DHODH (dihydroorotate dehydrogenase; IC50 = 5.2 nM)

Brequinar is a dihydroorotate dehydrogenase/DHODH inhibitor with antineoplastic properties. Brequinar inhibits the enzyme dihydroorotate dehydrogenase, thereby blocking de novo pyrimidine biosynthesis.
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%.

The ability of Brequinar/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.[2]

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Transplantation of BALB/c hearts into C3H mice was performed as previously described. Brequinar/BQR was administered once daily by i.p. injection, while uridine was administered twice daily. Mice were bled through the orbital vein using a microhematocrit capillary tube, and the blood was centrifuged for 10 min at 550 3 g. The percentage of packed cell volumes was determined with a microhematocrit capillary tube reader. All mice were killed 4 h after receiving their last dose of BQR or uridine.

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].

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)[2].

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.[3]

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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

Pharmacokinetic (PK) studies showed that brequina has a half-life of approximately 10 hours. [3]

[1]. Int J Antimicrob Agents. 2011 Nov;38(5):435-41.
[2]. J Immunol. 1998 Jan 15;160(2):846-53.

[3]. Nature. 2022 Apr;604(7904):134-140.

Brbequina sodium is the organic sodium salt of brbequina. It possesses a variety of activities, including anticoronavirus, antitumor, antiviral, EC 1.3.5.2 [dihydroorotate dehydrogenase (quinone)] inhibitor, immunosuppressant, pyrimidine synthesis inhibitor, and antimetabolite activity. It contains the brbequina (1-) domain.
Brbequina sodium is the sodium salt form of brbequina. Brbequina 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 brbequina (BQR) against Cantagalo virus replication. BQR is a potent inhibitor of cellular dihydroorotate dehydrogenase, an enzyme involved in the de novo pyrimidine synthesis pathway. Infection with 0.5 μM BQR reduced viral progeny production by more than 90%, with an EC50 (the concentration required to inhibit 50% viral replication) of 0.017 μM. BQR also inhibited the replication of other orthopoxviruses at similar concentrations. In the presence of the drug, the accumulation of early viral proteins returned to control levels, while the expression of late genes 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 uridine (URD) and BQR simultaneously, viral DNA replication, late gene expression, and the production of infectious progeny viruses all returned to control levels. These data suggest that BQR targets and inhibits viral DNA synthesis by depleting the cytosine pool, and that this inhibition can be bypassed via a salvage pathway when URD is added to the cell culture. [1] Intracellular pyrimidine nucleotides (PyNs) can be synthesized de novo from glutamine, CO2, and ATP, or salvage synthesized 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 effects 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). Urate in combination with BQR prevented BQR-induced anemia in BALB/c mice. Conversely, the same dose of uridine had no effect on the immunosuppressive activity of BQR. PyN levels were reduced in the bone marrow of mice treated with BQR, 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 hypothesize that its immunosuppressive activity may be partly attributable to this unexpected ability of BQR to inhibit lymphocyte tyrosine phosphorylation. [2]

C23H14F2NNAO2

397.36

397.089

C, 69.52; H, 3.55; F, 9.56; N, 3.53; Na, 5.79; O, 8.05

96201-88-6

Brequinar;96187-53-0

23663964

Typically exists as White to off-white solid at room temperature

550.9ºC at 760mmHg

>360°

287ºC

5.853

0

5

3

29

557

0

[Na].O=C(C1C(C)=C(C2C=CC(C3C(F)=CC=CC=3)=CC=2)N=C2C=1C=C(C=C2)F)O

PZOHOALJQOFNTB-UHFFFAOYSA-M

InChI=1S/C23H15F2NO2.Na/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)/q+1/p-1

Sodium 6-Fluoro-2-(2'-fluoro[1,1'-biphenyl]-4-yl)-3-methyl-4-quinolinecarboxylate

Dup785 NSC-368390 Dup 785 NSC368390 Dup-785 Brequinar SodiumBipenquinate

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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