Cell-based replication assays

An interesting and more recent expression system for the development of cell-based assays is based on BacMams. These recombinant baculoviruses containing mammalian cell-active expression cassettes seem to be an efficient strategy to speed up assay development. These viruses are produced in insect cells and transiently express but do not replicate in transduced mammalian cells. The expression level can be well controlled by titrating the amount of virus. Highly reproducible transient expression levels, which are a prerequisite to use transient transfection for HTS, might thus be an attractive alternative for some approaches. 

Bioassay-guided fractionation of an aqueous extract from a Philippine Islands collection of the soft coral Lohophytum spp. yielded cembranoid diterpenes, Fig. (3), which exhibited moderate HIV-inhibitory activity in a cell-based in vitro anti-HIV assay [44], while new isomalabaricane triterpenes. Fig. (4), have been isolated from the sponge Stelletta spp. [45]. Other anti-HIV diterpenes also included the dolabellane diterpenes isolated from the Brazilian brown algae Dictyota pfaffi [46] and Dictyota menstrualis [47]. To investigate the effect of these diterpenes in the reverse transcription of the viral genomic RNA, the recombinant HIV-1 RT was assayed in vitro in the presence of each compound. All compounds inhibited the RNA-dependent DNA-polymerase activity of HIV-1 RT and consequently virus replication. 

Physically-maintained sink conditions In cell-based assays, an aliquot is removed for analysis from the receiver wells to make time point measurements. This is replicated in Caco-2 but not in PAMPA. 

The majority of RSV inhibitors that have emerged from cell-based virus replication assays have been determined to be inhibitors of the virus-host fusion process. The discovery and basic SAR associated with these compounds will be described followed by a discussion of their mode of action that, quite remarkably given the structural diversity, appears to converge on interfering with the formation and/or function of the RSV F protein six-helix bundle complex. 

As another example of modification at the base moiety of HEPT, methylation and benzylation of the N-3 position were carried out.55 From the fact that both iV-alkylated products were totally inactive in the replication assay of HIV-1 in MT-4 cells (data not shown), it can be concluded that the presence of an NH is indispensable for activity. Also, it is quite interesting to see a lactam structure in common in the reported HIV-1-specific heterocyclic compounds (47-49) and in the HEPT analogues, all of which have no substituent at the nitrogen. 

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