HIV-1; Claficapavir (A1752) primarily targets HIV-1 nucleocapsid protein (NC), with a dissociation constant (Kd) of around 20 nM detected by SPR assay, showing high binding affinity
This compound has almost no inhibitory effect on HIV-1 reverse transcriptase (RT) and integrase (IN), and these two enzymes are not its functional targets [1].
Claficapavir targets the HIV-1 nucleocapsid protein (NC). It binds strongly to HIV-1 NC with a dissociation constant (Kd) of 20 nM. The compound inhibits the chaperone properties of NC, which are essential for viral replication. By binding to NC, Claficapavir interferes with correct Gag processing and blocks particular chaperone functions such as Psi RNA dimerization and cTAR DNA destabilization.

Claficapavir binds directly to HIV-1 NC, interfering with correct Gag processing and blocking particular chaperone functions of NC, such as Psi RNA dimerization and complementary trans-activation response element (cTAR) DNA destabilization[1].

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1. Anti-HIV activity assay: In MT-4 cells infected with HIV-1 NL4-3/EGFP strain, Claficapavir (A1752) dose-dependently suppresses viral replication. The IC50 value is 1.0–2.0 μM determined by p24 ELISA, and its antiviral potency is comparable to tenofovir [1]
2. Cytotoxicity assay: The compound shows no obvious cytotoxicity at concentrations up to 50 μM, and its CC50 is much higher than 50 μM, presenting a favorable therapeutic index [1]
3. Target binding assay: SPR and intrinsic tryptophan fluorescence quenching assays verify that it binds to HIV-1 NC in a dose-dependent manner with a Kd of about 20 nM [1]
4. NC chaperone function inhibition: It dose-dependently inhibits NC-mediated Psi RNA dimerization and the formation of NC-Psi RNA complexes. It also blocks NC-induced cTAR DNA destabilization, while traditional inhibitors DIBA and SAMT have minimal effects on this process [1]
5. Viral infectivity assay: Progeny viruses produced after drug treatment show sharply reduced infectivity. At 1 μM, the synthesis of early reverse transcription product (-)ssDNA is inhibited by 60%, and the inhibition rate reaches nearly 90% at 5 μM [1]
6. Gag processing assay: It disrupts normal cleavage of Gag polyprotein, leading to accumulation of abnormal proteins such as 30 kD CA-NC and CA-SP1, which hinders viral maturation [1]
7. Viral core stability assay: It causes excessive stabilization of viral cores and blocks viral uncoating, thereby interrupting the subsequent reverse transcription process [1]
8. Time-of-addition assay: The assay confirms that this compound acts at the late stage of HIV replication including viral assembly and maturation, consistent with lopinavir and SAMT [1]

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In vitro, Claficapavir demonstrates potent antiviral activity against HIV-1 with an IC50 of approximately 1 microM. The compound binds directly to HIV-1 NC with high affinity (Kd = 20 nM), inhibiting its chaperone properties. Claficapavir interferes with correct Gag processing and blocks NC-mediated functions including Psi RNA dimerization and complementary trans-activation response element (cTAR) DNA destabilization. Antiviral efficacy is confirmed in cell-based HIV-1 replication assays.

In vivo activity data for Claficapavir are limited. As a nucleocapsid inhibitor, it has the potential for antiviral efficacy in animal models of HIV-1 infection. However, HIV-1 does not infect standard rodents, limiting in vivo evaluation to specialized models such as humanized mice or non-human primates. The compound's strong binding affinity (Kd = 20 nM) and antiviral activity suggest potential for in vivo efficacy, but published in vivo data are scarce.

1. Surface Plasmon Resonance (SPR) binding assay: Immobilize purified HIV-1 NC protein on the sensor chip. Prepare running buffer containing serially diluted Claficapavir (A1752) and flow across the chip at a constant flow rate. Monitor real-time binding signals, fit kinetic data with the 1:1 Langmuir model to calculate binding affinity and Kd value [1]
2. Tryptophan fluorescence quenching assay: Prepare detection buffer containing 5 μM NC protein, then add test compound with gradient concentrations. Set the excitation wavelength at 280 nm and scan fluorescence emission from 310 nm to 450 nm. Evaluate the binding capacity between the compound and NC according to fluorescence quenching degree [1]
3. Psi RNA dimerization and gel shift assay: Synthesize Psi RNA via in vitro transcription. Pre-incubate NC protein with serially diluted Claficapavir (A1752), then add denatured Psi RNA for reaction. Separate products by non-denaturing PAGE, stain RNA and protein with corresponding dyes respectively, and observe bands of RNA monomers, dimers and protein-nucleic acid complexes [1]
4. cTAR DNA destabilization assay: Use cTAR DNA labeled with fluorophore and quencher at two terminals. Pre-incubate NC protein with test compounds of different concentrations, then add labeled DNA. Set excitation wavelength at 520 nm and emission wavelength at 560 nm, detect fluorescence signals to assess the inhibition on NC chaperone activity [1]
5. Reverse transcriptase activity assay: Add reverse transcriptase, templates and primers into the reaction system, then incubate with gradient concentrations of Claficapavir (A1752). Determine changes of enzyme activity via fluorometric detection [1]
6. Integrase activity assay: Add integrase, substrates and serially diluted test compounds into the reaction system. Detect integrase activity by colorimetric method to judge the inhibitory effect [1]

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In vitro enzyme/receptor binding assays for Claficapavir involve direct binding studies with purified HIV-1 nucleocapsid protein. Binding affinity (Kd) is determined using surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or fluorescence-based assays. The compound's ability to inhibit NC chaperone functions is assessed using RNA or DNA substrate assays, such as Psi RNA dimerization or cTAR DNA destabilization assays. IC50 values are determined from dose-response curves in these functional assays.

1. Cytotoxicity assay: Seed MT-4 cells into 96-well plates and incubate with gradient concentrations of Claficapavir (A1752) for 5 days. Add luminescent reagent to lyse cells and incubate, then detect luminescence signals with microplate reader to calculate cell viability and CC50 [1]
2. Antiviral activity assay: Infect MT-4 cells with HIV-1 NL4-3/EGFP at an MOI of 0.05, and simultaneously add test compounds with different concentrations. After 3 days of culture, detect HIV-1 p24 protein in cell supernatant by ELISA to quantify viral proliferation, and observe EGFP fluorescence as auxiliary verification [1]
3. Viral reinfection assay: Collect cell supernatant after drug treatment, then perform centrifugation, filtration and concentration to obtain purified progeny virus. Take equal amounts of virus to infect fresh MT-4 cells. After 48 hours, detect the proportion of EGFP-positive cells by flow cytometry to evaluate viral infectivity [1]
4. qPCR assay for reverse transcription products: Infect target cells with progeny virus. Extract total cellular DNA after 6 hours of incubation, and detect the level of early reverse transcription product (-)ssDNA by real-time quantitative PCR [1]
5. Viral RNA Northern blot assay: Extract total cellular RNA 3 hours after viral infection, and detect intracellular HIV-1 genomic RNA via Northern blot hybridization [1]
6. Transfection assay: Transfect 293FT cells with HIV-1 proviral DNA or lentiviral vectors. Replace culture medium with medium containing Claficapavir (A1752) 6 hours post-transfection. After 48 hours, detect cellular fluorescence, supernatant p24 content and progeny virus infectivity respectively [1]
7. Colony formation assay: Culture HT1080 cells infected with lentivirus. Conduct drug screening and crystal violet staining, then count cell colonies to determine viral titer [1]
8. Time-of-addition (TOA) assay: Add test compounds at different time points after HIV infecting MT-4 cells. Continuously culture for 24 hours and collect supernatant regularly to detect p24 content, so as to confirm the acting phase of the compound in viral replication cycle [1]
9. Western blot assay: Collect viral particles and perform protein denaturation. Separate proteins by SDS-PAGE and transfer to membranes. Incubate with specific antibodies against Gag, CA, NC and RT for imaging, and analyze Gag cleavage and viral protein expression [1]

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In vitro cell-based assays for Claficapavir use HIV-1-infected cell lines such as MT-4, CEM, or peripheral blood mononuclear cells (PBMCs). Cells are infected with HIV-1 and treated with serial dilutions of Claficapavir for 3-7 days. Antiviral activity is measured by quantifying HIV-1 p24 antigen production, reverse transcriptase activity, or viral RNA levels by qPCR. IC50 values are calculated from dose-response curves. Cytotoxicity is assessed in parallel using MTT or CellTiter-Glo assays.

In vivo animal studies for Claficapavir would typically use HIV-1-infected humanized mouse models or SHIV-infected non-human primates for efficacy evaluation. The compound would be administered via oral, intravenous, or intraperitoneal routes at various doses. Viral load (plasma HIV-1 RNA) and CD4+ T cell counts would be monitored over time. Pharmacokinetic parameters would be determined from plasma concentration-time profiles. Standard protocols for antiretroviral drug evaluation would apply.

Pharmacokinetic properties of Claficapavir are not well-characterized in published literature. As a small molecule (MW ~393.86 g/mol), it would be expected to have moderate oral bioavailability and good tissue penetration. The compound has a logP of 3.9, suggesting moderate lipophilicity. Detailed PK parameters such as half-life, clearance, volume of distribution, and bioavailability would require experimental determination in appropriate animal models. The compound is typically dissolved in DMSO for in vitro studies.

In vitro cell tests prove that Claficapavir (A1752) has no obvious toxicity to MT-4 cells at concentrations up to 50 μM, and its CC50 is much higher than 50 μM. The literature has no records of in vivo toxicity, LD50, plasma protein binding rate, long-term toxicity and genotoxicity of this compound [1].

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Toxicity data for Claficapavir are limited. As a research compound targeting HIV-1 NC, it is for research use only and not for human therapeutic use. Standard in vitro cytotoxicity assays in various cell lines would be performed alongside antiviral activity studies to determine the therapeutic index. In vivo tolerability studies in animal models would be required for preclinical development. The compound should be handled with standard laboratory safety precautions for research chemicals.

[1]. Identification and characterization of a new type of inhibitor against the human immunodeficiency virus type-1 nucleocapsid protein. Retrovirology. 2015 Nov 6:12:90. doi: 10.1186/s12977-015-0218-9.

1. Mechanism of action: Claficapavir (A1752) binds HIV-1 NC with high affinity and inhibits its nucleic acid chaperone functions, blocking Psi RNA dimerization and cTAR DNA destabilization. It disrupts normal cleavage of Gag polyprotein and forms structurally abnormal viral cores, leading to defective viral uncoating and interrupted reverse transcription. It acts at the late stage of HIV replication, which is different from reverse transcriptase and integrase inhibitors [1]
2. Drug advantages: It is a novel functional HIV-1 NC inhibitor, distinct from traditional zinc-ejecting NC inhibitors. It has higher target specificity and avoids the cytotoxicity and off-target effects of early similar compounds. It can effectively produce non-infectious virus particles and shows better efficacy than DIBA, SAMT and other analogs [1]
3. Research background: The continuous emergence of drug-resistant HIV strains limits the application of existing anti-HIV drugs. HIV-1 NC is an essential multifunctional protein for viral replication and a promising new drug target. This compound is screened out via cell-based models and provides a new direction for anti-HIV drug research [1]
4. Control drugs: Classic anti-HIV agents including tenofovir, zidovudine, raltegravir, lopinavir, DIBA and SAMT are used as positive or negative controls for activity and phase comparison in all experiments [1]

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Claficapavir (A1752) is a specific nucleocapsid protein inhibitor with an IC50 of approximately 1 microM against HIV-1. It binds strongly to HIV-1 NC with a Kd of 20 nM, inhibiting its chaperone properties. The compound interferes with correct Gag processing and blocks functions such as Psi RNA dimerization and cTAR DNA destabilization. It is used for HIV-1 replication studies and as a lead for antiretroviral drug development. It is for research use only.

C17H12CLNO4S2

393.86448097229

392.989

C, 51.84; H, 3.07; Cl, 9.00; N, 3.56; O, 16.25; S, 16.28

2055732-24-4

1286537

Yellow to orange solid powder

3.9

1

6

5

25

589

0

C1=CC(=CC=C1C2=CC=C(O2)/C=C\3/C(=O)N(C(=S)S3)CCC(=O)O)Cl

YZLFZFALAZYTCI-ZROIWOOFSA-N

InChI=1S/C17H12ClNO4S2/c18-11-3-1-10(2-4-11)13-6-5-12(23-13)9-14-16(22)19(17(24)25-14)8-7-15(20)21/h1-6,9H,7-8H2,(H,20,21)/b14-9-

3-[(5Z)-5-[[5-(4-chlorophenyl)furan-2-yl]methylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]propanoic acid

Claficapavir; A1752; A-1752; A 1752; 2055732-24-4; Claficapavir [INN]; H8ENW8T6SL;

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 : 200 mg/mL (507.79 mM)

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.)