3CLPRO (SARS-CoV 3C-like protease)

3CLPRO cleaves SARS-CoV-2.1's polyproteins 1a and 1ab. Non-structural proteins, including proteins, cannot be released to carry out their roles without the assistance of SARS-CoV-2 3CLPRO, which inhibits viral replication [1].

Treatment of Syrian Golden hamsters with PF-332 (250 mg/kg, twice daily) completely protected the animals against intranasal infection with the beta (B.1.351) and delta (B.1.617.2) SARS-CoV-2 variants. Moreover, treatment of SARS-CoV-2 (B.1.617.2) infected animals with PF-332 completely prevented transmission to untreated co-housed sentinels.[2]

Protein binding [2]
Plasma protein binding was measured by the rapid equilibrium dialysis (RED) method to determine the free fraction and the unbound percentage of PF-332 for various species. An equilibrium dialysis was conducted in duplicate for each sample. 200 μl of plasma spiked with PF-332 were added in the plasma chamber and 350 μl of PBS pH = 7.4 were added in the buffer chamber. The dialysis block was then incubated at 37 °C for 6 h with constant shaking at 400 rpm. After 6 h, aliquots of the plasma and the buffer chambers were collected, spiked to obtain a matching homogeneous matrix, and quantified by LC–MS/MS. [2]
Microsomal metabolic stability [2]
Mouse liver microsomes (CD-1 male strain) were purchased from GIBCO. Hamster (Syrian female strain) and human liver microsomes were purchased from Xenotech. 1 ml of liver microsomal (LM) suspension at 20 mg/ml was mixed with 19 ml of 100 mM phosphate buffer. The latter is a titer solution containing 1 (M) KH2PO4 and 1 (M) K2HPO4 diluted in 10-fold distilled water (30 ml buffer + 270 ml of water) to obtain 100 mM phosphate buffer with an adjusted pH at 7.40 ± 0.02. A solution of NADPH Regeneration System (NRS) was prepared using 13 mM NADP, 33 mM Glucose-6-phosphate, 33 mM MgCl2, and 4 U/ml buffer solution of glucose-6-phosphate dehydrogenase.
All plastic materials including tips are incubated at 37 °C overnight. The LM suspension and the NRS solution were incubated at 37 °C for ~15 min before use. 48 μl of buffer was added to the wells of the blank plate. 40 μl of the compound at 1 uM was added to the working plates, 8 μl of NRS solution was added in the 0, 5, 10, 20, 30, and 60 min plates. The reaction is then initiated by adding 32 μl of 1 mg/ml of LM suspension to each plate. The reaction is terminated by adding 240 μl ice-cold acetonitrile at the designated time points. At T = 0, the acetonitrile is added before the LM solution.
The plates are centrifuged (3500 rpm, 20 min, and 15 °C); 110 μl of distilled water are then added to 110 μl of the supernatant and analyzed using an LC–MS/MS.

SARS-CoV-2 in vitro antiviral assays[2]
The assay using Vero E6 cells was derived from a previously established SARS-CoV assay. In this assay, fluorescence of Vero E6-eGFP cells declines after infection with SARS-CoV-2 due to virus-induced cytopathogenic effect. In the presence of an antiviral compound, the cytopathogenicity is inhibited and the fluorescent signal maintained. Vero E6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with heat-inactivated 10% v/v fetal calf serum (FCS) and 500 μg/ml Geneticin and kept under 5% CO2 at 37 °C.[2]
The test compounds were serially diluted in assay medium (DMEM supplemented with 2% v/v FCS). Diluted compounds were then mixed with Vero E6-eGFP cells corresponding to a final density of 25,000 cells/well in 96-well blackview plates. The next day, cells were infected with the SARS-CoV-2 at a final MOI of approximately 0.05 TCID50/cell. Final dilution of the different strains was adapted in order to obtain a similar MOI between all variants of interest. The plates were incubated in a humidified incubator at 37 °C and 5% CO2. At 4 days post-infection (pi), the wells were examined for eGFP expression using an argon laser-scanning microscope. The microscope settings were excitation at 488 nm and emission at 510 nm and the fluorescence images of the wells were converted into signal values. Toxicity of compounds in the absence of virus was evaluated in a standard MTS assay as described previously.[2]
A549-Dual™ hACE2-TMPRSS2 cells were cultured in DMEM 10% FCS supplemented with 10 µg/ml blasticidin, 100 µg/ml hygromycin, 0.5 µg/ml puromycin and 100 µg/ml zeocin. For the antiviral assay, cells were seeded in assay medium (DMEM 2%) at a density of 15,000 cells/well. One day after, the compound was serially diluted in assay medium (DMEM supplemented with 2% v/v FCS) and cells were infected with their respective SARS-CoV-2 strain at a MOI of approximately 0.05. The MOI was kept comparable for the variant strains in the different experiments. On day 4 pi., differences in cell viability caused by virus-induced CPE or by compound-specific side effects were analyzed using MTS as described previously.[2]
The results of in vitro antiviral experiments were expressed as EC50 values defined as the concentration of compound achieving 50% inhibition of the virus-reduced eGFP signals as compared to the untreated virus-infected control cells.

SARS-CoV-2 infection model in hamsters[2]
The hamster infection model of SARS-CoV-2 has been described before16,20. Female Syrian hamsters were purchased from Janvier Laboratories and kept per two in individually ventilated isolator cages at 21 °C, 55% humidity and 12:12 day/night cycles. Housing conditions and experimental procedures were approved by the ethics committee of animal experimentation of KU Leuven (license P065-2020). For infection, female hamsters of 6–8 weeks old were anesthetized with ketamine/xylazine/atropine and inoculated intranasally with 50 µL containing 104 TCID50 of SARS-CoV-2 Beta variant B.1.351 (day 0). On day 4 pi, animals were euthanized for the sampling of the lungs and further analysis by i.p. injection of 500 μl Dolethal (200 mg/ml sodium pentobarbital). All caretakers and technicians were blinded to group allocation in the animal facility.[2]
Treatment regimen (beta variant study) Hamsters were treated by oral gavage with either the vehicle (n = 12) or PF-332 at 125 (n = 10) or 250 (n = 12) mg/kg/dose twice daily starting from D0, just before the infection with the Beta variant. All the treatments continued until day 3 pi. Hamsters were monitored for appearance, behavior, and weight. At day 4 pi, hamsters were euthanized by i.p. injection of 500 μl Dolethal (200 mg/ml sodium pentobarbital). Lungs were collected and viral RNA and infectious virus were quantified by RT-qPCR and end-point virus titration, respectively as described before17.[2]
Efficacy-transmission study (delta variant study) Two groups of index hamsters were infected intranasally with 50 µl containing 104 TCID50 of SARS-CoV-2 Delta variant and treated with either vehicle or PF-332 at 250 mg/kg/dose twice daily starting from D0. On day 1 pi (just after the morning dose), each index hamster was co-housed with a contact hamster (non-infected, non-treated hamsters) in one cage and the co-housing continued until day 3 pi The treatment of index hamsters was continued until day 2 pi. At day 3 pi, all the index hamsters were euthanized whereas all the contact hamsters were euthanized the day after (i.e., day 4 pi of index) as mentioned before and lungs were collected to assess viral loads.

[1]. KoenVandyck, et al. Considerations for the Discovery and Development of 3-Chymotrypsin-Like Cysteine Protease Inhibitors Targeting SARS-CoV-2 Infection. Curr Opin Virol. 2021 Aug:49:36-40. doi: 10.1016/j.coviro.2021.04.006.
[2]. The oral protease inhibitor (PF-07321332) protects Syrian hamsters against infection with SARS-CoV-2 variants of concern. Nat Commun . 2022 Feb 15;13(1):719. doi: 10.1038/s41467-022-28354-0.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. The coronavirus 3-chymotrypsin-like protease (3CLpro) controls virus replication and is therefore considered a major target and promising opportunity for rational-based antiviral discovery with direct acting agents. Here we review first-generation SARS-CoV-2 3CLpro inhibitors PF-07304814, GC-376, and CDI-45205 that are being delivered either by injection or inhalation due to their low intrinsic oral bioavailability. In addition, PF-07321332 is now emerging as a promising second-generation clinical candidate for oral delivery. A key challenge to the development of novel 3CLpro inhibitors is the poor understanding of the predictive value of in vitro potency toward clinical efficacy, an issue complicated by the involvement of host proteases in virus entry. Further preclinical and clinical validation will be key to establishing 3CLpro inhibitors as a bona fide class for future SARS-CoV-2 therapeutics for both hospitalized and outpatient populations.[1]

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Nirmatrelvir is administered alongside ritonavir, a potent inhibitor of CYP3A enzymes, in order to inhibit its metabolism and increase plasma nirmatrelvir concentrations. While therapeutically beneficial, the use of ritonavir poses a significant risk of drug interaction due to its potent inhibition profile - patients and clinicians should consult the prescribing information for Paxlovid (nirmatrelvir and ritonavir) to evaluate any potential for drug interaction with existing medications prior to the initiation of Paxlovid. Nirmatrelvir is an inhibitor of a cysteine residue in the 3C-like protease (3CLPRO) of SARS-CoV-2. This cysteine is responsible to the activity of the 3CLPRO of SARS-CoV-2 and potentially other members of the coronavirus family. The 3CLPRO, also known as the main protease or non structural protein 5, is responsible for cleaving polyproteins 1a and 1ab. These polyproteins contain the 3CLPRO itself, a papain-like (PL) cysteine protease, and 14 other nonstructural proteins. Without the activity of the 3CLPRO, nonstructural proteins (including proteases) cannot be released to perform their functions, inhibiting viral replication.

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Nirmatrelvir is an azabicyclohexane that is (1R,5S)-3-azabicyclo[3.1.0]hexane substituted by {(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}aminoacyl, 3-methyl-N-(trifluoroacetyl)-L-valinamide, methyl and methyl groups at positions 2S, 3, 6 and 6, respectively. It is the first orally administered inhibitor of SARS-CoV-2 main protease developed by Pfizer and used in combination with ritonavir for the treatment of COVID-19. It has a role as an EC 3.4.22.69 (SARS coronavirus main proteinase) inhibitor and an anticoronaviral agent. It is a nitrile, a member of pyrrolidin-2-ones, a secondary carboxamide, a pyrrolidinecarboxamide, a tertiary carboxamide, an organofluorine compound and an azabicyclohexane.

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Nirmatrelvir (PF-07321332) is an orally bioavailable 3C-like protease (3CLPRO) inhibitor that is the subject of clinical trial NCT04756531. 3CLPRO is responsible for cleaving polyproteins 1a and 1ab of SARS-CoV-2. Without the activity of the SARS-CoV-2 3CLPRO, nonstructural proteins (including proteases) cannot be released to perform their functions, inhibiting viral replication. In 2020, Pfizer was investigating another potential treatment for SARS-CoV-2, [PF-07304814]. Both drugs were inhibitors of SARS-CoV-2 3CLPRO, but nirmatrelvir has the advantage of being orally bioavailable. Nirmatrelvir is advantageous in that it can be prescribed to patients before they require hospitalization, while [PF-07304814] requires intravenous administration in hospital. In December 2021, the FDA granted an emergency use authorization to Paxlovid, a co-packaged product containing both nirmatrelvir and [ritonavir], for the treatment of certain patients with mild-to-moderate COVID-19. It was fully approved by the FDA on May 25, 2023. Paxlovid was approved for use in Canada in January 2022 for the treatment of adult patients with mild-moderate COVID-19 and later granted conditional marketing authorization by the European Commission on January 27, 2022.

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Paxlovid is a co-packaged combination of nirmatrelvir, a second generation protease inhibitor, and ritonavir, a pharmacological enhancer, that is used to treated infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) , the cause of the novel and severe coronavirus disease, 2019 (COVID-19). Paxlovid is given orally for 5 days in patients early in the course of infection and has not been linked to serum aminotransferase elevations or to clinically apparent liver injury.

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Nirmatrelvir is an orally bioavailable, peptidomimetic inhibitor of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) main protease (Mpro; 3C-like protease; 3CL protease; 3CLpro; nsp5 protease), with potential antiviral activity against SARS-CoV-2 and other coronaviruses. Upon oral administration, nirmatrelvir selectively targets, binds to, and inhibits the activity of SARS-CoV-2 Mpro. This inhibits the proteolytic cleavage of viral polyproteins, thereby inhibiting the formation of viral proteins including helicase, single-stranded-RNA-binding protein, RNA-dependent RNA polymerase, 20-O-ribose methyltransferase, endoribonuclease and exoribonuclease. This prevents viral transcription and replication.

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In the US, Europe, and Canada, nirmatrelvir, in combination with [ritonavir], is indicated for the treatment of mild-to-moderate coronavirus disease 2019 (COVID-19) in adults who are at high risk for progression to severe COVID-19, including hospitalization or death. In Europe, this therapeutic indication is approved under conditional marketing authorization.

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Paxlovid is indicated for the treatment of coronavirus disease 2019 (COVID-19) in adults who do not require supplemental oxygen and who are at increased risk for progressing to severe COVID 19.

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Paxlovid is a co-packaged combination of nirmatrelvir, a second generation protease inhibitor, and ritonavir, a pharmacological enhancer, that is used to treated infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) , the cause of the novel and severe coronavirus disease, 2019 (COVID-19). Paxlovid is given orally for 5 days in patients early in the course of infection and has not been linked to serum aminotransferase elevations or to clinically apparent liver injury.

C23H32F3N5O4EXACTMASS

499.5265

499.24

C, 55.30; H, 6.46; F, 11.41; N, 14.02; O, 12.81

2628280-40-8

Nirmatrelvir-d9;2861202-76-6

155903259

White to off-white solid powder

2.2

131Ų

[H][C@]12CN([C@H](C(=O)N[C@@H](C[C@]3([H])CCNC3=O)C#N)[C@@]1([H])C2(C)C)C(=O)[C@@H](NC(=O)C(F)(F)F)C(C)(C)C

LIENCHBZNNMNKG-OJFNHCPVSA-N

InChI=1S/C23H32F3N5O4/c1-21(2,3)16(30-20(35)23(24,25)26)19(34)31-10-13-14(22(13,4)5)15(31)18(33)29-12(9-27)8-11-6-7-28-17(11)32/h11-16H,6-8,10H2,1-5H3,(H,28,32)(H,29,33)(H,30,35)/t11-,12-,13-,14-,15-,16+/m0/s1

(1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide

Nirmatrelvir; PF-07321332; PF 07321332; P7R9A5P7H32; Nirmatrelvir; Paxlovid; PF-07321332; PF07321332; Nirmatrelvir [USAN]; UNII-7R9A5P7H32; F07321332; brand name Paxlovid;

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 : 100~140 mg/mL ( 200.18~280.26 mM )
Ethanol : 50 ~100 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.16 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.16 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.16 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10% DMSO+90% Corn Oil: ≥ 2.08 mg/mL (4.16 mM)

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