DHODH (dihydroorotate dehydrogenase)

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].

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].

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].

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].

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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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

Pharmacokinetic (PK) studies showed an approximately 10 h half-life of Brequinar. [5]

[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.

Brequinar is a quinolinemonocarboxylic acid that is quinoline substituted by 2'-fluoro[1,1'-biphenyl]-4-yl, methyl, carboxy and fluoro groups at positions 2, 3, 4, and 6, respectively. It is an inhibitor of dihydroorotate dehydrogenase, an enzyme that is required for de novo pyrimidine biosynthesis. The compound exhibits antineoplastic and antiviral properties. It has a role as an EC 1.3.5.2 [dihydroorotate dehydrogenase (quinone)] inhibitor, an immunosuppressive agent, an antineoplastic agent, an antiviral agent, a pyrimidine synthesis inhibitor, an anticoronaviral agent and an antimetabolite. It is a member of biphenyls, a member of monofluorobenzenes, a quinolinemonocarboxylic acid and a monocarboxylic acid. It is a conjugate acid of a brequinar(1-).
Brequinar is a synthetic quinolinecarboxylic acid analogue with antineoplastic properties. Brequinar inhibits the enzyme dihydroorotate dehydrogenase, thereby blocking de novo pyrimidine biosynthesis. This agent may also enhance the in vivo antitumor effect of antineoplastic agents such as 5-FU. (NCI04)

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In the present work, the antiviral activity of brequinar (BQR) against the replication of Cantagalo virus was evaluated. BQR is a potent inhibitor of cellular dihydroorotate dehydrogenase, an enzyme of the de novo pyrimidine biosynthetic pathway. Infection in the presence of 0.5μM BQR reduced virus progeny production by >90%, revealing an EC(50) (drug concentration required to inhibit 50% of virus replication) of 0.017μM. Replication of other orthopoxviruses was also inhibited by BQR at similar levels. In the presence of the drug, virus early proteins accumulated to control levels, whereas late gene expression was severely impaired. This result was confirmed by indirect immunofluorescence assays and analysis of time-regulated expression of a reporter gene under the control of a virus promoter. Both assays revealed nearly 90% inhibition of late gene expression. BQR also blocked virus DNA replication, which accounted for the subsequent inhibition of virus late gene expression. The ablation of virus DNA replication, late gene expression and infectious progeny production was restored to control levels when infected cells were co-treated with uridine (URD) and BQR. These data demonstrated that BQR targeted virus DNA synthesis by depleting the cellular pyrimidine pool, which was bypassed by the salvage pathway when URD was added to the cell cultures. [1]

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Brequinar is an inhibitor of dihydroorotate dehydrogenase, an enzyme that is required for de novo pyrimidine biosynthesis. Here we report that brequinar has activity against a broad spectrum of viruses. The compound not only inhibits flaviviruses (dengue virus, West Nile virus, yellow fever virus, and Powassan virus) but also suppresses a plus-strand RNA alphavirus (Western equine encephalitis virus) and a negative-strand RNA rhabdovirus (vesicular stomatitis virus). Using dengue virus serotype 2 (DENV-2) as a model, we found that brequinar suppressed the viral infection cycle mainly at the step of RNA synthesis. Supplementing the culture medium with pyrimidines (cytidine or uridine) but not purines (adenine or guanine) could be used to reverse the inhibitory effect of the compound. Continuous culturing of DENV-2 in the presence of brequinar generated viruses that were partially resistant to the inhibitor. Sequencing of the resistant viruses revealed two amino acid mutations: one mutation (M260V) located at a helix in the domain II of the viral envelope protein and another mutation (E802Q) located at the priming loop of the nonstructural protein 5 (NS5) polymerase domain. Functional analysis of the mutations suggests that the NS5 mutation exerts resistance through enhancement of polymerase activity. The envelope protein mutation reduced the efficiency of virion assembly/release; however, the mutant virus became less sensitive to brequinar inhibition at the step of virion assembly/release. Taken together, the results indicate that (i) brequinar blocks DENV RNA synthesis through depletion of intracellular pyrimidine pools and (ii) the compound may also exert its antiviral activity through inhibition of virion assembly/release. [2]

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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). The ability of BQR to induce anemia in BALB/c mice is prevented by the coadministration of uridine. In contrast, the immunosuppressive activity of BQR is unaffected by similar doses of uridine. PyN levels in the bone marrow, but not in the spleen, are depressed in mice treated with BQR. These observations suggest that the induction of anemia by BQR is due to depletion of intracellular PyN in hemopoietic stem cells located in the bone marrow. They also suggest that the mechanism of immunosuppression by BQR may be only marginally dependent on depletion of intracellular PyN in lymphocytes located in the periphery. We report a novel activity of BQR: inhibition of tyrosine phosphorylation, and hypothesize that the immunosuppressive activity may be due, in part, to this unsuspected ability of BQR to inhibit tyrosine phosphorylation in lymphocytes. [3]

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.)