Viral capsid protein VP1 (hydrophobic pocket formed by VP1, VP3 and VP2). No IC50, Ki, EC50, or DC50 values for target binding are provided in this manuscript [1].

Pleconaril (also known as VP 63843; Win 63843) is a capsid inhibitor and an antiviral drug that has the potential for the treatment of enterovirus infection and asthma. Pleconaril is effective in inhibiting replication with an IC50 of 50 nM. Pleconaril, administered either orally or intranasally, is active against viruses in the Picornaviridae family, including Enterovirus and Rhinovirus.It has shown useful activity against the dangerous strain enterovirus D68. Pleconaril has been used as treatment on a compassionateuse basis in neonates and immunodeficient patients with severe EV infections.

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Kinase Assay: Pleconaril (also known as VP 63843; Win 63843) is a capsid inhibitor and an antiviral drug that has the potential for the treatment of enterovirus infection and asthma. Pleconaril is effective in inhibiting replication with an IC50 of 50 nM.

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Cell Assay: Rhinovirus infections do not only cause common colds, but may also trigger severe exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Even though rhinoviruses have been the focus of extensive drug development efforts in the past, an anti-rhinoviral drug still has to make it to the market. In the past, the viral capsid protein VP1 has been shown to be an important target for the development of antiviral molecules. Furthermore, many different chemical scaffolds appear to possess the properties that are required to inhibit virus replication by this mechanism of action. I-6602, an analogue of the rhinovirus inhibitor pirodavir, was previously identified as a potent inhibitor of rhinovirus infection. Here, we describe the antiviral activity of its analogue ca603, a molecule with a modified linker structure, and corroborate its mechanism of action as a capsid binder.

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Pleconaril susceptibility was tested against various echovirus 11 (E11) strains and control viruses. The IC50 values: patient strains M07067754, M08084333, M09053042 all had IC50 ≥32 μM; prototype strain E11-PT IC50 = 0.0038 μM; clinical isolate 20750473 IC50 = 0.0035 μM; clinical isolate 20750433 IC50 = 0.042 μM; clinical isolate 20750473 (another) IC50 = 0.0033 μM; clinical isolate 21051708 IC50 = 0.0011 μM; clinical isolate 21051867 IC50 = 0.0031 μM; clinical isolate 21051929 IC50 = 0.0036 μM; clinical isolate 21052365 (genogroup C) IC50 = 0.05 μM; control CVB3 IC50 ≥32 μM; EV71 IC50 ≥32 μM; HRV16 IC50 = 0.0016 μM; E13 IC50 = 0.032 μM; CAV9 IC50 = 0.032 μM [1].
In vitro induction of resistance: a sensitive E11 clinical strain (20750473) was cultured with increasing concentrations of Pleconaril. Resistance was observed by passage 3 (strain 3A4) with an increase in IC50 to 1 μM. At passages 4-6, IC50 was ≥32 μM, confirming resistance. Sequencing revealed an I183M substitution after three passages, which was retained in further passages. When pleconaril was omitted, a V119I substitution appeared (strain 11B5) that allowed more efficient replication [1].
Structural modelling and PEARL interaction energy values: For sensitive strains E11-PT and 20750473, PEARL interaction values for Pleconaril were -483.6 and -362.6 respectively, indicating strong interaction; for resistant patient strain M07067754, the value was -270.9, indicating inefficient docking. Pocket factor interaction values were -281.0 (E11-PT), -237.8 (20750473), and -286.7 (M07067754), showing that pleconaril outcompetes the pocket factor in sensitive strains but not in the resistant strain [1].
Amino acid substitutions conferring resistance: Patient resistant strains carried V117I, V119M, and I188L mutations in VP1. Modelling suggested V119M confers resistance due to the protruding sulfate side chain of methionine. In vitro induced resistant strains carried I183M (and later V119I). Site-directed mutagenesis of V119M (single or double with I188L) yielded viable clones with IC50 ≥32 μM, confirming resistance [1].

Pleconaril, administered either orally or intranasally, is active against viruses in the Picornaviridae family, including Enterovirus and Rhinovirus.It has shown useful activity against the dangerous strain enterovirus D68. Pleconaril has been used as treatment on a compassionateuse basis in neonates and immunodeficient patients with severe EV infections.

In silico docking and modelling: The three-dimensional structure of Pleconaril was taken from PDB entry 1C8M. Docking of Pleconaril into the hydrophobic pocket of echovirus 11 capsid models was carried out using PATCHDOCK software. The top 20 results were ranked according to their energy profiles determined by PEARLS to select the most stable complex. Pockets were identified by POCKETFINDER. Interaction energy values (PEARL) for Pleconaril and the pocket factor were calculated to assess binding efficiency and competition. Amino acid sequences of VP1-4 of E11-PT were submitted to I-TASSER for homology modelling using PDB entry 1H8T as template. The top three-dimensional models were projected onto the known structure of the echovirus VP1-4 complex to create a model of the asymmetrical unit of the EV capsid. Steric clashes were removed via CHIRON. Visualization was performed with RASTOP, CHIMERA, and PDBsum [1].

Pleconaril inhibition assay: The assay was performed as described previously (Wildenbeest et al., 2012). Susceptibility to Pleconaril was expressed as the IC50 (50% inhibitory concentration). The method involved incubating viruses with serial dilutions of Pleconaril and determining the concentration that inhibited virus replication by 50% [1].
In vitro isolation of drug-resistant mutants: A pleconaril-sensitive E11 clinical strain (20750473, 100 TCID50 in 50 μL) was incubated in 10-fold dilutions of Pleconaril (100 to 0.0001 μg/mL) in duplicate in Eagle‘s minimal essential medium (EMEM) with 2% fetal calf serum for a maximum of 7 days. The virus growing at the highest pleconaril concentration was passaged further with Pleconaril in 10-fold dilutions (100 to 0.0001 μg/mL) in duplicate in EMEM/2% FCS. This process was repeated for six passages, after which the virus was passaged a further five times without Pleconaril. For each passage, viral isolates were stored for VP1 sequencing and in vitro susceptibility testing [1].
Site-directed mutagenesis: Seven mutant E11 clones were constructed by site-directed mutagenesis introducing single, double or triple mutations at residues 117, 119, 183, and 188 in VP1. Clones were linearized by XhoI digestion, RNA was transcribed with T7 RNA polymerase, and RNA was transfected into Vero cells using a transfection reagent. Supernatant was titrated by the TCID50 method and sequenced for verification [1].

Oral or intranasally

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Absorption, Distribution and Excretion
70% (oral)

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Protein Binding
> 99%

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Virol J.2015 Jul 14;12:106.J Gen Virol.2015 Mar;96(Pt 3):571-9.

Pleconaril is an antiviral drug manufactured by Schering-Plough, a viral capsid inhibitor used to prevent acute asthma attacks and common cold symptoms in asthmatic patients who have been exposed to picornaviruses. Its mechanism of action is to inhibit viral replication. Because Pleconaril has been found to induce CYP3A enzyme activity, increasing the risk of serious drug interactions, its use has not been approved by the U.S. Food and Drug Administration (FDA). It has been reported that Pleconaril is found in Isatis tinctoria, and relevant data are available. Pleconaril is a small molecule inhibitor active against picornaviruses, including enteroviruses and rhinoviruses. Pleconaril binds to hydrophobic pockets in the major capsid proteins, preventing the uncoating of the viral RNA genome. In enteroviruses, it also prevents viral attachment to host cells. Drug Indications It has been studied for the treatment of upper respiratory tract infections.

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Mechanism of Action
Priliconal binds to a hydrophobic pocket in viral protein 1 (the main protein that makes up the capsid of a small RNA virus). This stiffens and compresses the viral capsid, preventing its RNA from uncoating. Therefore, the virus cannot attach to host cells and cause infection.

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Pleconaril is a capsid inhibitor designed to dock within a hydrophobic pocket formed by the capsid proteins VP1, VP3 and VP2. The compound leads to stiffening of the capsid structure, preventing viral RNA release into the cell [1].
In 2002, the New Drug Application for Pleconaril as a drug against the common cold was rejected by the FDA, mainly because of safety reasons. It is now licensed by Schering-Plough and has been investigated for treatment of HRV-induced exacerbations of asthma and COPD (NCT00394914) and neonatal sepsis (NCT00031512) [1].
Resistance to Pleconaril can arise via mutations in the VP1 hydrophobic pocket (e.g., V119M, I183M). Cross-resistance with IVIg may occur as both target the viral capsid [1].

C18H18F3N3O3

381.35

381.13

C, 56.69; H, 4.76; F, 14.95; N, 11.02; O, 12.59

153168-05-9

Pleconaril-d4;2749329-20-0

1684

White to biege solid powder

1.3±0.1 g/cm3

481.2±55.0 °C at 760 mmHg

61-62°

244.8±31.5 °C

0.0±1.2 mmHg at 25°C

1.509

5.77

0

9

6

27

471

0

FC(C1=NC(C2C([H])=C(C([H])([H])[H])C(=C(C([H])([H])[H])C=2[H])OC([H])([H])C([H])([H])C([H])([H])C2=C([H])C(C([H])([H])[H])=NO2)=NO1)(F)F

KQOXLKOJHVFTRN-UHFFFAOYSA-N

InChI=1S/C18H18F3N3O3/c1-10-7-13(16-22-17(27-24-16)18(19,20)21)8-11(2)15(10)25-6-4-5-14-9-12(3)23-26-14/h7-9H,4-6H2,1-3H3

3-(3,5-dimethyl-4-(3-(3-methylisoxazol-5-yl)propoxy)phenyl)-5-(trifluoromethyl)-1,2,4-oxadiazole

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 : 76~100 mg/mL ( 199.29~262.23 mM )
Ethanol : 76 mg/mL
Water : ˂1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.56 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 25.0 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: ≥ 2.5 mg/mL (6.56 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 10% DMSO+90% Corn Oil: ≥ 2.5 mg/mL (6.56 mM)

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