Antifungal;TGF-β-Smad3
Caerulomycin A (CaeA), in inducing the generation of Tregs. It was observed that CaeA substantially up-regulated the pool of Tregs, as evidenced by an increased frequency of CD4(+) Foxp3(+) cells. In addition, CaeA significantly suppressed the number of Th1 and Th17 cells, as supported by a decreased percentage of CD4(+)/IFN-γ(+) and CD4(+)/IL-17(+) cells, respectively. Furthermore, we established the mechanism and observed that CaeA interfered with IFN-γ-induced STAT1 signaling by augmenting SOCS1 expression. An increase in the TGF-β-mediated Smad3 activity was also noted. Furthermore, CaeA rescued Tregs from IFN-γ-induced inhibition. These results were corroborated by blocking Smad3 activity, which abolished the CaeA-facilitated generation of Tregs. In essence, our results indicate a novel role of CaeA in inducing the generation of Tregs. This finding suggests that CaeA has enough potential to be considered as a potent future drug for the treatment of autoimmunity.[1]

Caerulomycin A (CaeA), in inducing the generation of Tregs. It was observed that CaeA substantially up-regulated the pool of Tregs, as evidenced by an increased frequency of CD4(+) Foxp3(+) cells. In addition, CaeA significantly suppressed the number of Th1 and Th17 cells, as supported by a decreased percentage of CD4(+)/IFN-γ(+) and CD4(+)/IL-17(+) cells, respectively. Furthermore, we established the mechanism and observed that CaeA interfered with IFN-γ-induced STAT1 signaling by augmenting SOCS1 expression. An increase in the TGF-β-mediated Smad3 activity was also noted. Furthermore, CaeA rescued Tregs from IFN-γ-induced inhibition. These results were corroborated by blocking Smad3 activity, which abolished the CaeA-facilitated generation of Tregs. In essence, our results indicate a novel role of CaeA in inducing the generation of Tregs. This finding suggests that CaeA has enough potential to be considered as a potent future drug for the treatment of autoimmunity.[1]

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Caerulomycin A significantly increases the frequency of CD4+ Foxp3+ Tregs in a dose-dependent manner when naïve CD4+ T cells are stimulated with anti-CD3/CD28 antibodies, both alone and in synergy with TGF-β. [1]
Caerulomycin A suppresses the differentiation of Th1 and Th17 cells, as evidenced by decreased percentages of CD4+/IFN-γ+ and CD4+/IL-17+ cells and reduced secretion of IFN-γ and IL-17. [1]
Caerulomycin A enhances TGF-β-induced phosphorylation of Smad3 and suppresses IFN-γ- and IL-6-induced phosphorylation of STAT1 in CD4+ T cells. [1]
Caerulomycin A upregulates SOCS1 expression and downregulates IFN-γ-induced expression of T-bet, Smad7, and FasL. [1]
Caerulomycin A increases mitochondrial membrane potential and downregulates CD44 expression in activated CD4+ T cells. [1]

Caerulomycin A ameliorates clinical symptoms of collagen-induced arthritis in DBA/1 mice, reducing inflammation, synovitis, and joint damage in a dose-dependent manner (1 and 10 mg/kg). [1]
Caerulomycin A increases the percentage of Foxp3+ Tregs in draining lymph nodes and reduces proinflammatory cytokines (IFN-γ, TNF-α, IL-6) in joint tissues. [1]
Caerulomycin A reduces serum levels of matrix metalloproteinase-3 (MMP-3) and decreases inflammatory activity as visualized by fluorescence imaging using ProSense and OsteoSense probes. [1]

Electrophoretic mobility shift assay (EMSA) was performed to assess STAT1 and Smad3 DNA-binding activity. Nuclear extracts were prepared from CD4+ T cells treated with Caerulomycin A and stimulated with IFN-γ or TGF-β. Labeled double-stranded oligonucleotides for STAT1 and Smad3 consensus sequences were used, and protein-DNA complexes were separated on polyacrylamide gels. [1]
Western blotting was used to analyze phosphorylation of STAT1, STAT3, STAT4, Smad3, JAK1, and JAK2, as well as expression of Smad7 and T-bet. Total protein levels were used as loading controls. [1]

Th1 and Th17 cells were treated with phorbol 12-myristate 13-acetate (40 nm) and ionomycin (1 μm) for 2 h. To block cytokine secretion, brefeldin A (10 μg/ml) was added. Later, and cells were incubated further for 3 h. Tregs, Th1, and Th17 cells were cultured with different concentrations of CaeA (0–0.15 μm). The modulation in the frequency of Tregs, Th1, and Th17 cells was analyzed by flow cytometry.[1]
A booster dose of bovine collagen type II in incomplete Freund adjuvant was injected on day 21. Later, CaeA (1 and 10 mg/kg body weight) and 0.5% carboxyl methyl cellulose emulsion in water was administered daily for 50 days, and animals were monitored every day for arthritis symptoms. [1]
The IFN-γ-mediated STAT1 response was measured by initially incubating CD4 T cells with CaeA (0–0.31 μm) for 24 h, followed by IFN-γ (200 units/ml) stimulation for 30 min. To evaluate the TGF-β-mediated Smad3 response, CD4 T cells were incubated initially with CaeA (0–0.31 μm) for 24 h, followed by IFN-γ (200 units/ml) treatment for 48 h. Later, cells were pulsed with TGF-β (2 ng/ml) for 1 h. [1]
CD4 T cells stimulated with anti-CD3 (2 μg/ml) and CD28 (1 μg/ml) Abs were cultured with CaeA (0–0.31 μm) for 72 h. Later, cells were pelleted and resuspended in JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) medium (2.5 μg/ml). The cultures were incubated at room temperature for 15–20 min in the dark. Later, cells were washed with flow cytometry staining buffer (2% FBS in PBS) and acquired immediately using a flow cytometer (FACSCalibur), and then the analysis was done using FACSDiva software. [1]

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Naïve CD4+ T cells were isolated from mouse splenocytes and stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies under polarizing conditions for Treg, Th1, or Th17 differentiation in the presence of Caerulomycin A (0–0.15 μM). [1]
Flow cytometry was used to analyze intracellular Foxp3, IFN-γ, and IL-17 expression. Cells were fixed, permeabilized, and stained with fluorescent antibodies. [1]
ELISA was performed to quantify IFN-γ, TNF-α, and IL-17 levels in culture supernatants using sandwich assay with biotin-conjugated secondary antibodies and streptavidin-HRP. [1]
Mitochondrial membrane potential was assessed using JC-1 dye, and fluorescence shift from green to red was measured by flow cytometry. [1]
Treg functional suppression assays were conducted by co-culturing Tregs with effector CD4+ T cells and analyzing CD69 expression and alloresponse in mixed lymphocyte reactions. [1]

Collagen-induced arthritis was established in male DBA/1 mice by intradermal immunization with bovine type II collagen emulsified in complete Freund’s adjuvant on day 0, followed by a booster on day 21. [1]
Caerulomycin A was administered daily at 1 or 10 mg/kg body weight, suspended in 0.5% carboxyl methyl cellulose emulsion, for 50 days starting after booster immunization. [1]
Disease severity was scored based on limb inflammation and swelling. Mice were sacrificed on day 30 for immunological assays and day 50 for histopathology and fluorescence imaging. [1]
In vivo fluorescence imaging was performed using ProSense 680/750 and OsteoSense 680/800 probes injected intravenously 24 hours before imaging under anesthesia. [1]

[1]. Caerulomycin A enhances transforming growth factor-β (TGF-β)-Smad3 protein signaling by suppressing interferon-γ (IFN-γ)-signal transducer and activator of transcription 1 (STAT1) protein signaling to expand regulatory T cells (Tregs). J Biol Chem. 2014 Jun 20;289(25):17515-28.

Caerulomycin A is a pyridine alkaloid, formed by the substitution of the 4-methoxy group and the 6-(E)-(hydroxyimino)methyl group at the 4-position of 2,2'-bipyridine. It was isolated from the marine actinomycete Actinoalloteichus cyanogriseus and possesses antitumor activity. It is both an antitumor drug and a marine metabolite and a bacterial metabolite. Caerulomycin A is an aldoxime compound, belonging to the aromatic ether class, a bipyridine compound, and also a pyridine alkaloid. It is derived from the hydride of 2,2'-bipyridine. Caerulomycin A has been reported to exist in Actinoalloteichus cyanogriseus, and relevant data are available. Caerulomycin A is a bipyridine compound initially known for its antifungal and antibacterial properties; recently, it has also been found to possess immunomodulatory activity that promotes the production of regulatory T cells (Tregs). [1] This mechanism involves upregulating SOCS1 to inhibit the IFN-γ-STAT1 signaling pathway and enhancing the TGF-β-Smad3 signaling pathway, thereby leading to an increase in Foxp3+ Treg cells and suppression of the Th1/Th17 response. [1] Penicillin A has shown potential as a treatment for autoimmune diseases such as rheumatoid arthritis by inducing Treg cell proliferation and antigen-specific tolerance. [1]

C12H11N3O2

229.23

229.085

C, 62.87; H, 4.84; N, 18.33; O, 13.96

21802-37-9

135514797

White to off-white solid powder

1.23g/cm3

400.4ºC at 760mmHg

195.9ºC

1.96

1

5

3

17

260

0

COC1=CC(C2=NC=CC=C2)=NC(/C=N/O)=C1

JCTRJRHLGOKMCF-RIYZIHGNSA-N

InChI=1S/C12H11N3O2/c1-17-10-6-9(8-14-16)15-12(7-10)11-4-2-3-5-13-11/h2-8,16H,1H3/b14-8+

(E)-4-methoxy-[2,2'-bipyridine]-6-carbaldehyde oxime

Carulomycin A; AC1NTHG8; AC1N-THG8; AC1N THG8 NSC-114341; NSC114341; NSC 114341; HE185727; HE 185727; HE-185727; Caerulomycin A

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 : ~150 mg/mL (~654.34 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.91 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 25.0 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: ≥ 2.5 mg/mL (10.91 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (10.91 mM)

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