PEDV 3CLpro (IC50 = 1.11 μM)

GC376 and Molnupiravir exhibit additive activity against SARS-CoV-2 at 72 hours and synergistic activity against SARS-CoV-2 at 48 hours.Introduction: The development of effective vaccines has partially mitigated the trend of the SARS-CoV-2 pandemic; however, the need for orally administered antiviral drugs persists. This study aims to investigate the activity of molnupiravir in combination with nirmatrelvir or GC376 on SARS-CoV-2 to verify the synergistic effect. Methods: The SARS-CoV-2 strains 20A.EU, BA.1 and BA.2 were used to infect Vero E6 in presence of antiviral compounds alone or in combinations using five two-fold serial dilution of compound concentrations ≤EC90. After 48 and 72 h post-infection, viability was performed using MTT reduction assay. Supernatants were collected for plaque-assay titration. All experiments were performed in triplicate, each being repeated at least three times. The synergistic score was calculated using Synergy Finder version 2. Results: All compounds reached micromolar EC90. Molnupiravir and GC376 showed a synergistic activity at 48 h with an HSA score of 19.33 (p < 0.0001) and an additive activity at 72 h with an HSA score of 8.61 (p < 0.0001). Molnupiravir and nirmatrelvir showed a synergistic activity both at 48 h and 72 h with an HSA score of 14.2 (p = 0.01) and 13.08 (p < 0.0001), respectively. Conclusion: Molnupiravir associated with one of the two protease-inhibitors nirmatrelvir and GC376 showed good additive-synergic activity in vitro[2].

GC376, a dipeptidyl bisulfite adduct salt, it is an inhibitor of 3CLpro (3C-like protease) with potent antiviral and coronavirus activity, notably against SARS-CoV.The unprecedented coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a serious threat to global public health. Development of effective therapies against SARS-CoV-2 is urgently needed. Here, we evaluated the antiviral activity of a remdesivir parent nucleotide analog, GS441524, which targets the coronavirus RNA-dependent RNA polymerase enzyme, and a feline coronavirus prodrug, GC376, which targets its main protease, using a mouse-adapted SARS-CoV-2 infected mouse model. Our results showed that GS441524 effectively blocked the proliferation of SARS-CoV-2 in the mouse upper and lower respiratory tracts via combined intranasal (i.n.) and intramuscular (i.m.) treatment. However, the ability of high-dose GC376 (i.m. or i.n. and i.m.) was weaker than GS441524. Notably, low-dose combined application of GS441524 with GC376 could effectively protect mice against SARS-CoV-2 infection via i.n. or i.n. and i.m. treatment. Moreover, we found that the pharmacokinetic properties of GS441524 is better than GC376, and combined application of GC376 and GS441524 had a synergistic effect. Our findings support the further evaluation of the combined application of GC376 and GS441524 in future clinical studies[3].

Porcine epidemic diarrhea virus (PEDV), being highly virulent and contagious in piglets, has caused significant damage to the pork industries of many countries worldwide. There are no commercial drugs targeting coronaviruses (CoVs), and few studies on anti-PEDV inhibitors. The coronavirus 3C-like protease (3CLpro) has a conserved structure and catalytic mechanism and plays a key role during viral polyprotein processing, thus serving as an appealing antiviral drug target. Here, we report the anti-PEDV effect of the broad-spectrum inhibitor GC376 (targeting 3Cpro or 3CLpro of viruses in the picornavirus-like supercluster). GC376 was highly effective against the PEDV 3CLpro and exerted similar inhibitory effects on two PEDV strains. Furthermore, the structure of the PEDV 3CLpro in complex with GC376 was determined at 1.65 Å. We elucidated structural details and analyzed the differences between GC376 binding with the PEDV 3CLpro and GC376 binding with the transmissible gastroenteritis virus (TGEV) 3CLpro. Finally, we explored the substrate specificity of PEDV 3CLpro at the P2 site and analyzed the effects of Leu group modification in GC376 on inhibiting PEDV infection. This study helps us to understand better the PEDV 3CLpro substrate specificity, providing information on the optimization of GC376 for development as an antiviral therapeutic against coronaviruses[1].

Evaluation of antiviral activity in Vero e6 cells[3]
Cell viability was determined using the Cell Titer-Glo kit following the manufacturer’s instructions. Briefly, Vero E6 cells were seeded in 96-well plates with opaque walls. After 12–16 h, the indicated concentrations of GC376 (0, 1, 5, 10, 50, 100, 500 µM), GS441524 (0, 1, 5, 10, 50, 100, 500 µM) and GC376 + GS441524 (0, 0.5, 2.5, 5, 25, 50, 250 µM) were added for 24 h. Cell Titer-Glo reagent was added to each well, and luminescence was measured using a GloMax 96 Microplate Luminometer.
Antiviral activity experiment was determined following a previous method. Briefly, Vero E6 cells were pretreated with the indicated concentrations of GC376 (0, 0.5, 1, 2, 4, 6, 8, 10 µM), GS441524 (0, 0.5, 1, 2, 4, 6, 8, 10 µM) and GC376 + GS441524 (0, 0.25, 0.5, 1, 2, 3, 4, 5 µM) or with vehicle solution (12% sulfobutylether-β-cyclodextrin, pH 3.5) alone for 1 h. The cells were then infected with HRB26 or HRB26M at an MOI of 0.005 and incubated for 1 h at 37°C. The cells were washed with PBS, and virus growth medium containing the indicated amounts of GC376, GS441524 and GC376 + GS441524 or vehicle solution alone was added. The supernatants were collected at 24 h p.i. for viral titration by a PFU assay in Vero E6 cells. Relative viral titres were calculated on the basis of the ratios to the viral titres in the mock-treated counterparts. The data were analyzed using GraphPad Prism 7.0. The results are shown as the mean values with standard deviations of three independent experiments.
In 96-well clear flat-bottom plates, Vero E6 cells (3000 cells/well) were seeded and incubated for 24 hours at 37°C with 5% CO₂. Following incubation, a multiplicity of infection (MOI) of 0.1 was used to infect cells. Adsorption of SARS-CoV-2 was allowed to occur for one hour at 37°C. After the virus inoculum was removed, the cells were covered with media that contained molnupiravir (0.62–50 μM), nirmatrelvir (0.62–50 μM), and GC376 (0.21–16.7 μM) diluted three times. Every plate contained mock-infected cells, infected positive controls (SARS-CoV-2 alone), and negative controls (compounds alone). Following 48 and 72 hours of incubation at 37°C with 5% CO₂, the viability of the cells was assessed using the MTT reduction assay.

Female BALB/c mice
111 or 55.5 mg/kg
i.m.

---

In vivo toxicity study of GC376 and GS441524[3]
The toxicity studies were performed in 4- to 6-week-old female BALB/c mice. BALB/c mice were assigned to four groups (five mice per group), one mock group (i.m. administration of solvent) and three i.m. administered groups: GC376 (40 mM/l, 100 µl), GS441524 (40 mM/l, 100 µl) and GC376 + GS441524 (20 mM/l, 100 µl), respectively. Mice in the mock and experimental groups were weighed daily for 15 days. In addition, blood samples were collected at 0, 5, 10 and 15 days after administration. Various blood chemistry values or blood cell counts were performed at Wuhan Servicebio Biological Technology Co., Ltd. The data were analyzed using GraphPad Prism 7.0.

---

In vivo antiviral study of GC376 and GS441524[3]
Firstly, groups of six 4- to 6-week-old female BALB/c mice were treated i.m. with a loading dose of GC376 (40 or 8 mM/l, 100 µl), GS441524 (40 or 8 mM/l, 100 µl) and GC376 + GS441524 (20 or 4 mM/l, 100 µl), followed by a daily maintenance dose. Alternatively, mice were treated intranasally with a single treatment (GC376, 20 mM/l, 50 µl; GS441524, 20 mM/l, 50 µl; GC376 + GS441524, 10 mM/l, 50 µl) or a combination of GC376 (20 mM/l, 50 µl, i.n. and 40 mM/l, 100 µl, i.m.), GS441524 (20 mM/l, 50 µl, i.n. and 40 mM/l, 100 µl, i.m.) and GC376 + GS441524 (10 mM/l, 50 µl, i.n. and 20 mM/l, 100 µl, i.m.), followed by a daily maintenance dose. As a control, mice were administered vehicle solution (12% sulfobutylether-β-cyclodextrin, pH 3.5) daily. One hour after administration of the loading dose of GC376, GS441524 and GC376 + GS441524 or vehicle solution, each mouse was inoculated intranasally with103.6 PFU of HRB26M in 50 μl. Three mice from each group were euthanized on days 3 and 5 p.i. The nasal turbinates and lungs were collected for viral detection by qPCR and PFU assay according to previously described methods]. The amount of vRNA for the target SARS-CoV-2 N gene was normalized to the standard curve from a plasmid containing the full-length cDNA of the SARS-CoV-2 N gene. The assay sensitivity was 1000 copies/ml. The data were analyzed using Microsoft Excel 2016 and GraphPad Prism 7.0.

---

Pharmacokinetics study of GC376 and GS441524 in BALB/c mice and SD rats[3]
Healthy SPF BALB/c mice (7-8 weeks) and SD rats (4-6 weeks) were used in a single-dose PK study. At time point zero, the BALB/c mice and SD rats of groups A, B and C (each group including twenty BABL/c mice or five SD rats) received i.m. injections of GC376 (111 mg/kg), GS441524 (67 mg/kg) and GC376 + GS441524 (55.5 + 33.5 mg/kg), which are the same doses according to in vivo antiviral study. The blood was collected at 0, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h and placed in a precooled polypropylene centrifuge tube containing 3.0 µl of 40% EDTAK2. Then, the whole blood was centrifuged at 7800 g/min for 10 min at 4°C. Plasma was collected and stored in a freezer at −80°C. Plasma drug concentration was analyzed using LC-MS/MS. Pharmacokinetic parameters were calculated using WinNonlin software (version 6.4), and a non-atrioventricular model was used for data fitting. The data were analyzed using Microsoft Excel 2016 and GraphPad Prism 7.0.

[1]. Viruses. 2020 Feb 21;12(2):240.

[2]. Microorganisms. 2022 Jul 21;10(7):1475.

[3]. Emerg Microbes Infect. 2021 Dec;10(1):481-492

GC-376 is a 3C-like protease (3CLpro or Mpro) inhibitor that stops the cleavage and activation of functional viral proteins required for replication and transcription in host cells. The compound is known as a direct acting antiviral (DAA) for coronaviruses, and was initially developed using structure-guided design to combat MERS-Cov infections. In addition, GC-376 displays potent activity against various coronaviruses such as feline, ferret, and mink Mpro coronavirus. A prodrug of [GC-373], GC-376 has recently been used to test inhibition of SARS-Cov-2 Mpro in vitro, and results show potent inhibition of this target. These results suggest that GC-376, and its metabolites may have therapeutic potential for Covid-19.

C21H30N3NAO8S

507.5330

507.165

C, 49.70; H, 5.96; N, 8.28; Na, 4.53; O, 25.22; S, 6.32

1416992-39-6

1416992-39-6 (sodium);1417031-79-8 (free acid);

71481119

Solid powder

4

8

12

34

783

2

CC(C)C[C@@H](C(=O)N[C@@H](CC1CCNC1=O)C(O)S(=O)(=O)[O-])NC(=O)OCC2=CC=CC=C2.[Na+]

BSPJDKCMFIPBAW-JPBGFCRCSA-M

InChI=1S/C21H31N3O8S.Na/c1-13(2)10-16(24-21(28)32-12-14-6-4-3-5-7-14)19(26)23-17(20(27)33(29,30)31)11-15-8-9-22-18(15)25;/h3-7,13,15-17,20,27H,8-12H2,1-2H3,(H,22,25)(H,23,26)(H,24,28)(H,29,30,31);/q;+1/p-1/t15?,16-,17-,20?;/m0./s1

sodium;(2S)-1-hydroxy-2-[[(2S)-4-methyl-2-(phenylmethoxycarbonylamino)pentanoyl]amino]-3-(2-oxopyrrolidin-3-yl)propane-1-sulfonate

GC376 sodium; GC-376; GC 376; GC-376 sodium; GC376; GC376 sodium; H1NMJ5XDG5; (betaS)-alpha-Hydroxy-beta-[[(2S)-4-methyl-1-oxo-2-[[(phenylmethoxy)carbonyl]amino]pentyl]amino]-2-oxo-3-pyrrolidinepropanesulfonic acid sodium salt; Sodium (2S)-2-((S)-2-(((benzyloxy)carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-(2-oxopyrrolidin-3-yl)propane-1-sulfonate; GC-376; sodium;(2S)-1-hydroxy-2-[[(2S)-4-methyl-2-(phenylmethoxycarbonylamino)pentanoyl]amino]-3-(2-oxopyrrolidin-3-yl)propane-1-sulfonate; GC 376 sodium

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: ~100 mg/mL (~197.0 mM)
Ethanol: ~100 mg/mL (~197.0 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.

---

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).

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)]
*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)

---

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
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

---

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.

---

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