Respiratory Syncytial Virus (RSV) fusion (F) protein. It inhibits the pre- to postfusion conformational changes of the RSV F protein. [2]
Active against RSV A and RSV B clinical isolates with a mean EC50 of 0.43 nM. [2]

Presatovir is a brand-new, orally accessible RSV fusion inhibitor that was found after a lead optimization effort on a hit that came from a high-throughput screen for phenotypic RSV antivirals. Against a variety of RSV A and B clinical isolates, presetovir demonstrates strong action, with an average EC50 value of 0.43 nM[1]. Pre-to post-triggered conformational changes of the RSV F protein are inhibited by GS-5806, indicating a potential mechanism of antiviral activity[2].

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GS-5806 blocks RSV fusion protein-mediated cell-cell fusion. Mutations conferring drug resistance map to the RSV F gene, confirming the F protein as the molecular target. [2]
GS-5806 inhibits the pre- to postfusion conformational changes of a purified, truncated RSV F protein (ΔTM-RSV F) in vitro. When triggered by exposure to low-ionic-strength buffer, ΔTM-RSV F forms rosette-like structures observable by electron microscopy. In the presence of a 5-fold molar excess of GS-5806, the number of rosettes formed was significantly reduced (average 23 rosettes per image) compared to DMSO control (108 rosettes) or an inactive analog GSC-1 (106 rosettes) (P < 0.002). [2]
The inhibitory effect of GS-5806 on rosette formation was dose-dependent, with fewer rosettes observed at increasing concentrations of the compound. [2]
GS-5806 inhibits the insertion of ΔTM-RSV F protein into liposomes. When triggered in the presence of liposomes, ΔTM-RSV F inserts into the lipid bilayer. In GS-5806-treated samples, only 4% ± 3% of liposomes contained ΔTM-RSV F, compared to 13% ± 4% in DMSO controls. Additionally, the average number of ΔTM-RSV F molecules per liposome was reduced to 7 ± 3 in GS-5806-treated samples versus 25 ± 10 in controls. [2]
A resistance variant with a threonine-to-alanine amino acid change at position 400 of the RSV F protein (T400A) showed reduced susceptibility to GS-5806. The ΔTM-RSV F T400A protein formed similar numbers of rosettes in the presence of DMSO (44 ± 9) or GS-5806 (46.6 ± 8), indicating loss of inhibition. In liposome insertion assays, the percentage of liposomes containing ΔTM-RSV F T400A was similar between GS-5806-treated (8% ± 3%), DMSO-treated (14% ± 3%), and GSC-1-treated (16% ± 2%) samples, and the average number of protein molecules deposited per liposome (∼30) was similar across all treatments. [2]
Unlike other known RSV entry inhibitors such as TMC-353121 and BMS-433771, GS-5806 did not influence the formation of six-helix bundles when isolated peptides were mixed in its presence. Differential scanning calorimetry, isothermal titration calorimetry, and circular dichroism did not detect direct interaction of GS-5806 with isolated six-helix bundles. This suggests GS-5806 interferes with an earlier step in the conformational transition of the RSV F protein. [2]

In a cotton rat model of RSV infection, prestatovir dose-dependent (0–30 mg/kg) exhibits antiviral effectiveness. Preclinical species exhibit oral bioavailability ranging from 46 to 100%, with Sprague-Dawley rats demonstrating the compound's entry into lung tissue. Presatovir oral therapy administered in many doses seems safe for adults, and in healthy human subjects experimentally infected with RSV, the high dose group shows a strong antiviral activity and a decrease in the severity of the disease. In order to inform future dose decisions, a PK-PD connection can be developed in a group receiving a lower dose of Presatovir[1].

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Rosette Formation Inhibition Assay: Purified ΔTM-RSV F protein, stored in high-ionic-strength buffer (500 mM NaCl, 250 mM imidazole, 20 mM Tris, pH 8.0) to maintain its pre-triggered conformation, was used. The pre- to postfusion conformational change was initiated by overnight dialysis at 4°C against low-ionic-strength buffer (10 mM HEPES, pH 8.0) in the presence of a 5-fold molar excess of GS-5806, an inactive analog (GSC-1), or 0.1% DMSO. Samples were then applied to electron microscopy grids, and the number of rosettes formed was quantified from 6-8 randomly selected EM images by visual inspection. [2]
Liposome Insertion Assay: Liposomes were prepared in low-ionic-strength buffer at a high concentration (∼8 mM, 3,000-fold excess relative to RSV F) to promote insertion and avoid rosette formation. ΔTM-RSV F protein was triggered by mixing with these liposomes in the presence of GS-5806, GSC-1, or DMSO. Samples were applied to EM grids, and the number of liposomes containing ΔTM-RSV F molecules and the number of molecules per liposome were quantified from 7 randomly selected EM images for each experiment. [2]
Six-Helix Bundle Interaction Assays: The potential direct interaction of GS-5806 with isolated six-helix bundles was assessed using multiple biophysical techniques. Differential scanning calorimetry, isothermal titration calorimetry, and circular dichroism spectroscopy were performed on six-helix bundle peptides in the presence and absence of GS-5806 to detect any binding or conformational changes. [2]

The study references that GS-5806 is active against a diverse collection of RSV A and RSV B clinical isolates in cell-based assays, with a mean EC50 of 0.43 nM. Resistance selection studies in cell culture identified the T400A mutation in the RSV F gene. [2]

[1]. Discovery of an oral respiratory syncytial virus (RSV) fusion inhibitor (GS-5806) and clinical proof of concept in a human RSV challenge study. J Med Chem. 2015 Feb 26;58(4):1630-1643.

[2]. GS-5806 inhibits pre- to postfusion conformational changes of the respiratory syncytial virus fusion protein. Antimicrob Agents Chemother. 2015 Nov;59(11):7109-12.

Presatovir has been used in clinical trials investigating respiratory syncytial virus (RSV) infection. Presatovir is an orally administered RSV fusion protein (F protein) inhibitor with potential antiviral activity. After oral administration of GS-5806, this drug specifically binds to the F protein on the viral surface, thereby inhibiting RSV F protein-mediated fusion of the virus with the host cell membrane and preventing viral entry into the cell. This can block RSV replication, reduce viral load, and alleviate disease severity. The RSV F protein is a viral surface glycoprotein that plays a crucial role in RSV fusion with and entry into target cells.

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GS-5806 is a small-molecule inhibitor of respiratory syncytial virus replication that blocks viral entry by targeting the RSV fusion protein. [2]
The compound prevents the fusion protein from undergoing the conformational changes required for fusion of the viral envelope with the host cell membrane. [2]
Unlike some other RSV fusion inhibitors that bind to the six-helix bundle, GS-5806 appears to inhibit an earlier step in the fusion process, preventing the pre- to postfusion transition. [2]
The T400A resistance mutation maps to a region of the F protein distinct from the six-helix bundle binding site of other inhibitors, consistent with a different mechanism or binding site for GS-5806. [2]

C24H30CLN7O3S

532.06

531.181

1353625-73-6

58029842

Off-white to light yellow solid powder

1.5±0.1 g/cm3

1.735

1.79

2

8

5

36

905

2

CC1=CN2C(=CC(=N2)[C@@H]3CCCCN3C(=O)C4=C(C=CC(=C4)Cl)NS(=O)(=O)C)N=C1N5CC[C@@H](C5)N

GOFXWTVKPWJNGD-UWJYYQICSA-N

InChI=1S/C24H30ClN7O3S/c1-15-13-32-22(27-23(15)30-10-8-17(26)14-30)12-20(28-32)21-5-3-4-9-31(21)24(33)18-11-16(25)6-7-19(18)29-36(2,34)35/h6-7,11-13,17,21,29H,3-5,8-10,14,26H2,1-2H3/t17-,21-/m0/s1

N-[2-[(2S)-2-[5-[(3S)-3-aminopyrrolidin-1-yl]-6-methylpyrazolo[1,5-a]pyrimidin-2-yl]piperidine-1-carbonyl]-4-chlorophenyl]methanesulfonamide

GS-5806 GS 5806 GS5806

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 : ~6 mg/mL (~11.28 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 0.6 mg/mL (1.13 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 6.0 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: ≥ 0.6 mg/mL (1.13 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 6.0 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: ≥ 0.6 mg/mL (1.13 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 6.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 0.48 mg/mL (0.90 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 5: ≥ 0.48 mg/mL (0.90 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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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 (Please use freshly prepared in vivo formulations for optimal results.)