Antiviral; HRV (human rhinovirus) 3C protease
Rupintrivir targets the human rhinovirus (HRV) 3C protease, a cysteine protease that is essential for viral replication. As a peptidomimetic irreversible inhibitor, rupintrivir binds covalently to the active site of the 3C protease, blocking the processing of viral polyproteins into functional proteins. This inhibition prevents the formation of the viral replication complex and halts viral replication. Rupintrivir also exhibits immunomodulatory properties.

Rupintrivirvr (AG7088), in H1-HeLa and MRC-5 cell protection assays, inhibited the replication of all HRV serotypes (48 of 48) tested, as well as that of related picornaviruses, such as coxsackieviruses A21 and B3, enterovirus 70, and echovirus 11. The mean 50% effective concentration (EC50) was 0.023 μM (range, 0.003 to 0.081 μM) and the mean EC90 was 0.082 μM (range, 0.018 to 0.261 μM).
In vitro, rupintrivir exhibits antiviral activity against 48 HRV serotypes with a mean EC50 of 0.023 μM in H1-HeLa and MRC-5 cell protection assays. It is a selective inhibitor of HRV 3C protease with an EC50 of 5 nM. Rupintrivir inhibits EV71 and EV-D68 replication. The compound's antiviral activity has been confirmed in various cell-based assays measuring viral replication and cytopathic effect. Rupintrivir also exhibits immunomodulatory properties.

Rupintrivirvr (AG7088) decreases the TH-2 cytokine IL-4 that is induced by RV in ex vivo HDM-sensitized mouse lung slices (PCLS)[2].
In vivo, rupintrivir has shown potential in clinical studies for reducing viral load and alleviating symptoms in patients with the common cold and other HRV-related illnesses. However, specific in vivo efficacy data in animal models are not extensively detailed in the available literature. The compound's irreversible inhibition of HRV 3C protease makes it a valuable tool in antiviral research. It has been evaluated in clinical trials for the treatment of rhinovirus infections.

Enzyme assays.[1]
The proteolytic activity of HRV 14 3C protease was measured by a continuous fluorescence resonance energy transfer assay as described previously. In brief, cleavage of the substrate peptide was monitored by the appearance of fluorescent emission at 490 nm (following excitation at 336 nm) in a Perkin-Elmer LS50-B spectrophotometer. Data were analyzed with the nonlinear regression analysis program ENZFITTER, which calculates a first-order rate constant for the inactivation of HRV 14 3C protease. Protease selectivity assays were performed with commercially available proteases (at approximately 10 nM concentrations) essentially as described by the supplier.

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Analysis of proteolytic processing. [1]
The ability of Rupintrivirvr (AG7088) to inhibit HRV 14 3C-mediated proteolytic processing was assessed by polyacrylamide gel electrophoresis (PAGE) of radiolabeled sodium dodecyl sulfate (SDS)-solubilized lysates of HRV 14-infected cells. Initially, H1-HeLa cells were infected with HRV 14 at an MOI of 10. Eight and one-half hours after infection, the cells were washed with PBS and the medium was replaced with methionine- and cysteine-deficient medium. At 9 h after infection, appropriate concentrations of compounds were added. After a 30-min exposure to compounds, 50 μCi of [35S]Met-[35S]Cys (Expre35S 35S protein label) was added. One hour later, the monolayers were washed twice with cold PBS and lysed in 250 μl of radioimmunoprecipitation assay buffer (50 mM Tris-Cl [pH 8.0], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS), sonicated, and stored at −70°C for subsequent analysis. Proteins present in the solubilized cell lysates were resolved by 12% PAGE. Following electrophoresis, gels were stained with Coomassie brilliant blue, destained, and treated with Amplify. Gels were air dried overnight in cellulose sheets and exposed to film at −80°C.
In vitro enzyme/receptor binding assays for rupintrivir are protease inhibition assays using purified HRV 3C protease. The enzyme is incubated with a fluorogenic peptide substrate and varying concentrations of rupintrivir, and the rate of substrate cleavage is measured fluorometrically. The IC50 for inhibition of 3C protease activity is determined from dose-response curves. Irreversible inhibition is confirmed by pre-incubation and dilution experiments.

Time-of-addition assay. [1]
Subconfluent monolayers of H1-HeLa cells in six-well plates were infected with HRV 14 at an MOI of 15. After 1 h of adsorption, cell monolayers were washed three times with phosphate-buffered saline (PBS) and replenished with medium. AG7088 (0.5 μM) or WIN 51711 (3.0 μM) was added at concentrations 20-fold above the EC50 (as determined by the cell protection assay) at the time of infection and at various times thereafter. Eight hours after infection, samples were processed by three freeze-thaw cycles followed by sonication for 15 s and clarification by centrifugation (5 min at 15,000 × g at 4°C). Clarified cell and supernatant lysates were stored at −70°C for subsequent analysis for infectious virus.

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HCMV antiviral assay. [1]
The antiviral activity of AG7088 against HCMV AD169 replication in MRC-5 cells was determined by an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody MAb directed against the HCMV major immediate-early gene product. Briefly, following a 2-h virus adsorption, the inoculum was removed and medium containing the appropriate concentrations of compound was added. Five days after infection, the MRC-5 monolayers were incubated with MAb 810, followed by goat anti-mouse antibody conjugated with horseradish peroxidase, and viral antigen was then detected spectrophotometrically at 650 nm with the tetramethylbenzidine liquid substrate system. The EC50 was calculated as the concentration of compound that reduced the optical density to 50% of that of the virus control. The CC50 was measured by the XTT reduction method as described above.

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RV infection of PCLS[2]
Two PCLS per well were infected with 1 × 105 IU/mL of RV for 48 h in culture medium at cell culture conditions (33 °C, 5% CO2, and 100% humidity). To determine the replication dependent immune response, UV-inactivated virus (260 nm for 1 h) from the same batch was used under the same conditions. Pharmacological intervention was performed by infection with RV (1 × 10~5 IU/mL) in the presence of 100 nM rupintrivir.
In vitro cellular assays for rupintrivir are performed using HRV-infected cell lines, such as H1-HeLa or MRC-5 cells. Cells are infected with HRV and treated with varying concentrations of rupintrivir. Viral replication is assessed by measuring the cytopathic effect, viral RNA levels, or plaque formation. Cytotoxicity is evaluated using MTT assays to determine the selectivity index. The EC50 for inhibition of viral replication is determined from dose-response curves.

Mice were sensitized with HDM. Precision-cut lung slices (PCLS) were prepared from HDM-sensitized or non-sensitized mice. Lung slices were infected ex vivo with RV or RV together with rupintrivir. Modulation of immune responses was evaluated by cytokine secretion 48 h post infection. Results: In vivo HDM sensitization resulted in a TH-2/TH-17-dominated cytokine response that persisted in PCLS ex vivo. RV infection of PCLS from non-sensitized mice resulted in the induction of an antiviral and pro-inflammatory immune response, as indicated by the secretion of IFN-α, IFN-β, IFN-γ, TNF-α, MCP-1, IP-10, IL-10, and IL-17A. In contrast, PCLS from HDM-sensitized mice showed an attenuated antiviral response, but exaggerated IL-4, IL-6, and IL-10 secretion upon infection. Rupintrivir inhibited exaggerated pro-inflammatory cytokine IL-6 and TH-2 cytokine IL-4 in HDM-sensitized mice.[2]
In vivo animal experiments for rupintrivir are conducted in animal models of rhinovirus infection, such as mice or cotton rats infected with HRV. Animals are administered rupintrivir via oral or intranasal administration, and viral load in the respiratory tract is measured by quantitative PCR. Clinical signs such as sneezing, nasal discharge, and lung inflammation are assessed. However, detailed protocols are not extensively documented in the available literature.

Rupintrivir has a molecular weight of 598.66 g/mol and a molecular formula of C31H39FN4O7. It is a peptidomimetic inhibitor with a purity of ≥98%. The compound should be stored under recommended conditions. Detailed pharmacokinetic parameters such as half-life, Cmax, and bioavailability have been characterized in clinical studies. As an antiviral agent, rupintrivir has been evaluated for oral administration.

Rupintrivir has been evaluated in clinical studies and has been reported to be well-tolerated. Common adverse effects may include mild to moderate gastrointestinal disturbances, headache, or other treatment-emergent adverse events. No significant safety concerns have been reported in clinical trials. Comprehensive safety data are available from clinical studies. The compound is a research tool and is not an approved drug.

[1]. In vitro antiviral activity of AG7088, a potent inhibitor of human rhinovirus 3C protease. Antimicrob Agents Chemother. 1999 Oct;43(10):2444-50.

[2]. Rupintrivir reduces RV-induced TH-2 cytokine IL-4 in precision-cut lung slices (PCLS) of HDM-sensitized mice ex vivo. Respir Res. 2019 Oct 22;20(1):228.

[3]. Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 3. Structure-activity studies of ketomethylene-containing peptidomimetics. J Med Chem. 1999 Apr 8;42(7):1203-12.

Rupintrivir is an investigational rhinovirus 3C protease inhibitor for the treatment of human rhinovirus (HRV) infection. In H1-HeLa cell protection assays, rupintrivir was effective against all 48 HRV serotypes. It is designed to combat the common cold caused by rhinovirus. Rupintrivir is a peptide mimicry inhibitor with activity against human rhinovirus. It is an irreversible 3C protease inhibitor that blocks viral protein processing, thereby inhibiting viral replication. Its use for the treatment of viral infections is under investigation. Mechanism of Action: The 3C protease (3CP) is a core enzyme in rhinovirus replication. AG7088 prevents rhinovirus replication in respiratory cells by binding to and inhibiting this enzyme, thus preventing the onset of cold symptoms.
Rupintrivir is a potent and selective irreversible inhibitor of human rhinovirus 3C protease. It is also known as AG7088. Rupintrivir exhibits antiviral activity with a mean EC50 of 0.023 μM against 48 HRV serotypes. It also exhibits immunomodulatory properties. Rupintrivir has shown potential in clinical studies for reducing viral load and alleviating symptoms in patients with the common cold. It is a research compound and is not an approved drug.

C31H39FN4O7

598.66

598.28

C, 62.19; H, 6.57; F, 3.17; N, 9.36; O, 18.71

223537-30-2

6440352

White to off-white solid powder

1.213g/cm3

866.7ºC at 760mmHg

170-171°C

477.9ºC

1.87E-30mmHg at 25°C

1.537

3.935

3

9

16

43

1010

4

CCOC(=O)/C=C/[C@H](C[C@@H]1CCNC1=O)NC(=O)[C@H](CC2=CC=C(C=C2)F)CC(=O)[C@H](C(C)C)NC(=O)C3=NOC(=C3)C

CAYJBRBGZBCZKO-BHGBQCOSSA-N

InChI=1S/C31H39FN4O7/c1-5-42-27(38)11-10-24(16-21-12-13-33-29(21)39)34-30(40)22(15-20-6-8-23(32)9-7-20)17-26(37)28(18(2)3)35-31(41)25-14-19(4)43-36-25/h6-11,14,18,21-22,24,28H,5,12-13,15-17H2,1-4H3,(H,33,39)(H,34,40)(H,35,41)/b11-10+/t21-,22+,24+,28-/m0/s1

ethyl (S,E)-4-((2R,5S)-2-(4-fluorobenzyl)-6-methyl-5-(5-methylisoxazole-3-carboxamido)-4-oxoheptanamido)-5-((S)-2-oxopyrrolidin-3-yl)pent-2-enoate

AG-7088; AG7088; AG 7088; 223537-30-2; ag7088; Ruprintrivir; Rupintrivir-d4; RGE5K1Q5QW;

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 : ~50 mg/mL (~83.52 mM)

Solubility in Formulation 1: ≥ 1.67 mg/mL (2.79 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 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: 10% DMSO+90% Corn Oil: ≥ 1.67 mg/mL (2.79 mM)

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