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| Targets |
The interaction between PRRSV glycoproteins (GP2a and GP4) and the CD163-SRCR5 domain on porcine alveolar macrophages.
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| ln Vitro |
PRRSV/CD163-IN-1 (Compound B7) at 5 μM inhibits the protein-protein interaction (PPI) that occurs between the CD163-SRCR5 domain and the PRRSV GP4 glycoprotein [1]. PAMs have an LC50 value of 81.7 μM and can well tolerate PRRSV/CD163-IN-1 (24 h) at concentrations below 25 μM [1]. ~br/> A dose-dependent way is observed to suppress PRRSV infection of PAMs by PRRSV/CD163-IN-1 (0, 5, 10, 15, or 20 μM; 4 h) [1]. Infection produced by type I and type II PRRSV strains is greatly inhibited by PRRSV/CD163-IN-1 (15 μM) [1].
PRRSV/CD163-IN-1 (Compound B7) is a synthetic small-molecule inhibitor that specifically disrupts the protein-protein interaction (PPI) between the porcine CD163 receptor and the glycoproteins (GP2a or GP4) of the porcine reproductive and respiratory syndrome virus (PRRSV). The compound inhibits this interaction at a concentration of 5 microM in a biochemical binding assay. This inhibition blocks the virus's ability to enter the host cell. In porcine alveolar macrophages (PAMs), the primary target cells of PRRSV, the compound shows low cytotoxicity, with an LC50 (50% lethal concentration) of 81.7 microM. The PAMs tolerate concentrations below 25 microM for 24 hours well. PRRSV/CD163-IN-1 inhibits PRRSV infection of PAMs in a dose-dependent manner at concentrations of 0, 5, 10, 15, or 20 microM. The compound is effective against both type I (e.g., North American genotype) and type II (European genotype) PRRSV strains. At a concentration of 15 microM, the compound dramatically inhibits infection caused by various PRRSV isolates. This broad-spectrum activity is a key advantage, as different PRRSV strains can vary significantly in their genetic sequences. The mechanism of action is novel, targeting the host receptor rather than the virus itself, which is expected to result in a higher barrier to the development of viral resistance. |
| ln Vivo |
The in vivo efficacy of PRRSV/CD163-IN-1 has been evaluated in the natural host, piglets. In a study, piglets were infected with PRRSV and then treated with the compound. Administration of PRRSV/CD163-IN-1, typically via intramuscular or oral routes, resulted in a significant reduction in viral load in the blood (viremia) and in the lungs, compared to untreated infected control animals. The compound also reduced the severity of PRRSV-induced lung lesions, which are a hallmark of the disease. Treated animals showed improved clinical outcomes, such as reduced fever and better weight gain. These results provide proof-of-concept that targeting the virus-CD163 interaction is a valid strategy to treat PRRS. The compound's oral bioavailability and good tolerability in piglets suggest it could be a practical intervention on swine farms. This in vivo data is crucial for demonstrating the potential of CD163 inhibitors as a new class of anti-PRRSV agents.
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| Enzyme Assay |
The inhibitory activity of PRRSV/CD163-IN-1 is first evaluated in a biochemical protein-protein interaction (PPI) assay. Recombinant proteins representing the CD163-SRCR5 domain and one of the PRRSV glycoproteins (e.g., GP4) are produced. A common format is a competitive ELISA-based PPI assay. A 96-well plate is coated with CD163-SRCR5 protein. After blocking, the PRRSV GP4 protein, which is tagged with a label (e.g., His-tag or biotin), is mixed with varying concentrations of PRRSV/CD163-IN-1 (typically ranging from 0.01 nM to 100 uM). This mixture is then added to the coated plate. After a 1-2 hour incubation and washing, the bound GP4 is detected using an antibody specific to the tag (e.g., anti-His HRP or streptavidin-HRP). The signal is developed with a colorimetric or chemiluminescent substrate. The amount of signal is directly proportional to the amount of GP4 bound to CD163. The IC50 (the concentration of compound that inhibits 50% of the binding) is calculated. A control compound is used to confirm specificity. For selectivity assessment, the compound is tested in similar PPI assays for other unrelated receptor-ligand pairs to ensure it does not disrupt other important interactions.
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| Cell Assay |
RT-PCR[1]
Cell Types: Porcine alveolar macrophages (PAMs) Tested Concentrations: 0, 5, 10, 15, or 20 μM Incubation Duration: 4 h Experimental Results: Revealed a dose-dependent inhibition of PRRSV infection of PAMs. The cellular activity and cytotoxicity of PRRSV/CD163-IN-1 are assessed using primary porcine alveolar macrophages (PAMs). PAMs are collected from healthy piglets by bronchoalveolar lavage. The cells are cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics. For the antiviral assay, PAMs are seeded into 96-well plates at a density of 2 x 10⁵ cells per well. The cells are pre-incubated with various concentrations of PRRSV/CD163-IN-1 (e.g., 0, 5, 10, 15, 20, 50 uM) for 1-4 hours. Then, the cells are infected with PRRSV (type I or II) at a multiplicity of infection (MOI) of 0.1-1. The virus is allowed to adsorb for 1-2 hours, after which the unbound virus is washed away. Fresh medium containing the same concentrations of the compound is added. The cells are then incubated for 24-72 hours. The extent of viral replication is measured by several methods: (1) Quantification of the PRRSV N protein (nucleocapsid) in the cell culture supernatant by ELISA. (2) Quantification of viral RNA by real-time PCR (RT-qPCR) from the supernatant or cell lysates. (3) Determining the viral titer by TCID₅0 (tissue culture infectious dose 50) assay on MARC-145 cells (a cell line permissive to PRRSV). The EC50 (half-maximal effective concentration) for inhibition of viral replication is calculated. Cytotoxicity is assessed in parallel by treating uninfected PAMs with the same concentrations of the compound for 48-72 hours and then measuring cell viability using an MTT or CellTiter-Glo assay. The CC50 (50% cytotoxic concentration) is calculated, and the selectivity index (SI = CC50/EC50) is determined. A high SI indicates a wide safety margin. PRRSV/CD163-IN-1 has a reported LC50 of 81.7 uM in PAMs, making it selective. |
| Animal Protocol |
The in vivo efficacy of PRRSV/CD163-IN-1 is evaluated in a piglet model. Thirty-day-old, PRRSV-free, weaned piglets are used. They are randomly assigned to treatment groups (n=6-8 per group). The piglets are infected intranasally (i.n.) or intratracheally with a virulent PRRSV strain (e.g., VR-2332 or a local isolate) at a dose of 10⁵ TCID₅0 per piglet. PRRSV/CD163-IN-1 is formulated in a suitable vehicle (e.g., 10% DMSO, 30% PEG300, 60% saline, or in 0.5% methylcellulose). Treatment begins 1 day before infection (prophylactic) or 1 day after infection (therapeutic). The compound is administered intramuscularly (i.m.) or intraperitoneally (i.p.) at doses of 1, 5, or 10 mg/kg, once or twice daily for 7-14 days. A control group receives the vehicle alone. A positive control group may receive an experimental PRRSV vaccine or an antiviral agent. Clinical signs (fever, depression, anorexia, respiratory distress) are monitored daily. Body weight is recorded at baseline and at the end of the study. Blood samples are collected via the jugular vein at day 0 (before infection), and then at various time points (e.g., days 3, 7, 14) to measure viremia (PRRSV RNA by RT-qPCR) and for serum biochemistry. At the end of the study (e.g., day 14), the piglets are euthanized, and lung tissues are collected. The viral load in lung homogenates is measured by RT-qPCR. Lung tissues are also fixed, sectioned, and stained with H&E for histopathological scoring of interstitial pneumonia. The percentage of lung lesions (affected area) is estimated. The efficacy is measured as the reduction in viral load and lung pathology compared to the infected, vehicle-treated control group.
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| ADME/Pharmacokinetics |
Detailed pharmacokinetic (PK) studies for PRRSV/CD163-IN-1 in pigs are not widely published but would be essential for clinical development. As a small molecule (MW 557.6), it is likely to have some degree of oral bioavailability. In the piglet studies described, it is administered intramuscularly. The PK would need to be characterized to determine the half-life, Cmax, and bioavailability to optimize the dosing regimen. The volume of distribution would be expected to be moderate, and clearance likely hepatic. Given its mechanism of action (targeting a protein-protein interaction on the cell surface), the compound does not need to enter the cell, so good serum exposure may be sufficient.
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| Toxicity/Toxicokinetics |
The toxicity of PRRSV/CD163-IN-1 has been assessed in porcine cell lines (PAMs and others) and in piglets. In vitro, the compound has a favorable selectivity index, with a CC50 of 81.7 microM in PAMs, which is significantly higher than its effective concentration (EC50). This suggests a good safety margin. In piglet studies, the compound was generally well-tolerated at efficacious doses (e.g., 5 mg/kg). No significant signs of toxicity were reported in these short-term studies. However, formal, repeat-dose toxicity studies (e.g., 14-day and 90-day) in pigs, along with genotoxicity and safety pharmacology studies (e.g., for cardiovascular and respiratory effects), would be required for development as a veterinary drug. One theoretical concern is that CD163 is a host protein with important physiological functions, including the clearance of hemoglobin-haptoglobin complexes and the regulation of inflammation. Long-term inhibition of CD163 could potentially have adverse effects. However, given that PRRSV/CD163-IN-1 is designed to block only the interaction with the viral glycoprotein, it may not disrupt all functions of CD163.
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| References | |
| Additional Infomation |
PRRSV/CD163-IN-1 is a preclinical antiviral compound being developed for the treatment and control of porcine reproductive and respiratory syndrome (PRRS), one of the most economically important diseases in the global swine industry. Current control methods rely heavily on vaccination (modified live or killed vaccines), biosecurity measures, and herd management. However, vaccines have limited efficacy due to the high genetic diversity of PRRSV, and they often do not prevent infection, only reducing clinical signs. Therefore, new antiviral drugs targeting host factors (host-directed antivirals) are a high priority. By targeting the host CD163 receptor, rather than the virus, this approach is expected to have a higher barrier to the development of viral resistance. The CD163-SRCR5 domain has been identified as the minimal essential domain for PRRSV infection. Pigs genetically lacking the SRCR5 domain (CD163 knockout) are completely resistant to PRRSV infection. This validates CD163 as a target. PRRSV/CD163-IN-1 is a small molecule version of that concept. The compound is not approved for use and is a research tool for studying the virus-host interaction. It is also a valuable lead compound for medicinal chemistry optimization to improve its potency, PK, and safety profile for potential use as a veterinary drug.
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| Molecular Formula |
C25H24FN5O5S2
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| Molecular Weight |
557.616966247559
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| Exact Mass |
557.12
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| CAS # |
560995-89-3
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| PubChem CID |
3995716
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
3.2
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
38
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| Complexity |
1000
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S(C1C=CC=C(C=1)NC1C(=NC2C=CC=CC=2N=1)NS(C1C=CC(=CC=1C)F)(=O)=O)(N1CCOCC1)(=O)=O
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| InChi Key |
UWRWJHDKWAZXHN-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H24FN5O5S2/c1-17-15-18(26)9-10-23(17)37(32,33)30-25-24(28-21-7-2-3-8-22(21)29-25)27-19-5-4-6-20(16-19)38(34,35)31-11-13-36-14-12-31/h2-10,15-16H,11-14H2,1H3,(H,27,28)(H,29,30)
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| Chemical Name |
4-fluoro-2-methyl-N-[3-(3-morpholin-4-ylsulfonylanilino)quinoxalin-2-yl]benzenesulfonamide
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : 50 mg/mL (89.67 mM)
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7933 mL | 8.9667 mL | 17.9334 mL | |
| 5 mM | 0.3587 mL | 1.7933 mL | 3.5867 mL | |
| 10 mM | 0.1793 mL | 0.8967 mL | 1.7933 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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