Pirodavir (R77975) (R 77975), the prototype of broad-spectrum anti-picornavirus compounds, is a potent human rhinovirus (HRV) capsid-binding inhibitor.

Pirodavir是一种高效、广谱的抗肠道病毒化合物。对100株人类鼻病毒(HRV)中的80株，在64 ng/mL的浓度下具有抑制作用。同一研究中，Pirodavir也能有效抑制16种肠道病毒，平均80%抑制浓度(IC80)为1,300 ng/mL。对于肠道病毒71，Pirodavir的半抑制浓度(IC50)为5,420 nM，90%抑制浓度(IC90)大于13,350 nM。Pirodavir抑制了56种实验室鼻病毒株和3种临床分离株。在<100 nM的IC50下，Pirodavir对59%的血清型和分离株有抑制效果。Pirodavir浓度在16和4μg/mL时，分别使细胞生长减少66%（标准误0.75）和28%（标准误0.25）。低浓度(1μg/mL)的Pirodavir对细胞生长没有抑制作用。在37°C下，Pirodavir对对数期细胞生长的50%细胞毒性浓度为7μg/mL。在抗病毒测定条件下（密集的HeLa细胞在33°C时），50%细胞毒性浓度大于50μg/mL。

The extract and binding assay buffer consists of 25 mM sodium phosphate, 10 mM potassium fluoride, 10 mM sodium molybdate, 10% glycerol, 1.5 mM EDTA, 2 mM dithiothreitol, 2 mM CHAPS, and 1 mM phenylmethylsulfonyl fluoride (pH 7.4), at room temperature. Intracellular receptors produced in this fashion exhibit reproducible interaction with known ligands at the published affinity. These preparations are subjected to extensive quality control experiments before the assays, covering receptor response, specificity, size, and reference ligand affinity. Receptor assays are performed with a final volume of 250 μL containing from 50-75 μg of extract protein, plus 1-2 nM [3H]Dex at 84 Ci/mmol and varying concentrations of competing ligand (0 to 10 μM). Assays are set up using a 96-well minitube system, and incubations are carried out at 4°C for 18 h. Equilibrium under these conditions of buffer and temperature is achieved by 6-8 h. Nonspecific binding is defined as that binding remaining in the presence of 1000 nM unlabeled Dex. At the end of the incubation period, 200 μL of 6.25% hydroxyapatite are added in wash buffer (binding buffer in the absence of dithiothreitol and phenylmethylsulfonyl fluoride). Specific ligand binding to receptor is determined by a hydroxyapatite-binding assay. Hydroxyapatite absorbs the receptor-ligand complex, allowing for the separation of bound from free radiolabeled ligand. The mixture is vortexed and incubated for 10 min at 4°C and centrifuged, and the supernatant is removed. The hydroxyapatite pellet is washed two times in wash buffer. The amount of receptor-ligand complex is determined by liquid scintillation counting of the hydroxyapatite pellet after the addition of 0.5 mM EcoScint A scintillation cocktail from National Diagnostics[1].

Pirodavir (R 77975) is dissolved in DMSO (10 mg/mL) and stored, and then diluted in growth medium before use[2]. HeLa cells are seeded at a concentration of approximately 180,000 cells per dish in six-well plates containing 4 mL of growth medium. Growth medium consist of Eagle's basal medium, supplemented with 5% fetal calf serum, 2% sodium bicarbonate, and 1% glutamine. After 24 h of incubation at 37°C in a humidified CO2 atmosphere, the growth medium is removed and replaced by the test solutions (fresh growth medium with or without various concentrations of the antiviral compounds). To assess the cytotoxicity of the antiviral compounds (e.g., Pirodavir), the number of living cells are determined present in triplicate cultures at the time of Pirodavir addition and every 24 h for 3 days. Following trypsinization, the number of viable cells for each drug concentration is counted in triplicate with a Coulter Counter[2].