Drug primary activity assays

Proteolytic enzymes in the respiratory mucosa play important role(s) in the regulation of lung inflammation and remodelling [123, 124], Pulmonary proteolytic enzymes, however, also comprise one of the barriers which pulmonary-administered protein/peptide drugs have to overcome in order to achieve adequate bioavailability [125]. Intriguingly, the pulmonary enzymatic barrier is an aspect that has been little investigated and is poorly understood. Inconsistencies in the data available to date are most likely a result of the use of different techniques (e.g., PCR, immunotechniques and enzyme activity assays), different species and different cell (pheno)types, for example primary cells vs. cell lines. 

FIGURE 11.1 Three levels of compound testing in a drug discovery-development program. Level 1 entails testing every compound made for primary activity. Level 2 can be done on selected compounds for scaffold (chemical type) characterization. Level 3 may involve labor-intensive assays to fully characterize mechanism of action and define all activities relevant for possible candidate selection. The actual types of assay are different for agonists (Table 11.1) and antagonists (Table 11.2). 

As mentioned earlier, the early clinical development of tumor-targeted anticancer agents requires the use of non-traditional methods such as molecular drug endpoints (Western blots, activity assays) in tumor or surrogate tissue, and functional imaging studies. A review of the literature [27] showed that such methods were not routinely incorporated into the study design for early trials of anticancer agents, and rarely formed the primary basis for... 

Kunkel, E. J., Plavec, L, Nguyen, D., Melrose, J., Rosier, E. S., Kao, L. T., Wang, Y., Hytopoulos, E., Bishop, A. C., Bateman, R., et al. (2004). Rapid structure-activity and selectivity analysis of kinase inhibitors by Bio Map analysis in complex human primary cell-based models. ASSAY Drug Dev. Technol. 2 431-441. 

Despite the complexity of the experiments and the enormous data manipulation necessary, complex biological pathways, as well as new drug targets are being identified by this method. Examples include screens for compounds that arrest cells in mitosis, that block cell migration, and that block the secretory pathway [50], or assays with primary T cells from PLP TCR transgenic mice for their inhibitory activity on the proliferation and secretion of proinflammatory cytokines in PLP-reactive T cells [51], and identification of small-molecule inhibitors of histone acetyltransferase activity [52]. 

Because the chemical structure of a molecule encodes its biological properties, structure has long served as the primary variable and determinant for the discovery of new drugs by medicinal chemists. For this reason, systematic structural modification has been the primary tool of choice to isolate and enhance a desired biologic activity. Moreover, with the relatively recent development of in vitro receptor-binding assays, combinatorial methods of chemical synthesis, and computer graphics, the overall approach to structural modification has become increasingly sophisticated. 

Primary assays are devised to incorporate physiological or enzymatic targets for screening biological activity of potential drug compounds, ilie biological assays are then reconfirmed in specific biochemical and whole cell assays to characterize the target-compound interaction. 

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