberine Chloride Reduce SARS-CoV-2 Replication In Vitro.

Epidemic RNA viruses seem to arise year after year leading to countless infections and devastating disease. SARS-CoV-2 is the most recent of these viruses, but there will undoubtedly be more to come. While effective SARS-CoV-2 vaccines are being deployed, one approach that is still missing is effective antivirals that can be used at the onset of infections and therefore prevent pandemics. Here, we screened FDA-approved compounds against SARS-CoV-2. We found that atovaquone, a pyrimidine biosynthesis inhibitor, is able to reduce SARS-CoV-2 infection in human lung cells. In addition, we found that berberine chloride, a plant-based compound used in holistic medicine, was able to inhibit SARS-CoV-2 infection in cells through direct interaction with the virion. Taken together, these studies highlight potential avenues of antiviral development to block emerging viruses. Such proactive approaches, conducted well before the next pandemic, will be essential to have drugs ready for when the next emerging virus hits.

A Bioluminescent 3CLPro Activity Assay to Monitor SARS-CoV-2 Replication and Identify Inhibitors.

Our therapeutic arsenal against viruses is very limited and the current pandemic of SARS-CoV-2 highlights the critical need for effective antivirals against emerging coronaviruses. Cellular assays allowing a precise quantification of viral replication in high-throughput experimental settings are essential to the screening of chemical libraries and the selection of best antiviral chemical structures. To develop a reporting system for SARS-CoV-2 infection, we generated cell lines expressing a firefly luciferase maintained in an inactive form by a consensus cleavage site for the viral protease 3CLPro of coronaviruses, so that the luminescent biosensor is turned on upon 3CLPro expression or SARS-CoV-2 infection. This cellular assay was used to screen a metabolism-oriented library of 492 compounds to identify metabolic vulnerabilities of coronaviruses for developing innovative therapeutic strategies. In agreement with recent reports, inhibitors of pyrimidine biosynthesis were found to prevent SARS-CoV-2 replication. Among the top hits, we also identified the NADPH oxidase (NOX) inhibitor Setanaxib. The anti-SARS-CoV-2 activity of Setanaxib was further confirmed using ACE2-expressing human pulmonary cells Beas2B as well as human primary nasal epithelial cells. Altogether, these results validate our cell-based functional assay and the interest of screening libraries of different origins to identify inhibitors of SARS-CoV-2 for drug repurposing or development.

Purine and pyrimidine biosynthesis inhibitors efficiently suppress equine arteritis virus replication

Purine and pyrimidine biosynthesis inhibitors efficiently suppress equine arteritis virus replication

In vitro activity of olorofim against clinical isolates of the Fusarium oxysporum and Fusarium solani species complexes.

Invasive fusariosis is associated with marked morbidity and mortality in immunocompromised hosts, and clinical outcomes are poor with conventional therapy. Olorofim (F901318) is an investigational antifungal in the orotomide class that selectively targets fungal dihydroorotate dehydrogenase (DHODH) causing inhibition of pyrimidine biosynthesis. We evaluated the in vitro activity of olorofim against 61 clinical isolates of the Fusarium oxysporum and Fsolani species complexes (FOSC and FSSC, respectively), the most prevalent causes of invasive fusariosis. Clinical isolates of FOSC (n=45) and FSSC (n=16) were identified using DNA sequence analysis of the translation elongation factor 1-alpha (TEF1α) and RNA polymerase II second largest subunit (RPB2). Antifungal susceptibility testing was performed by CLSI M38 broth microdilution for olorofim, amphotericin B, isavuconazole, posaconazole, voriconazole and micafungin. Olorofim demonstrated good in vitro activity against both FOSC and FSSC. Against the 45 FOSC isolates, olorofim MICs ranged between 0.03-0.5mg/L and 0.06->4mg/L at the 50% and 100% inhibition endpoints, respectively. Against FSSC isolates, olorofim MIC ranged between 0.25-1mg/L and 1->4mg/L at 50% and 100% inhibition, respectively. While amphotericin B also demonstrated similar in vitro activity (MIC ranges 1-4 and 0.25-4mg/L against FOSC and FSSC, respectively), neither the triazoles nor micafungin demonstrated consistent in vitro activity against Fusarium isolates at clinically relevant concentrations. The investigational agent olorofim demonstrated good in vitro activity against FOSC and FSSC clinical isolates. Further studies are warranted to determine how well this in vitro activity translates into in vivo efficacy.

Replication of Equine arteritis virus is efficiently suppressed by purine and pyrimidine biosynthesis inhibitors

RNA viruses are responsible for a large variety of animal infections. Equine Arteritis Virus (EAV) is a positive single-stranded RNA virus member of the family Arteriviridae from the order Nidovirales like the Coronaviridae. EAV causes respiratory and reproductive diseases in equids. Although two vaccines are available, the vaccination coverage of the equine population is largely insufficient to prevent new EAV outbreaks around the world. In this study, we present a high-throughput in vitro assay suitable for testing candidate antiviral molecules on equine dermal cells infected by EAV. Using this assay, we identified three molecules that impair EAV infection in equine cells: the broad-spectrum antiviral and nucleoside analog ribavirin, and two compounds previously described as inhibitors of dihydroorotate dehydrogenase (DHODH), the fourth enzyme of the pyrimidine biosynthesis pathway. These molecules effectively suppressed cytopathic effects associated to EAV infection, and strongly inhibited viral replication and production of infectious particles. Since ribavirin is already approved in human and small animal, and that several DHODH inhibitors are in advanced clinical trials, our results open new perspectives for the management of EAV outbreaks.

Reporter-Guided Transposon Mutant Selection for Activation of Silent Gene Clusters in Burkholderia thailandensis.

Most natural product biosynthetic gene clusters that can be observed bioinformatically are silent. This insight has prompted the development of several methodologies for inducing their expression. One of the more recent methods, termed reporter-guided mutant selection (RGMS), entails creation of a library of mutants that is then screened for the desired phenotype via reporter gene expression. Herein, we apply a similar approach to Burkholderia thailandensis and, using transposon mutagenesis, mutagenize three strains, each carrying a fluorescent reporter in the malleilactone (mal), capistruin (cap), or an unidentified ribosomal peptide (tomm) gene cluster. We show that even a small library of <500 mutants can be used to induce expression of each cluster. We also explore the mechanism of activation and find that inhibition of pyrimidine biosynthesis is linked to the induction of the mal cluster. Both a transposon insertion into pyrF as well as small-molecule-mediated inhibition of PyrF trigger malleilactone biosynthesis. Our results pave the way toward the broad application of RGMS and related approaches to Burkholderia spp.

Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de novo pyrimidine biosynthesis inhibition

There is an increasing interest in the field of cancer therapy for small compounds targeting pyrimidine biosynthesis, and in particular dihydroorotate dehydrogenase (DHODH), the fourth enzyme of this metabolic pathway. Three available DHODH structures, featuring three different known inhibitors, were used as templates to screen in silico an original chemical library from Erevan University. This process led to the identification of P1788, a compound chemically related to the alkaloid cerpegin, as a new class of pyrimidine biosynthesis inhibitors. In line with previous reports, we investigated the effect of P1788 on the cellular innate immune response. Here we show that pyrimidine depletion by P1788 amplifies cellular response to both type-I and type II interferons, but also induces DNA damage as assessed by γH2AX staining. Moreover, the addition of inhibitors of the DNA damage response led to the suppression of the P1788 stimulatory effects on the interferon pathway. This demonstrates that components of the DNA damage response are bridging the inhibition of pyrimidine biosynthesis by P1788 to the interferon signaling pathway. Altogether, these results provide new insights on the mode of action of novel pyrimidine biosynthesis inhibitors and their development for cancer therapies.

Anfractuous assemblies of IMP dehydrogenase and CTP synthase: new twists on regulation?

CTP synthase (CTPS) and IMP dehydrogenase (IMPDH) catalyse the rate-limiting steps of de novo CTP and guanosine nucleotide biosynthesis, respectively, and form filament assemblies in response to inhibitors. A recent study explores the morphology and dynamics of these assemblies using fluorescence and super-resolution confocal microscopy with cell lines expressing CTPS1 and IMPDH2 fusion proteins. The formation and dismantling of mixed assemblies depends on nucleotide levels, suggesting a co-regulation function.

Gemcitabine, a broad-spectrum antiviral drug, suppresses enterovirus infections through innate immunity induced by the inhibition of pyrimidine biosynthesis and nucleotide depletion.

Gemcitabine, an anti-cancer chemotherapy drug, has additionally shown the antiviral activity against a broad range of viruses and we also have previously reported its synergistic antiviral activity with ribavirin against enteroviruses. As a cytidine analog, gemcitabine has been reported to have an inhibitory activity on the pyrimidine biosynthesis. In addition, a few inhibitors of the pyrimidine biosynthesis have shown to induce the innate immunity in a yet-to-be-determined manner and inhibit the virus infection. Thus, we also investigated whether the anti-enteroviral activity of gemcitabine is mediated by innate immunity, induction of which is related with the inhibition of the pyrimidine synthesis. In this study, we found that the addition of exogenous cytidine, uridine and uridine mono-phosphate (UMP) effectively reversed the antiviral activity of gemcitabine in enterovirus-infected as well as enteroviral replicon-harboring cells, demonstrating gemcitabine’s targeting of the salvage pathway. Moreover, the expression of several interferon (IFN)-stimulated genes (ISGs) was significantly induced by the treatment of gemcitabine, which was also suppressed by the co-treatment with cytidine. These results suggest that the antiviral activity of gemcitabine involves ISGs induced by the inhibition of the pyrimidine biosynthesis.

Synergy and Target Promiscuity Drive Structural Divergence in Bacterial Alkylquinolone Biosynthesis

Microbial natural products are genetically encoded by dedicated biosynthetic gene clusters (BGCs). A given BGC usually produces a family of related compounds that share a core but contain variable substituents. Though common, the reasons underlying this divergent biosynthesis are in general unknown. Herein, we have addressed this issue using the hydroxyalkylquinoline (HAQ) family of natural products synthesized by Burkholderia thailandensis. Investigations into the detailed functions of two analogs show that they act synergistically in inhibiting bacterial growth. One analog is a nanomolar inhibitor of pyrimidine biosynthesis and at the same time disrupts the proton motive force. A second analog inhibits the cytochrome bc1 complex as well as pyrimidine biogenesis. These results provide a functional rationale for the divergent nature of HAQs. They imply that synergy and target promiscuity are driving forces for the evolution of tailoring enzymes that diversify the products of the HAQ biosynthetic pathway.

Original Chemical Series of Pyrimidine Biosynthesis Inhibitors That Boost the Antiviral Interferon Response.

ABSTRACTDe novo pyrimidine biosynthesis is a key metabolic pathway involved in multiple biosynthetic processes. Here, we identified an original series of 3-(1H-indol-3-yl)-2,3-dihydro-4H-furo[3,2-c]chromen-4-one derivatives as a new class of pyrimidine biosynthesis inhibitors formed by two edge-fused polycyclic moieties. We show that identified compounds exhibit broad-spectrum antiviral activity and immunostimulatory properties, in line with recent reports linking de novo pyrimidine biosynthesis with innate defense mechanisms against viruses. Most importantly, we establish that pyrimidine deprivation can amplify the production of both type I and type III interferons by cells stimulated with retinoic acid-inducible gene 1 (RIG-I) ligands. Altogether, our results further expand the current panel of pyrimidine biosynthesis inhibitors and illustrate how the production of antiviral interferons is tightly coupled to this metabolic pathway. Functional and structural similarities between this new chemical series and dicoumarol, which was reported before to inhibit pyrimidine biosynthesis at the dihydroorotate dehydrogenase (DHODH) step, are discussed.

Respiratory syncytial virus infection in macaques is not suppressed by intranasal sprays of pyrimidine biosynthesis inhibitors

There is imperious need for efficient therapies against ubiquitous and life-threatening respiratory viruses, foremost among them being the human respiratory syncytial virus (hRSV). Several research groups who performed functional screens for broad-spectrum antivirals identified compounds targeting the de novo pyrimidine biosynthesis pathway. Despite their strong antiviral activity in vitro, whether such antimetabolites are effective in vivo remains highly controversial. Here, we evaluated two potent pyrimidine biosynthesis inhibitors developed in our laboratory, IPPA17-A04 and GAC50, in a model of mild hRSV-infection in cynomolgus macaques. In this model, hRSV replication is restricted to the epithelium of the upper respiratory tract, and is compatible with a topical treatment by intranasal sprays. The local administration of palivizumab, a neutralizing anti-hRSV antibody used in clinics, significantly reduced virus replication. In contrast, pyrimidine biosynthesis inhibitors did not show any inhibitory effect on hRSV growth when delivered topically as experimented in our model. Our results should help to better define the potential applications of this class of antimetabolites in the treatment of viral infections.

Viral Polymerase-Helicase Complexes Regulate Replication Fidelity To Overcome Intracellular Nucleotide Depletion.

ABSTRACTTo date, the majority of work on RNA virus replication fidelity has focused on the viral RNA polymerase, while the potential role of other viral replicase proteins in this process is poorly understood. Previous studies used resistance to broad-spectrum RNA mutagens, such as ribavirin, to identify polymerases with increased fidelity that avoid misincorporation of such base analogues. We identified a novel variant in the alphavirus viral helicase/protease, nonstructural protein 2 (nsP2) that operates in concert with the viral polymerase nsP4 to further alter replication complex fidelity, a functional linkage that was conserved among the alphavirus genus. Purified chikungunya virus nsP2 presented delayed helicase activity of the high-fidelity enzyme, and yet purified replication complexes manifested stronger RNA polymerization kinetics. Because mutagenic nucleoside analogs such as ribavirin also affect intracellular nucleotide pools, we addressed the link between nucleotide depletion and replication fidelity by using purine and pyrimidine biosynthesis inhibitors. High-fidelity viruses were more resistant to these conditions, and viral growth could be rescued by the addition of exogenous nucleosides, suggesting that mutagenesis by base analogues requires nucleotide pool depletion. This study describes a novel function for nsP2, highlighting the role of other components of the replication complex in regulating viral replication fidelity, and suggests that viruses can alter their replication complex fidelity to overcome intracellular nucleotide-depleting conditions.IMPORTANCE Previous studies using the RNA mutagen ribavirin to select for drug-resistant variants have highlighted the essential role of the viral RNA-dependent RNA polymerase in regulating replication fidelity. However, the role of other viral replicase components in replication fidelity has not been studied in detail. We identified here an RNA mutagen-resistant variant of the nsP2 helicase/protease that conferred increased fidelity and yet could not operate in the same manner as high-fidelity polymerases. We show that the alphavirus helicase is a key component of the fidelity-regulating machinery. Our data show that the RNA mutagenic activity of compounds such as ribavirin is coupled to and potentiated by nucleotide depletion and that RNA viruses can fine-tune their replication fidelity when faced with an intracellular environment depleted of nucleotides.