DHODH (dihydroorotate dehydrogenase)
Brequinar (BQR) is a potent inhibitor of cellular dihydroorotate dehydrogenase (DHODH), the fourth enzyme and a rate-limiting step in the de novo pyrimidine biosynthetic pathway. [1]

Brequinar has a 17 nM EC50 and decreases the formation of virus progeny by over 90%. Additionally, other orthopoxviruses are inhibited and virus DNA replication is blocked by Brequinar (5 μM). Brequinar has a strong effect on the late stage of the virus cycle, although it has no influence on the expression of early virus genes[1]. Brequinar, which has an EC50 of 78 nM in the CFI test, decreases the amount of envelope protein synthesis and the viral titer in a dose-dependent manner. Brequinar (5 μM) prevents the synthesis of viral RNA. Brequinar has an antiviral action, however pyrimidine neutralizes it. In cell culture, viruses resistant to brequinar can be chosen. Both the WT and NS5 mutant replicons' luciferase activity are suppressed by bequinar (5 μM)[2]. PyNTP rise is successfully inhibited by brequinar sodium. Brequinar sodium has an IC50 of 0.26 μM, which substantially suppresses cell proliferation. Brequinar sodium inhibits p56lck autophosphorylation with an IC50 of 70 μM; the corresponding inhibition values for 25, 50, and 100 μM of Brequinar sodium are 39, 41, and 60%. Additionally, at an IC50 of 70 μM, bequinar sodium prevents the phosphorylation of histone 2B, the exogenous substrate, by p56lck; at 25, 50, 100, and 200 μM, the inhibition is 10, 43, 59, and 86%. The Brequinar Brequinar sodium has an IC50 of 105 μM, which suppresses autophosphorylation of p59fyn by 0, 17, 48, and 65% at 25, 50, 100, and 200 μM, respectively. Moreover, at an IC50 of 20 μM, Brequinar sodium suppresses the phosphorylation of histone 2B by p59fyn; at 10, 25, 50, 100, and 200 μM, the corresponding inhibitions are 26, 54, 79, 83, and 84%[3].

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Brequinar (BQR) exhibited potent antiviral activity against Cantagalo virus (CTGV), a vaccinia virus strain, in BSC-40 cells. Treatment with 0.5 µM BQR reduced infectious virus progeny production by >90% at 24 hours post-infection (hpi). The 50% effective concentration (EC50) for inhibition of CTGV replication was calculated to be 0.017 ± 0.05 µM. The 50% cytotoxic concentration (CC50) in BSC-40 cells was >75 µM, resulting in a selective index (CC50/EC50) of >4410. [1]
The antiviral activity of BQR (at 5 µM) extended to other orthopoxviruses, including vaccinia virus strains WR, IOC, Wyeth, and cowpox virus (CPXV), severely inhibiting infectious particle production. [1]
BQR had no direct virucidal effect on purified CTGV particles when incubated with the drug prior to infection. [1]
In one-step growth curve analysis using a multiplicity of infection (MOI) of 1 and 30 µM BQR, the production of infectious CTGV particles was completely abolished from the initial hours of infection. [1]
Western blot analysis showed that BQR (30 µM) did not affect the accumulation of the viral early protein F11, but completely abrogated the accumulation of CTGV structural (late) proteins. [1]
Using a recombinant CTGV expressing β-galactosidase under an early/late promoter, BQR (30 µM) showed minimal effect on reporter activity at 6 hpi (early phase) but strongly inhibited activity at 24 hpi (late phase), similar to the DNA synthesis inhibitor cytosine arabinoside (Ara-C). [1]
Indirect immunofluorescence assays confirmed severe inhibition (approximately 89.12%) of late viral protein (D8 and structural proteins) expression and a drastic reduction (82.9%) in the formation of viral factories (virosomes) in the cytoplasm of BQR-treated cells at 20 hpi. [1]
Slot-blot analysis demonstrated that BQR (30 µM) severely repressed CTGV DNA replication, which normally begins at 6-8 hpi in untreated cells. [1]
The antiviral effects of BQR on CTGV DNA replication, late protein expression, and infectious progeny production were completely rescued to normal levels when infected cells were co-treated with 30 µM BQR and exogenous uridine (URD) at 100 µM or 300 µM, which provides pyrimidines via the salvage pathway. [1]

Compared to untreated BALB/c mice, mice treated with Brequinar sodium (10–20 mg/kg/day) exhibited a 31% decrease in the percentage of packed cell volume. In bone marrow cells, brequinar sodium lowers UTP and CTP levels by 30 and 25%, respectively. When uridine (1000–2000 mg/kg/day) and bequinar sodium (10–20 mg/kg/day) are taken together, anemia is avoided and the hematocrit levels stay at values (61-63%) that are similar to those of untreated controls [3].

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In the dengue virus AG129 mouse model, orally administered Brequinar (3 mg/kg twice daily) showed no efficacy, likely due to the presence of plasma uridine (~6 µM) that rescues viral replication [2]

Immunoprecipitated p59fyn or p56lck from CTLL-4 cells or LSTRA cells (5×106) is preincubated with various concentrations of BQR in the PTK buffer (50 mM HEPES (pH 7.4), 10 mM MgCl2, and 10 mM MnCl2) on ice for 10 min. Exogenous substrate, histone 2B (2 μg), is added and, after 10 min, the reaction is initiated by addition of 10 μCi [γ-32P]ATP. After incubation at 20°C for 10 min, the reaction mixture is subjected to electrophoresis in a 12.5% SDS-polyacrylamide gel. Phosphorylation of the kinase and the exogenous substrate is analyzed by autoradiography[3].

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Recombinant full-length NS5 proteins of DENV-2 (wild-type and E802Q mutant) were expressed and purified for de novo RdRp activity assays [2]
Equal amounts of WT and mutant NS5 proteins were incubated with a DENV-2 subgenomic RNA template in the presence or absence of Brequinar; RdRp products were analyzed on a denaturing polyacrylamide gel and quantified [2]
The mutant NS5 (E802Q) showed approximately 2-fold higher RdRp activity compared to WT NS5 [2]

Intracellular pyrimidine nucleotides (PyN) can be synthesized de novo from glutamine, CO2, and ATP, or they can be salvaged from preformed pyrimidine nucleosides. The antiproliferative and immunosuppressive activities of brequinar sodium (BQR) are thought to be due to the inhibition of the activity of dihydroorotate dehydrogenase, which results in a suppression of de novo pyrimidine synthesis. Here we describe the effects of the pyrimidine nucleoSide, uridine, on the antiproliferative and immunosuppressive activities of BQR. In vitro reduction of PyN levels in Con A-stimulated T cells and inhibition of cell proliferation by low concentrations of BQR (< or =65 microM) are reversed by uridine. However, uridine is unable to reverse the effects of high concentrations of BQR (> or =65 microM)[3].

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Virus Yield Reduction Assay: BSC-40 cells were infected with 1000 plaque-forming units (PFU) of CTGV for 2 hours (adsorption). The inoculum was removed, and cells were treated with various concentrations of Brequinar (BQR) (0.005 µM to 10 µM) or 0.1% DMSO (control) for 24 hours. Cells were then harvested, lysed, and virus titers in the lysates were determined by standard plaque assay on fresh BSC-40 cell monolayers. [1]
Virucidal Assay: Purified CTGV particles (4 x 10^6 PFU/mL) were incubated with different concentrations of BQR (0.1, 1, 5 µM) or 0.1% DMSO for 1 hour at room temperature with occasional shaking. The mixture was then titrated by plaque assay to determine if the drug directly inactivated virus particles. [1]
Cytotoxicity Assay (Neutral Red Uptake): BSC-40 cells were seeded in 96-well plates and incubated with concentrations of BQR ranging from 0.01 µM to 75 µM for 24 hours. Control cells received 0.1% DMSO. Cell viability was assessed using the neutral red uptake assay. Dye taken up by viable cells was extracted with a methanol/acetic acid solution and quantified by measuring absorbance at 490 nm. [1]
One-Step Growth Analysis: BSC-40 cells were infected with CTGV at an MOI of 1 and treated with 30 µM BQR or 0.1% DMSO. At various time points post-infection (3, 6, 8, 16, 18, 20, 24 h), cells were harvested and processed for virus titration by plaque assay to generate a growth curve. [1]
Western Blot Analysis: BSC-40 cells were infected with CTGV at an MOI of 1 and treated with 30 µM BQR or 0.1% DMSO. In some assays, uridine (100 µM or 300 µM) was co-added. At specified times post-infection, cells were harvested in SDS sample buffer. Proteins were separated by SDS-PAGE (11.5% gel), transferred to nitrocellulose membranes, and probed with specific primary antibodies (e.g., rabbit anti-VACV structural proteins, mouse anti-α-tubulin, rabbit anti-F11). Detection was performed using appropriate horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. [1]
Reporter Gene (β-Galactosidase) Activity Assay: BSC-40 cells were infected with recombinant CTGV-βGal (MOI=1) and treated with 0.1% DMSO, 30 µM BQR, or 40 µg/mL Ara-C. At 6 and 24 hpi, cells were harvested in water and lysed. β-galactosidase activity was measured by adding the substrate O-nitrophenyl-β-D-galactopyranoside. The reaction was stopped with Na2CO3 upon color development, and absorbance was measured at 420 nm. [1]
Indirect Immunofluorescence Assay (IFA): BSC-40 cells grown on coverslips were infected with CTGV (MOI=1) and treated with 30 µM BQR or 0.1% DMSO for 20 hours. Cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and blocked. They were then stained with primary antibodies (rabbit anti-VACV structural proteins, mouse anti-D8) followed by species-specific fluorescent secondary antibodies. Cell nuclei and viral DNA were stained with DAPI. Samples were analyzed by fluorescence microscopy. Infected cells and virosomes were quantified by counting. [1]
Viral DNA Replication Analysis (Slot-Blot): BSC-40 cells were infected with CTGV (MOI=1) and treated with 30 µM BQR with or without uridine (100 µM or 300 µM). Cells were harvested at different times post-infection. Cell extracts were prepared, and DNA was applied to nylon membranes using a slot-blot apparatus. Membranes were hybridized with a 32P-labeled DNA probe corresponding to the HindIII D fragment of the vaccinia virus genome. Viral DNA accumulation was detected by autoradiography and quantified by densitometry. [1]

Method 1: Brequinar sodium was dissolved in 0.9% NaCl for i.p. injections and in distilled water for in vitro studies. [1]
Method 2: Brequinar sodium was formulated as a 10 mg/mL solution in saline. [2]
Mice; 15,25, 50 mg/kg, ip injection
Eight to ten-week-old female BALB/C mice were randomized and not blinded. Molnupiravir was resuspended in corn oil and 10% DMSO, with dosing twice a day as oral gavage. Brequinar was resuspended in 10% DMSO and sterile saline, with dosing daily as intraperitoneal injection. Our pharmacokinetic (PK) studies showed an approximately 10 h half-life of Brequinar, and dosing was either started at 12 h before infection or 24 h after infection as indicated.
Mice were anaesthetized by intraperitoneal injection with 50 μl of a mix of xylazine (0.38 mg per mouse) and ketamine (1.3 mg per mouse) diluted in PBS. Mice were intranasally inoculated with 1 × 105 p.f.u. of the Beta variant of SARS-CoV-2 in 50 μl. Challenged mice were weighed on the day of infection and daily for 2 days after infection; there were no significant changes in weights observed. For prophylactic dosing, 2 days after infection, or therapeutic dosing 3 days after infection, five mice were killed from each treatment and control group, lungs were collected to determine viral titre by a plaque assay, and fixed in 4% paraformaldehyde for 24 h before sectioning and staining with haematoxylin and eosin by UMSOM Histology Core5. Pathological scoring on blinded haematoxylin and eosin-stained sections was performed for each mouse and analysed for inflammation.[5]

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In the dengue AG129 mouse model, Brequinar was administered orally at 3 mg/kg twice daily; no efficacy was observed [2]
Pharmacokinetic studies in mice indicated that an oral dose of 25 mg/kg achieved a maximum plasma concentration of 231 µM, but high plasma protein binding (99.06%) resulted in a free drug concentration of 2.17 µM [2]

Pharmacokinetic (PK) studies showed that the half-life of brequina was approximately 10 hours. [5]

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After oral administration of 25 mg/kg brequina to mice, the peak plasma concentration reached 231 µM. [2]
The predicted plasma protein binding rate was 99.06%, resulting in a free drug concentration of 2.17 µM. [2]
The plasma uridine level in mice treated with brequina was approximately 6 µM, which may have counteracted the antiviral effect of the drug in vivo. [2]

The 50% cytotoxic concentration (CC50) of Brequinar (BQR) in BSC-40 cells was >75 µM, as determined by a 24-hour neutral red uptake assay. Even at the highest concentration tested (75 µM), cell viability remained at around 80%. [1]

[1]. Potent antiviral activity of brequinar against the emerging Cantagalo virus in cell culture. Int J Antimicrob Agents. 2011 Nov;38(5):435-41.

[2]. Characterization of dengue virus resistance to brequinar in cell culture. Antimicrob Agents Chemother. 2010 Sep;54(9):3686-95.

[3]. In vitro and in vivo mechanisms of action of the antiproliferative and immunosuppressive agent, brequinar sodium. J Immunol. 1998 Jan 15;160(2):846-53.

[4]. Bifunctional Naphtho[2,3-d][1,2,3]triazole-4,9-dione Compounds Exhibit Antitumor Effects In Vitro and In Vivo by Inhibiting Dihydroorotate Dehydrogenase and Inducing Reactive Oxygen Species Production. J Med Chem. 2020 Jun 4.

[5]. Pyrimidine inhibitors synergize with nucleoside analogues to block SARS-CoV-2. Nature. 2022 Apr;604(7904):134-140.

Brequina is a quinoline monocarboxylic acid with the structure quinoline, substituted at positions 2, 3, 4, and 6 with 2'-fluoro[1,1'-biphenyl]-4-yl, methyl, carboxyl, and fluorine atoms, respectively. It is an inhibitor of dihydroorotate dehydrogenase, an enzyme essential for the de novo synthesis of pyrimidines. This compound possesses antitumor and antiviral activity. It is an EC 1.3.5.2 [dihydroorotate dehydrogenase (quinone)] inhibitor, immunosuppressant, antitumor drug, antiviral drug, pyrimidine synthesis inhibitor, anticoronavirus drug, and antimetabolite. It belongs to the biphenyl, monofluorobenzene, quinoline monocarboxylic acid, and monocarboxylic acid classes. It is the conjugate acid of brequina (1-). Brequina is a synthetic quinoline carboxylic acid analog with antitumor properties. Brequina inhibits dihydroorotate dehydrogenase, thereby blocking the de novo synthesis of pyrimidines. This drug may also enhance the in vivo antitumor effects of antitumor drugs such as 5-fluorouracil. (NCI04)

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This study evaluated the antiviral activity of brequina (BQR) against cantagalo virus replication. BQR is a potent inhibitor of dihydroorotate dehydrogenase (an enzyme in the de novo pyrimidine synthesis pathway). Infection in the presence of 0.5 μM BQR reduced viral progeny yield by more than 90%, with an EC50 (the concentration required to inhibit 50% of viral replication) of 0.017 μM. BQR also exhibited similar inhibitory effects on the replication of other orthopoxviruses. In the presence of the drug, the accumulation of early viral proteins returned to control levels, while late gene expression was severely suppressed. This result was confirmed by indirect immunofluorescence analysis and time-regulated expression analysis of reporter genes under viral promoter control. Both analyses showed that late gene expression was suppressed by nearly 90%. BQR also blocked viral DNA replication, which explains the subsequent suppression of late viral gene expression. When infected cells were treated with both uridine (URD) and BQR, viral DNA replication, late gene expression, and the production of infectious progeny viruses were restored to control levels. These data suggest that BQR targets and inhibits viral DNA synthesis by consuming the cytosine pool, and that this inhibition can be bypassed by a salvage pathway when URD is added to the cell culture. [1]

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Brecquina is a dihydroorotate dehydrogenase inhibitor, which is essential for the de novo synthesis of pyrimidines. This paper reports that brecquina is active against a variety of viruses. The compound inhibits not only flaviviruses (dengue virus, West Nile virus, yellow fever virus, and Poissan virus), but also positive-sense RNA alphaviruses (western equine encephalitis virus) and negative-sense RNA rhabdoviruses (vesicular stomatitis virus). Using dengue virus type 2 (DENV-2) as a model, we found that brecquina mainly inhibits the viral infection cycle during the RNA synthesis phase. The inhibitory effect of the compound can be reversed by adding pyrimidines (cytidine or uridine) rather than purines (adenine or guanine) to the culture medium. DENV-2 was continuously cultured in a medium containing brequina to produce a partially resistant virus to the inhibitor. Sequencing of these resistant viruses revealed two amino acid mutations: one mutation (M260V) was located on a helix of the viral envelope protein II domain, and the other mutation (E802Q) was located on the primer loop of the non-structural protein 5 (NS5) polymerase domain. Functional analysis showed that the NS5 mutation exerted resistance by enhancing polymerase activity. The envelope protein mutation reduced the assembly/release efficiency of viral particles; however, the mutant virus was less sensitive to the inhibitory effect of brequina in the viral particle assembly/release step. In summary, the results indicate that (i) brequina blocks DENV RNA synthesis by depleting the intracellular pyrimidine pool; and (ii) the compound may also exert its antiviral activity by inhibiting the assembly/release of viral particles. [2]

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Intracellular pyrimidine nucleotides (PyN) can be synthesized de novo from glutamine, CO2 and ATP, or recycled from pre-formed pyrimidine nucleosides. The antiproliferative and immunosuppressive activities of brequina sodium (BQR) are thought to be due to its inhibition of dihydroorotate dehydrogenase activity, thereby inhibiting the de novo synthesis of pyrimidines. This article describes the effect of the pyrimidine nucleoside uridine on the antiproliferative and immunosuppressive activities of BQR. In vitro experiments showed that low concentrations of BQR (≤65 μM) reduced PyN levels in Con A-stimulated T cells and inhibited cell proliferation, while uridine reversed these effects. However, uridine could not reverse the effects of high concentrations of BQR (≥65 μM). The ability of BQR to induce anemia in BALB/c mice could be inhibited by co-administration of uridine. Conversely, the same dose of uridine had no effect on the immunosuppressive activity of BQR. PyN levels were decreased in the bone marrow of BQR-treated mice, while PyN levels in the spleen were unaffected. These observations suggest that BQR-induced anemia is due to the depletion of PyN in bone marrow hematopoietic stem cells. They also suggest that the immunosuppressive mechanism of BQR may depend only slightly on the depletion of PyN in peripheral lymphocytes. We report a novel activity of BQR: inhibition of tyrosine phosphorylation, and speculate that its immunosuppressive activity may be partly attributable to this unexpected ability of BQR to inhibit lymphocyte tyrosine phosphorylation. [3]

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Braequina (BQR) is an immunosuppressant and antiproliferative drug that has been used for transplant therapy. [1]
Its mechanism of action against CTGV is attributed to the inhibition of de novo pyrimidine synthesis by cells through blocking dihydroorotate dehydrogenase (DHODH), leading to depletion of the intracellular pyrimidine pool required for viral DNA synthesis. Its antiviral effect is completely reversed when exogenous uridine (a precursor to the pyrimidine rescue pathway) is provided, which supports the above mechanism. [1]
This study suggests that, due to the local nature of CTGV skin lesions, BQR could be considered for future evaluation as a local antiviral therapy, potentially avoiding systemic immunosuppression. [1]

C23H15F2NO2

375.3675

375.107

C, 73.59; H, 4.03; F, 10.12; N, 3.73; O, 8.52

96187-53-0

Brequinar sodium;96201-88-6

57030

White to off-white solid powder

1.3±0.1 g/cm3

550.9±50.0 °C at 760 mmHg

317 °C

287.0±30.1 °C

0.0±1.6 mmHg at 25°C

1.645

6.69

1

5

3

28

551

0

O=C(C1=C(C)C(C2=CC=C(C3=CC=CC=C3F)C=C2)=NC4=CC=C(F)C=C14)O

PHEZJEYUWHETKO-UHFFFAOYSA-N

InChI=1S/C23H15F2NO2/c1-13-21(23(27)28)18-12-16(24)10-11-20(18)26-22(13)15-8-6-14(7-9-15)17-4-2-3-5-19(17)25/h2-12H,1H3,(H,27,28)

6-fluoro-2-(2'-fluoro-[1,1'-biphenyl]-4-yl)-3-methylquinoline-4-carboxylic acid

Brequinar; Bipenquinate; DUP 785; NSC368390; DUP785; DUP-785; NSC 368390; DUP785; 6-Fluoro-2-(2'-fluoro-[1,1'-biphenyl]-4-yl)-3-methylquinoline-4-carboxylic acid; Brequinar [INN]; brequinarum; 6-fluoro-2-[4-(2-fluorophenyl)phenyl]-3-methylquinoline-4-carboxylic acid; Biphenquinate; 6-fluoro-2-(2'-fluorobiphenyl-4-yl)-3-methylquinoline-4-carboxylic acid; NSC-368390;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~25 mg/mL (~66.60 mM)

Solubility in Formulation 1: 2.08 mg/mL (5.54 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.54 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)