IFNAR2 (Interferon alpha/beta receptor 2). It functions as an agonist that binds to IFNAR2 and activates the JAK/STAT signaling pathway. [2]

JAK1; IFNAR2

CDM-3008, also known as RO8191, exhibits antiviral activity that is reliant on IFNAR2/JAK1 but not IFNAR1/Tyk2. IFNAR2 agonist RO8191 is known to activate IFN signaling in mice [1]. HCV replicon activity was significantly suppressed by RO8191 (0.08, 0.4, 2, 10 μM; for 72 hours) in a dose-dependent way. The HCV NS3 and NS4A protein levels are mostly decreased by RO8191 in the replicon cells' perinuclear area [1]. Viral proteins including NS3, NS4A/B, and NS5A/B vanish when exposed to RO8191 (0.08–10 μM) over 72 hours [1].

---

CDM-3008 inhibits hepatitis B virus (HBV) replication in primary cultured human hepatocytes (PXB cells). In PXB cells infected with HBV genotype C, treatment with CDM-3008 for 7 days reduced cellular HBV DNA levels in a dose-dependent manner, with a half-maximal inhibitory concentration (IC50) of 0.1 μM. [2]
CDM-3008 treatment significantly reduced the levels of covalently closed circular DNA (cccDNA) in HBV-infected PXB cells at concentrations of 10-100 μM. [2]
CDM-3008 treatment significantly decreased both HBsAg (at 10-100 μM) and HBeAg (at 0.1-100 μM) levels in the cell culture medium of HBV-infected PXB cells. [2]
The anti-HBV activity of CDM-3008 is dependent on IFNAR2. Knockdown of IFNAR2 using specific siRNA in HCV replicon cells suppressed the CDM-3008-induced expression of the interferon-stimulated gene (ISG) OAS1. [2]
In HepG2-derived Hep38.7-Tet cells, which are relatively defective in ISG expression, the IC50 of CDM-3008 for anti-HBV activity was greater than 100 μM, more than 1000 times higher than in PXB cells. This suggests its anti-HBV activity is primarily mediated by ISGs. [2]
CDM-3008 and entecavir (ETV) showed an additive effect on inhibiting HBV replication. In PXB cells, combined treatment with 0.1 μM CDM-3008 and 0.1 nM ETV significantly reduced HBV DNA levels compared to either treatment alone. [2]
Microarray analysis of PXB cells treated with CDM-3008 (30 μM) or IFNα (10 ng/ml) for 4 and 8 hours showed that CDM-3008 induced a similar pattern of gene expression, particularly activating the interferon signaling pathway. The number of genes upregulated by CDM-3008 (257 at 4h, 245 at 8h) was higher than those upregulated by IFNα (170 at 4h, 182 at 8h). [2]
CDM-3008 induced the expression of multiple ISGs, including OAS1 (involved in pgRNA degradation), ISG20 (involved in degrading RNA-DNA complexes), and APOBEC3F and APOBEC3G (involved in cccDNA degradation). qPCR analysis confirmed significant upregulation of these genes compared to DMSO control. [2]
CDM-3008 induced stronger feedback inhibition of the JAK/STAT pathway than IFNα. It more potently upregulated suppressors of cytokine signaling, including SOCS1, SOCS2, SOCS3, and CISH. It also downregulated IFNAR2 expression at 4 hours. [2]
In a time-course experiment, OAS1 expression in PXB cells treated with CDM-3008 peaked at 8 hours, decreased at 24 hours, and increased again at 48 hours, showing a pattern of feedback regulation. [2]
CDM-3008 induced a stronger state of refractoriness to further interferon stimulation than IFNα. When PXB cells were pre-treated with CDM-3008 for 24 hours and then stimulated with IFNβ, the increase in OAS1 expression was not significant, whereas pre-treatment with IFNα allowed for a significant increase upon IFNβ stimulation. [2]
CDM-3008 specifically induced several genes not highly induced by IFNα, including SDS, SOCS3, C11orf96, SAA2, and CISH. qPCR confirmed the specific upregulation of SOCS2, SOCS3, CISH, WFDC2, and SLPI. [2]
3D-PCR analysis suggested that CDM-3008 treatment leads to the degradation of cccDNA molecules that have undergone deamination. While random C-to-T and G-to-A transitions were found in cccDNA from DMSO-treated cells, specific mutations (C-to-A at position 45, A-to-C at position 83) were enriched in the remaining cccDNA from CDM-3008-treated cells, indicating edited cccDNA was preferentially degraded. [2]

In the liver of 6-week-old C57BL/6J mice, RO8191 (CDM-3008; 30 mg/kg; oral) dramatically increased the expression of the antiviral genes Oas1b, Mx1, and Pkr [1].

---

Oral administration of RO8191 induces ISG expression in mouse liver. Twenty-four hours after oral administration of 30 mg/kg RO8191 to mice, real-time RT-PCR analysis of liver tissue showed significant induction of multiple ISGs, including Oas1b, Mx1, Pkr, Ifit3, Isg15, Mda5, Rig-I, Socs1, Stat1, and Usp18. [1]
RO8191 does not significantly induce inflammatory cytokines and chemokines in mouse liver. [1]
Oral administration of RO8191 reduces HCV titer in a humanized liver mouse model. In chimeric mice with human hepatocytes infected with HCV genotype 1b, daily oral administration of RO8191 at 30 mg/kg for 14 days reduced serum HCV RNA levels. [1]

Surface Plasmon Resonance (SPR) Binding Assay: The direct interaction between RO8191 and the extracellular domain (ECD) of IFNAR2 was evaluated using SPR spectroscopy. Recombinant IFNAR2 ECD protein was immobilized on a sensor chip surface using amine coupling. To stabilize the binding site, the protein was mixed with 2 μM RO8191 during immobilization. RO8191 at concentrations of 0.31 μM and 0.63 μM, and PEG-IFN-α2a as a positive control, were injected as analytes over the sensor chip. Background signal from a control flow cell was subtracted. Kinetic parameters were obtained by globally fitting the resulting sensorgrams to a 1:1 binding model using dedicated software. The analysis yielded KD values of 480.5 nM and 484.5 nM for the two concentrations of RO8191, demonstrating a direct, dose-dependent interaction. An inactive control compound did not show binding. [1]

Anti-HBV Activity Assay in PXB Cells: Primary human hepatocytes (PXB cells) were infected with HBV genotype C for 28 days. Cells were then treated with serial dilutions of CDM-3008 (0.0003–100 μM) for 7 days. Cellular DNA was purified, and HBV DNA and cccDNA (after T5 exonuclease treatment) were quantified by qPCR using specific primers and probes. HBsAg and HBeAg levels in the culture medium were measured by ELISA. Cell viability was assessed using the RealTime-Glo MT Cell Viability Assay. [2]
IFNAR2 Knockdown and ISG Expression: HCV replicon cells were transfected with IFNAR2 siRNA or control siRNA for 2 days. Cells were then treated with 30 μM CDM-3008 for 8 hours. Total RNA was purified, and OAS1 mRNA expression was measured by qPCR to confirm the dependency of CDM-3008's effect on IFNAR2. [2]
Microarray Gene Expression Analysis: PXB cells (HBV-infected or uninfected) were treated with 30 μM CDM-3008 or 10 ng/ml IFNα for 4 or 8 hours. Total RNA was purified and analyzed using Illumina Human HT-12 v4 Expression BeadChips. Data were processed using Bioconductor and analyzed with Ingenuity Pathways Analysis (IPA). [2]
qPCR for Gene Expression Validation: PXB cells were treated with DMSO, 30 μM CDM-3008, or 10 ng/ml IFNα for 4 or 8 hours. Total RNA was reverse transcribed, and the expression levels of specific genes (e.g., SOCS1, STAT2, IFNAR1, IFNAR2, ISG15, USP18, OAS1, ISG20, APOBEC3F, APOBEC3G, SOCS2, SOCS3, CISH, WFDC2, SLPI) were quantified by qPCR using specific primers and SYBR Green or TaqMan probes. [2]
3D-PCR for APOBEC3 Activity: PXB cells infected with HBV for 28 days were treated with 100 μM CDM-3008 or 10,000 IU/ml IFNα for 7 days. Cellular DNA was purified. cccDNA was amplified by PCR, followed by nested PCR for the HBx region with a gradient of denaturation temperatures (80–92 °C). Products were separated by gel electrophoresis, cloned, and sequenced to detect G-to-A or C-to-T transitions indicative of APOBEC3-mediated deamination. [2]
Additive Effect Assay with Entecavir: HBV-infected PXB cells were treated with 0.1 μM CDM-3008, 0.1 nM entecavir (ETV), or a combination of both for 7 days. Cellular HBV DNA levels were measured by qPCR to assess the additive effect. [2]
OAS1 Time-Course Expression: PXB cells were treated with 30 μM CDM-3008 or 10 ng/ml IFNα for 0, 8, 24, and 48 hours. OAS1 mRNA expression was measured by qPCR to analyze the kinetics of ISG induction and feedback regulation. [2]
Interferon Refractoriness Assay: PXB cells were pretreated with 30 μM CDM-3008 or 10 ng/ml IFNα for 24 hours. Cells were then treated with 10 ng/ml IFNβ for 4 hours. OAS1 mRNA expression was measured by qPCR to assess the level of feedback inhibition induced by the pretreatment. [2]

Mouse ISG Induction Study:** Six-week-old C57BL/6J mice were orally administered a single dose of 30 mg/kg RO8191 or vehicle (10% dimethyl sulfoxide and 10% Cremophor). Twenty-four hours after administration, mice were euthanized, and livers were harvested. Total RNA was extracted from liver tissue, and the mRNA expression levels of murine ISGs were measured using real-time RT-PCR. Data were analyzed using Student's t-test, with 4 mice per group. [1]
* **Humanized Liver Mouse Model Efficacy Study:** Chimeric mice with human hepatocytes (generated by transplanting human primary hepatocytes into SCID mice carrying a uPA transgene) were infected with HCV genotype 1b. Five weeks post-infection, with stable HCV viremia (~10^6 copies/mL), mice were treated for 14 days. One group received daily oral administration of 30 mg/kg RO8191. A second group received subcutaneous injections of 30 mg/kg PEG-IFN-α2a twice weekly. Serum samples were collected at various time points, and HCV RNA was quantified using real-time RT-PCR. [1]

---

Mouse ISG Induction Study: Six-week-old C57BL/6J mice were orally administered a single dose of 30 mg/kg RO8191 or vehicle (10% dimethyl sulfoxide and 10% Cremophor). Twenty-four hours after administration, mice were euthanized, and livers were harvested. Total RNA was extracted from liver tissue, and the mRNA expression levels of murine ISGs were measured using real-time RT-PCR. Data were analyzed using Student's t-test, with 4 mice per group. [1]
Humanized Liver Mouse Model Efficacy Study: Chimeric mice with human hepatocytes (generated by transplanting human primary hepatocytes into SCID mice carrying a uPA transgene) were infected with HCV genotype 1b. Five weeks post-infection, with stable HCV viremia (~10^6 copies/mL), mice were treated for 14 days. One group received daily oral administration of 30 mg/kg RO8191. A second group received subcutaneous injections of 30 mg/kg PEG-IFN-α2a twice weekly. Serum samples were collected at various time points, and HCV RNA was quantified using real-time RT-PCR. [1]

In PXB cells, CDM-3008 did not show dose-dependent cytotoxicity at concentrations up to 100 μM after 7 days of treatment, as measured by the RealTime-Glo MT Cell Viability Assay. [2]
In Hep38.7-Tet cells, CDM-3008 showed no significant cytotoxicity up to 100 μM after 6 days of treatment, as measured by XTT assay. [2]
The study notes that CDM-3008 has problems with solubility and metabolic stability that may affect its use as a drug. A derivative, CDM-3032, was developed with improved solubility and metabolic stability in mouse and human hepatic microsomes. [2]

[1]. An Orally Available, Small-Molecule Interferon Inhibits Viral Replication. Sci Rep. 2012;2:259.

[2]. An interferon-like small chemical compound CDM-3008 suppresses hepatitis B virus through induction of interferon-stimulated genes. PLoS One. 2019 Jun 12;14(6):e0216139.

CDM-3008 (also known as RO8191) is a small chemical compound that functions as an interferon-like molecule. It acts as an agonist for IFNAR2, activating the JAK/STAT pathway and inducing the expression of interferon-stimulated genes (ISGs). [2]
Unlike IFNα, which requires injection, CDM-3008 is potentially available for oral administration and can be produced at low cost, offering significant advantages. [2]
Orally administered CDM-3008 is thought to be absorbed from the intestine and delivered to the liver through the portal vein, potentially reducing side effects compared to systemically injected IFNα. [2]
CDM-3008 induces ISG expression specifically through IFNAR2, whereas IFNα requires a heterodimer of IFNAR1 and IFNAR2. This specificity may reduce side effects associated with IFNAR1 downstream signaling. [2]
CDM-3008 shows stronger feedback inhibition of the JAK/STAT pathway than IFNα, which may help in regulating potential side effects. [2]
The compound is also known as a "cccDNA modulator" due to its ability to reduce cccDNA levels in HBV-infected cells, likely through the induction of APOBEC3 family proteins. [2]

C14H5F6N5O

373.212822675705

373.039

691868-88-9

2768133

Off-white to yellow solid powder

3.7

0

11

1

26

531

0

FC(C1C=C(C(F)(F)F)N=C2C=1C=CC1=NC(C3=NN=CO3)=CN21)(F)F

GRHYZVJEXKTJOS-UHFFFAOYSA-N

InChI=1S/C14H5F6N5O/c15-13(16,17)7-3-9(14(18,19)20)23-11-6(7)1-2-10-22-8(4-25(10)11)12-24-21-5-26-12/h1-5H

2-[2,4-bis(trifluoromethyl)imidazo[1,2-a][1,8]naphthyridin-8-yl]-1,3,4-oxadiazole

RO8191 CDM-3008 RO 8191 CDM3008 RO-8191

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

DMSO : ~5 mg/mL (~13.40 mM)

Solubility in Formulation 1: 0.5 mg/mL (1.34 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 sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.0 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.

---

Solubility in Formulation 2: 0.5 mg/mL (1.34 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.0 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.

---

View More

Solubility in Formulation 3: ≥ 0.5 mg/mL (1.34 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 5.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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