Cellular antiviral assay

The inhibitory effects of the pyrrolinone derivatives were evaluated using enzyme inhibition and cellular activation assays. Compound 6 (Fig. 4.3-5) showed an IC50 of 10 nM, compared to 0.6 nM for the related peptide inhibitor 5 (L682,679). However, the synthetic agent 6 showed better cell transport capacity. In a cellular antiviral assay, 5 and 6 showed CIC95 values (the concentration that inhibits 95% of virus multiplication in the cellular cultures) of 6.0 and 1.5 pM, respectively. Smith and Hirschmann proposed that the improved cellular uptake properties of polypyrrolinones are due to a reduction in the inhibitor solvation. Solvation is an impediment to transport because extraction of a molecule into a lipid bilayer from an aqueous phase is... 

A sensitive method to measure toxic effects of drugs is to monitor cellular metabolism by incorporation of 3H-thymidine or 14C-protein hydrolysate. 14C-protein is used in preference to 3H-thymidine when it is necessary to avoid competition in uptake between the labeled thymidine and an unlabeled nucleoside reverse-transcriptase inhibitor. These methods are used mainly to monitor sublethal toxicity after initial screening or when comparing structure-activity relationships. The assays described here are carried out using 6-mL culture tubes (Note 1). These assays are carried out in parallel with the antiviral assay. 

Several diverse classes of potent CCR5 antagonists, Figure 7 have recently been reported [2], Scaffolds reported to support high potency against CCR5 in antiviral cellular assays included the aminocyclohexyl (compound 13) [45], 4,4-disubstituted piperidine (compound 14, GSK163929) [46,47], tetrahydro-2/T-1,3-oxazine (compound 15) [48], pyrrolidine (compound 16) [49], cyclopropyl (compound 17) [50] and 2,3-dihydro-lH-indene (compound 18) [51]. 

As lymphocytes, monocytes, and macrophages are the primary targets for viral infection, the penetration of antiviral agents is important. Lymphocytes and monocytes are indicated as peripheral blood mononuclear cells (PBMC). The preparation of control PBMC from blood was reported in considerable detail by Jemal et al. [38], In addition, a validated assay for the determination of ATA in PBMC was developed. The determination of protease inhibitors in hmnan PBMC was reported by several groups [39-40]. After LLE, the analytes were analysed by LC-MS. Both methods enable the intra-cellular determination of the analytes and can be applied for TDM and pharmacokinetic studies. 

In spite of obtaining a 1000 fold improvement in IC50, the best compounds did not show a significant therapeutic index (TD50/EC50 of > 10-fold) in cellular assays and therefore did not warrant claims of antiviral activity. The lack of cellular activity despite IC50 s in the 3-10 nM range necessitated a close examination of possible explanations. 

Antifungal and cytotoxic a-methoxy-y-pyrone derivative from Polyangium sp. (Myxobacteria), f-ssHasNOa Mr 379.46. P. crystallizes from butyl methyl ether as colorless crystals with mp. 92-93°C, uv,, 244 nm, 252 nm. The pyrone part of P. has a close relationship to aureothin and spectinabilin from streptomycetes. In addition to the antifungal activity, e.g., against Us-tilago maydis (MHK 19 pg/L) a cytotoxic activity has also been observed which mimics an antiviral activity in cellular assays. The reason for this is an inhibition of NADH ubiquinone oxidoreductase in complex I of the respiratory chain. 

The antiviral activities of natural products, including ingredients, fractions and extracts, need to be evaluated by various anti-viral models, including in vitro and in vivo models. Besides, the analysis of assay results of natural products is also very important. In the present section, the models to evaluate the in vitro anti-viral activities of potential natural products have been summarized, and the evaluation methods of several representative models have been introduced. The antiviral models include molecular level models and cellular level models (Table 3.2 Chattopadhyay et al. 2009). 

Cellular level models usually exploit the virus s ability to infect and replicate in specific cell lines in cell culture systems. The ceU culture system provides a rapid method to grow viruses at higher titres to test the antiviral activity of compounds/extracts. It is based on the observation that virus infection and multiplication results in cytopathic effect (CPE) due to either release of virus or induction of apoptosis as a result of host immune responses. Inhibition of CPE in presence of test compound could be due to inhibition of virus replication. The assay results combined with molecular level assay results will guide the later study in animal models. 

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