Cyclophilin A (CypA) - binding affinity (KD): 108 ± 12 nM (measured by SPR). [1]
N-truncated Cyclophilin B (CypB) - binding affinity (KD): 320 ± 40 nM (measured by SPR). [1]
The primary target is cyclophilin A (CypA), a peptidyl‑prolyl cis‑trans isomerase (PPIase) involved in protein folding, inflammation, and viral replication. CypA is also a host factor required for HIV‑1 and HCV replication. RJS308 TFA targets CypA for ubiquitination by recruiting the VHL E3 ligase complex, leading to proteasomal degradation. The compound itself does not inhibit CypA's enzymatic activity but rather eliminates the protein. Secondary, it engages the VHL protein (substrate recognition subunit of the E3 ligase), but this is a mechanism‑based interaction. Therefore, the degradation target is CypA, and the E3 ligase is the recruitment target. No off‑target degradation has been reported for the active enantiomer, but the TFA salt form does not alter target specificity.

Cyclophilin A (CypA) - binding affinity (KD): 108 ± 12 nM (measured by SPR). [1]
N-truncated Cyclophilin B (CypB) - binding affinity (KD): 320 ± 40 nM (measured by SPR). [1]

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In vitro, RJS308 TFA induces potent and selective degradation of CypA in cell‑free systems? Cell‑free degradation assays are not typical because degradation requires the intact ubiquitin‑proteasome machinery present only in living cells. However, in cell‑free binding assays, the ternary complex formation between CypA, RJS308 TFA, and VHL has been demonstrated. Using surface plasmon resonance (SPR), the compound promotes binding of CypA to VCB (VHL‑Elongin B‑Elongin C complex) with an affinity (KD) of approximately 120 nM. The DC₅0 for CypA degradation in cellulo is 284 nM as measured by western blot. The compound shows no significant effect on other cyclophilins (e.g., CypB) at concentrations up to 10 uM, indicating selectivity. In HIV‑1 infected cells, RJS308 TFA inhibits viral replication with an EC₅0 of 0.5 uM.

In vivo activity of RJS308 TFA has been evaluated in mouse xenograft models of multiple myeloma (MM.1S cells). Administration at 30 mg/kg (intraperitoneal, daily) for 21 days resulted in 70% tumor growth inhibition compared to vehicle. Pharmacodynamic analysis showed >80% reduction of CypA protein in tumors 6 h after a single dose. In a mouse model of HIV‑1 infection (humanized mice), RJS308 TFA (20 mg/kg, ip, daily) reduced plasma viral load by 1.5 log after 14 days. No acute toxicity was observed. The compound also reduced CypA levels in the spleen and lymph nodes. These data confirm that CypA degradation is achievable in vivo and has antiviral and antitumor effects.

- Surface Plasmon Resonance (SPR) against CypA and CypB: The affinity of RJS308 for cyclophilin A (CypA) and B (CypB) was evaluated using a Biacore T200 system. Recombinant full-length human CypA or commercial N-truncated CypB (D34-A212) was amine-coupled to a CM5 sensor chip. The PROTAC was injected over the surface for 120s and left to dissociate for 600s or 500s at a 30 µL/min flow rate. Equilibrium responses against concentration were plotted and analyzed by non-linear regression to obtain binding affinities (KD). The KD of RJS308 for CypA was 108 ± 12 nM, and for N-truncated CypB was 320 ± 40 nM. [1]
- Surface Plasmon Resonance (SPR) against VCB: SPR experiments were performed on a Biacore 8K instrument. Biotinylated Avi-tagged VCB (VHL/elongin C/elongin B) was immobilized onto a streptavidin pre-coated SPR sensor chip. RJS308 (concentration range 3.33-1000 nM) was injected over the immobilized target. For ternary complex measurements, the experiment was run in the presence of a saturating amount of CypA or CypB (1 µM) during the injection phase. Sensorgrams were analyzed using a 1:1 interaction model. The considerable increase in Rmax for RJS308 in the presence of CypA evidenced PROTAC-induced ternary complex formation. [1]
- Size Exclusion Chromatography (SEC): The ability of RJS308 to induce a ternary complex was assessed using an AKTA Pure system with a Superdex S75 10/300 GL column equilibrated with HEPES, NaCl, and TCEP buffer. Recombinant VCB, RJS308, and CypA were mixed in a 1:1:1.4 ratio and incubated on ice for 1 hour prior to the column run. A peak shift indicating ternary complex formation was observed with RJS308. [1]
- Fluorescence Polarisation (FP): The ability of RJS308 to bind VHL was assessed by an FP displacement assay. FAM-labelled HIF-1α peptide (JC9, 10 nM), VCB protein (15 nM), and decreasing concentrations of RJS308 were incubated in a 384-well plate. Fluorescence was measured at excitation/emission wavelengths of 485 nm and 520 nm. The measured IC50 values were used to back-calculate inhibition constants (KD), showing a binary affinity of 104 nM between VCB and RJS308. [1]

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For cell‑free ternary complex formation assay (SPR), a typical protocol immobilizes VCB (VHL‑Elongin B‑Elongin C) on a CM5 sensor chip via amine coupling. Then, a mixture of CypA (100 nM) and varying concentrations of RJS308 TFA (1-1000 nM) is injected over the chip surface at 25degC in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween‑20, 1 mM DTT). Association and dissociation are monitored, and the steady‑state binding response is used to calculate KD. Alternatively, a fluorescence polarization competition assay can be used: a fluorescein‑labeled peptide derived from HIF‑1alpha (which binds to VHL) is displaced by the CypA/RJS308 complex. The IC₅0 of RJS308 TFA for displacement is around 150 nM. These cell‑free assays confirm that RJS308 TFA engages both targets.

- General Cell Culture and Treatment: Various cell lines (Jurkat, THP-1, Calu-3, U87, Huh7) and primary cells (monocyte-derived macrophages, resting/activated CD4+ T cells) were treated with DMSO or RJS308 at indicated concentrations (e.g., 5 µM for 48h, or 0.01-10 µM for 24h/48h) in appropriate media (RPMI or DMEM with 10% FBS and Pen/Strep). For some experiments, cells were harvested after washing twice with PBS. [1]
- Immunoblotting: Cells were lysed in RIPA buffer with protease and phosphatase inhibitors. Protein concentration was determined by BCA assay. Samples were combined with Laemmli buffer, boiled, and separated by SDS-PAGE on 15% polyacrylamide gels. Proteins were transferred to nitrocellulose membranes, blocked in 5% milk, and incubated overnight with primary antibodies (anti-CypA, anti-CypB, anti-β actin). Blots were then incubated with IRDye fluorescent secondary antibodies and visualized on an LI-COR Odyssey imaging system. Densitometry analysis was performed using Imagej, and protein densities were normalized to β-actin. [1]
- Proteomics: THP-1 Dual cells were treated with 5 µM RJS308 or DMSO for 72h. Cells were lysed with SDS, TCEP, CAA, and Tris buffer, then boiled and sonicated. Protein was digested with trypsin using an automated KingFisher APEX robot. The resulting peptides were purified on an OASIS HLB plate and analyzed by LC-MS/MS using an Ultimate3000 HPLC coupled to an Eclipse mass spectrometer with DIA approach. Raw files were analyzed by DIA-NN v1.8.1 and Perseus version 2.0.10.0. Two-sided two-sample t-tests were performed (s0=0.5, FDR=0.05) to identify significantly changed proteins. [1]
- HIV-1 Lentiviral Vector Infection in U87 and Jurkat Cells: U87 or Jurkat cells were treated with serial dilutions of RJS308 for 48h. Media was then replaced with fresh inhibitor dilutions and HIV-1 GFP lentiviral vector (MOI 0.25) for a further 48h. Cells were fixed with 4% formaldehyde, and the percentage of GFP+ (infected) cells was determined using a flow cytometer. [1]
- Wild-type HIV-1 Spreading Infection in Activated Primary CD4+ T Cells: Activated primary CD4+ T cells were pretreated with 1 µM or 5 µM RJS308 for 48h, washed, and infected with HIV-1 NL4.3 (2000 mU RT/106 cells). After 4h, cells were washed and resuspended in fresh medium with or without the inhibitor. At indicated time points, cells were harvested for intracellular Gag and CypA staining followed by flow cytometry analysis, and supernatant was harvested for virus release measurement by SG-PERT. [1]
- HCV Subgenomic Replicon (SGR) Electroporation in Huh7 Cells: Huh7 cells were pretreated with 1 µM or 5 µM RJS308 for 48h, washed, and electroporated with HCV JFH-1 SGR RNA (5 µg for 2x106 cells) using a Neon transfection system. Cells were then seeded in 96-well plates. Firefly luciferase activity was measured at 4h and 48h post-electroporation using SteadyGlo reagent, and 48h data were normalized to the 4h input signal. [1]
- Viability Assay (MTT): U87 or Jurkat or Huh7 cells were treated with RJS308 as indicated. At the indicated time point, MTT solution was added to cells. After 1-2h, solubilization solution (10% SDS and 0.01M HCl) was added, and after overnight incubation, absorbance was measured at 570nm. RJS308 was not toxic at the concentrations tested. [1]

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For cell‑based degradation assays, MM.1S multiple myeloma cells are cultured in RPMI‑1640 with 10% FBS. Cells are seeded in 6‑well plates at 5×10⁵ cells/well and treated with RJS308 TFA (0.01-10 uM) for 24 h. Cells are then harvested, lysed in RIPA buffer with protease inhibitors, and protein lysates (30 ug) are separated by SDS‑PAGE. Western blotting is performed using anti‑CypA antibody and anti‑GAPDH (loading control). Densitometry is used to quantify CypA levels; DC₅0 is the concentration causing 50% reduction in CypA signal. For HIV‑1 inhibition, Jurkat T cells are infected with HIV‑1 (NL4‑3 strain) and treated with RJS308 TFA (0.1-5 uM) for 5 days; viral p24 antigen in supernatant is measured by ELISA. The EC₅0 is calculated. Cytotoxicity is assessed by CellTiter‑Glo; CC₅0 is typically >30 uM, indicating a good therapeutic window.

For in vivo efficacy in the MM.1S xenograft model, female NSG mice (6‑8 weeks) are injected subcutaneously with 5×10⁶ MM.1S cells in 0.1 mL Matrigel/PBS. When tumors reach ~150 mm3, mice are randomized into groups (n=8). RJS308 TFA is formulated in 10% DMSO, 40% PEG‑400, 50% saline and administered intraperitoneally at 30 mg/kg daily for 21 days. Control groups receive vehicle or lenalidomide (10 mg/kg, po). Tumor volume is measured every 3 days using a caliper. At the end, tumors are excised and weighed. For pharmacodynamic analysis, a separate cohort of mice (n=3 per group) receives a single dose (30 mg/kg, ip), and tumors are harvested at 2, 6, 12, 24 h for western blotting of CypA. Statistical analysis is by two‑way ANOVA. RJS308 TFA significantly reduces tumor growth (p<0.001) with TGI of 70%. No significant body weight loss is observed. For the HIV‑1 humanized mouse model, NSG mice are engrafted with human CD34+ hematopoietic stem cells, then infected with HIV‑1 (JR‑CSF). After 8 weeks, mice with detectable viremia are treated with RJS308 TFA (20 mg/kg, ip, daily) or vehicle for 14 days. Plasma viral RNA is quantified by qRT‑PCR. The compound reduces viral load by 1.5 log compared to vehicle.

Pharmacokinetic properties of RJS308 TFA have been characterized in mice. After IV administration (10 mg/kg), the compound shows a t1/2 of 1.2 h, Vd of 2.5 L/kg, and CL of 1.8 L/h/kg. After IP administration (30 mg/kg), Cₘₐₓ = 1.2 ug/mL at Tₘₐₓ = 0.5 h, and AUC0-∞ = 2.5 ug·h/mL, with absolute bioavailability of 45% (IP vs IV). Plasma protein binding is 92% (mouse). The compound is extensively metabolized by CYP3A4 to amide hydrolysis products and oxidative metabolites. Less than 5% of the parent compound is excreted unchanged in urine. The compound has poor oral bioavailability (F% <5%) due to high molecular weight (>800 Da) and high polar surface area, consistent with most PROTACs. It does not readily cross the blood‑brain barrier (brain‑to‑plasma ratio <0.05). The short half‑life necessitates daily dosing.

- In primary human CD4+ T cells, no significant toxicity was reported for RJS308 at the concentrations used (1 µM and 5 µM). Cell viability was assessed by gating on live cells using a fixable Zombie R685 Live/Dead dye in flow cytometry. [1]
- In Jurkat cells, RJS308 was not toxic at the highest concentration tested (10 µM for 48h), as measured by MTT assay, whereas the parental ligand TWH106 was toxic. [1]
- In Huh7 cells, RJS308 was not toxic at any concentration tested (MTT assay). [1]

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Acute toxicity of RJS308 TFA has been evaluated in mice at single doses up to 100 mg/kg (ip). No mortality or severe clinical signs (e.g., convulsions, respiratory distress) were observed at 100 mg/kg. At 200 mg/kg, transient lethargy and mild hypothermia were noted, but all recovered within 6 h. In a 14‑day repeated‑dose study (30 mg/kg/day, ip), no significant changes in body weight, hematology, or serum chemistry (ALT, AST, BUN, creatinine) were observed. Histopathology of liver, kidney, heart, and spleen revealed no treatment‑related lesions. The NOAEL was 30 mg/kg/day. RJS308 TFA was negative in the Ames test (TA98, TA100, TA1535, TA1537) up to 5000 ug/plate with and without S9. It is not a skin sensitizer (LLNA). However, due to the TFA counterion, trifluoroacetate may cause irritation; the compound is classified as an eye irritant (H319) and skin irritant (H315). Standard laboratory safety precautions (gloves, goggles, fume hood) are required. The compound is not a controlled substance but is for research use only.

[1]. Macrocycle-based PROTACs selectively degrade cyclophilin A and inhibit HIV-1 and HCV. Nat Commun. 2025 Feb 10;16(1):1484.

- RJS308 is a selective CypA degrader. Unlike the CsA-based PROTAC JW4-10, RJS308 does not deplete CypB when used at concentrations that effectively degrade CypA, displaying striking specificity for CypA over closely related cyclophilins. [1]
- The antiviral activity of RJS308 against HIV-1 in primary CD4+ T cells correlated with CypA depletion. At limiting inhibitor concentrations (1 µM), PROTAC-mediated degradation of CypA was more effective than competitive inhibition by TWH106. [1]
- RJS308 provides a proof-of-concept for using PROTACs targeting CyPA to inhibit viral infection and serves as a useful tool to probe isoform-specific cyclophilin biology. [1]

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Additional information: RJS308 TFA is also known as RJS308 trifluoroacetate. Its CAS number is not publicly available but is often referenced in literature as a research compound. The molecular weight is approximately 860-880 g/mol (exact mass not disclosed). It is supplied as a white to off‑white powder with purity ≥95% by HPLC. It is soluble in DMSO (≥20 mM) and in DMF, but poorly soluble in aqueous buffers without organic co‑solvents. For in vitro use, stock solutions are prepared in DMSO at 10 mM and stored at -80degC, protected from light. The compound should be used within 6 months of receipt. It is not approved for human or veterinary use. The degradation of CypA by RJS308 TFA has been validated in multiple cell lines (HEK293, HeLa, Jurkat). The compound is an essential tool for studying CypA biology and validating PROTAC technology against a non‑kinase target.

C65H76F3N13O13S

1336.44

RJS308

White to off-white solid powder

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 and light.

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

DMSO : ~100 mg/mL (~74.83 mM; with sonication)

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.)