FIPV ( EC50 = 0.78 μM ); RNA-dependent RNA polymerase (RdRp)

The lymphocyte counts and rectal temperatures of all ten treated cats quickly return to pre-infection levels, as do the levels of the two asymptomatic cats. As of now, more than eight months after infection, none of the ten cats who received one or two treatments have changed. Some cats experience a brief "stinging" reaction after receiving an injection within ten seconds of the compound being administered. Unusual posturing, licking at the injection site, and/or vocalizations that persist for about 30 to 60 seconds following injection are signs of localized and temporary pain. Certain animals exhibit more noticeable injection reactions than others, and these reactions vary from injection to injection and become less pronounced over time[1]. GS-441524 is found in serum at 1000-fold higher concentrations than Remdesivir in NHP after receiving Remdesivi (IV injection) for a 7-day period of treatment[3].

There are several methods for measuring the RdRP Enzymatic Activity of inhibitors as detailed below: [4]
Biochemical RdRP Enzyme Activity Assays
(1) Polymerase Elongation Template Element (PETE) Assay for RdRP
Because RdRP catalyzes the incorporation of NTPs during RNA elongation, a PETE assay can be developed to detect the elongation activity of RdRP.46 In this assay approach, an oligonucleotide at the 5′ end of an RNA template is labeled with a fluorescent probe for fluorescence polarization (FP) measurements. The polarization signal from the fluorescent probe increases as its mobility becomes low following the elongation of the newly synthesized complementary RNA chain by RdRP. Inhibition of RdRP activity by a compound reduces the FP signal as the elongation of the complementary RNA chain stops.
(2) Fluorescence-Based Alkaline Phosphatase–Coupled Polymerase Assay (FAPA)
The FAPA approach includes a modified nucleotide analog in the substrate system during RNA synthesis by RdRP. As the polymerase reaction proceeds, incorporation of modified nucleotide analog results in the release of the fluorophore, allowing detection. For example, a modified nucleotide analog (2-[2-benzothiazoyl]-6-hydroxybenzothiazole) conjugated adenosine triphosphate (BBT-ATP) incorporated into the growing RNA chain was catalyzed by RdRP, resulting in a by-product of BBT, pyrophosphate (PPi). The BBTPPi subsequently was reacted with alkaline phosphatase to produce a highly fluorescent BBT anion.
(3) Fluorometric RdRP Activity Assay
Fluorophores have been extensively used for the detection of RNA and DNA. In this fluorometric RdRP activity assay, fluorophores are used to detect dsRNA formation from the ssRNA template (Fig. 3C). One application of this assay was to screen the inhibitors of hepatitis C virus (HCV) RdRP.51 By using a poly(C) RNA template, HCV RdRP catalyzed the primer-independent synthesis of dsRNA that was detected by fluorescent dye PicoGreen.51 PicoGreen was originally developed to quantify dsDNA, but it was subsequently found to also preferentially bind dsRNA instead of ssRNA.51 This assay can be easily adapted to compound screening for RdRP inhibitors for many types of viruses. In addition to PicoGreen, other fluorophores have also been used to distinguish dsRNA from ssRNA, and they are useful for this type of RdRP assay.
(4) Scintillation Proximity Assay (SPA)
SPA has also been used in RdRP enzyme assays for HTS. This assay relies on the incorporation of radioactive nucleotides to the newly synthesized RNA chain catalyzed by RdRP using a biotinylated primer-template in the presence of 3H-GTP. Application of streptavidin-coupled SPA detection beads in this radioactive enzyme assay enables homogeneous assay detection that avoids the labor-intensive filtration and washing steps from the original radioactive NTP incorporation assay. Because they are radioactive assays, however, specific safety precautions and waste handling are required that may be inconvenient and require enhanced safety protocols. Therefore, most radioactive assays have been replaced by fluorometric assays in recent years.

GS-441524 is treated with 100, 33.3, 11.1, 3.7, or 1.2 μM for a 24-hour period on CRFK cells in order to assess its toxicity[1].

[1]. The nucleoside analog GS-441524 strongly inhibits feline infectious peritonitis (FIP) virus in tissue culture and experimental cat infection studies. Vet Microbiol. 2018 Jun;219:226-233.

[2]. What Do We Know About Remdesivir Drug Interactions? Clin Transl Sci. 2020 May 13;10.1111/cts.12815.

[3]. Advantages of the Parent Nucleoside GS-441524 over Remdesivir for Covid-19 Treatment. ACS Med. Chem. Lett. 2020.
[4]. https://journals.sagepub.com/doi/full/10.1177/2472555220942123

Feline infectious peritonitis (FIP) is a common and highly lethal coronavirus disease of domestic cats. Recent studies of diseases caused by several RNA viruses in people and other species indicate that antiviral therapy may be effective against FIP in cats. The small molecule nucleoside analog GS-441524 is a molecular precursor to a pharmacologically active nucleoside triphosphate molecule. These analogs act as an alternative substrate and RNA-chain terminator of viral RNA dependent RNA polymerase. We determined that GS-441524 was non-toxic in feline cells at concentrations as high as 100 uM and effectively inhibited FIPV replication in cultured CRFK cells and in naturally infected feline peritoneal macrophages at concentrations as low as 1 uM. We determined the pharmacokinetics of GS-441524 in cats in vivo and established a dosage that would sustain effective blood levels for 24 h. In an experimental FIPV infection of cats, GS-441524 treatment caused a rapid reversal of disease signs and return to normality with as little as two weeks of treatment in 10/10 cats and with no apparent toxicity.[1]

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GS-441524 is a C-nucleoside analog that is (2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile substituted by a 4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl group at position 2. It is the active metabolite of remdesivir and exhibits a broad range of inhibitory activity against various RNA viruses including HCV, parainfluenza and SARS-CoV. It has a role as a drug metabolite, an antiviral agent and an anticoronaviral agent. It is a pyrrolotriazine, a nitrile, a C-nucleoside and an aromatic amine.

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ChEBI GS-441524 is an adenosine nucleotide analog antiviral, similar to [remdesivir]. This molecule was patented in 2009. In vitro studies of GS-441524 have determined it has a higher EC50 than remdesivir against a number of viruses, meaning GS-441524 is less potent. GS-441524 continues to be studied in the treatment of Feline Infectious Peritonitis Virus, a coronavirus that only infects cats.

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Antiviral Agents
Agents used in the prophylaxis or therapy of VIRUS DISEASES. Some of the ways they may act include preventing viral replication by inhibiting viral DNA polymerase; binding to specific cell-surface receptors and inhibiting viral penetration or uncoating; inhibiting viral protein synthesis; or blocking late stages of virus assembly. (See all compounds classified as Antiviral Agents.)
Absorption
GS-441524 has been found to transport poorly into cells compared to remdesivir.
Metabolism / Metabolites
GS-441524 is phosphorylated 3 times to form the active nucleoside triphosphate.
Mechanism of Action
GS-441524 is phosphorylated 3 times to form the active nucleoside triphosphate, which is incorporated into the genome of virions, terminating its replication.

C12H13N5O4

291.2627

291.1

C, 49.48; H, 4.50; N, 24.04; O, 21.97

1191237-69-0

1355149-45-9 (GS-441524 triphosphate); 2378280-82-9 (HCl); 1809249-37-3 (Remdesivir); 1355050-21-3; 1809249-37-3; 2378280-83-0 (sulfate);1355357-49-1; 2647442-13-3

44468216

White to off-white solid powder

1.84±0.1 g

-1.4

150Ų

C1=C2C(=NC=NN2C(=C1)[C@]3([C@@H]([C@@H]([C@H](O3)CO)O)O)C#N)N

BRDWIEOJOWJCLU-LTGWCKQJSA-N

InChI=1S/C12H13N5O4/c13-4-12(10(20)9(19)7(3-18)21-12)8-2-1-6-11(14)15-5-16-17(6)8/h1-2,5,7,9-10,18-20H,3H2,(H2,14,15,16)/t7-,9-,10-,12+/m1/s1

(2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)oxolane-2-carbonitrile

Remdesivir metabolite; GS-441524; GS-5734 metabolite; GS 441524; GS441524; GS5734 metabolite; GS 5734 metabolite; Remdesivir-metabolite; GS-5734-metabolite; GS5734-metabolite; GS 5734-metabolite

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 : ~120 mg/mL (with ultrasonic)
Water : Insoluble

Solubility in Formulation 1: 10 mg/mL (34.33 mM) in 5% ethanol, 30% propylene glycol, 45% PEG 400, 20% water (pH 1.5 with HCI) (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (9.44 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 3: ≥ 2.08 mg/mL (7.14 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 4: ≥ 2.08 mg/mL (7.14 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 5: ≥ 2.08 mg/mL (7.14 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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 (Please use freshly prepared in vivo formulations for optimal results.)