Primary activity assays

Whether a project aims at delivering an agonist, an antagonist, or any other biologically active molecule, it needs a robust in vitro assay with adequate capacity and turnaround time to furnish frequent, quality potency data to drive design. 

It is widely accepted that key physicochemical properties give an indication of the drug-likeness of a molecule [8-10], and thus provide medicinal chemists with a fundamental imderstanding when trying to delineate the desired chemical space against a particular target These properties typically include lipophilicity which is closely linked to other properties like solubility, clearance, plasma protein binding, and permeability. 

Various in vitro assays are widely available for profiling distribution, metabolism, and pharmacokinetics (DMPK, also referred to as ADME absorption, distribution, metabolism, and excretion). Such properties of molecules are measured to ultimately predict their in vivo behavior. The metabolic stability of molecules is assessed routinely in drug discovery units by way of medium- to high-through-put assays using hepatic microsomes or hepatocytes obtained from different species (usually rat and/or human). Permeability assays (e.g., utilizing Caco-2 or MDCK cells) together with an assessment of efflux potential are also useful to troubleshoot unexpectedly low cell activity or can help select candidates for subsequent in vivo studies. 

Depending on the desired route of administration, experiments to determine the exposure in rodents (most likely rats and mice) and later in dog are necessary to predict likely exposure in man. 

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Figure 29) was tested in several cell lines in a one-dose in vitro primary cytotoxicity assay, and passed the criteria for activity (20-29% growth percentages) next, it was scheduled for evaluation against the full panel of 60 human tumor cell lines at minimum of five concentrations at 10-fold dilution, showing very favorable cytotoxicity <2003EJM781>. 

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. 

Taken together, these classes of compounds can cause a significant enrichment of false positives among the most active compounds. This is illustrated by the sudden spike in the number of actives at the highest levels of inhibition (the most active 0.2%) in a recent primary HTS assay run here at Wyeth see Figure 6.1. In the study by SiUs... 

The 5 k actives with percent inhibition of 25 to 40% and that feU into clusters of less than five compounds were treated separately using BCUT diversity analysis, as described in Section 6.2.5. A cell-based selection biased by primary activity from six bins per each of six axes yielded 1258 compounds. The combined selection from filtering, clustering, and diversity totaled 6986 compounds representing 3337 ring scaffolds and was submitted for confirmation assays. Note that the full set of 16 k filtered actives contained 9254 ring hashcodes, so the selected set covers 36.1% of the represented scaffolds. Because of the presence of duplicate samples in the corporate screening collection, 7275 samples were pulled and assayed. 

Various bioassay systems are available in the literature for the screening of potentially chemopreventive compounds. Table 1 lists the bioassay systems for chemoprevention, as well as anti-inflammatory activities often associated with chemoprevention, reported for terpenoids and described in this review. The assays for anti-carcinogenic compounds might be classified as in vitro and in vivo primary screening assays, in vivo assays of several tumor models for various target organs, and mechanism-based in vitro assays. 

In vitro primary screening assay AM Anti-mutagen activity... 

CK-MB can be measured in numerous ways. Immunoassays developed in recent years have improved on the analytical and clinical sensitivity and specificity of the earlier immunoinhibition and immunoprecipitation assays. These assays now (1) measure CK-MB directly and provide mass measurements, (2) are easily automated, and (3) provide rapid results (<30 minutes). Mass assays reliably measure low CK-MB concentrations in both samples with low total enzyme activity (<100 U/L) and with high total enzyme activity (>10,000 U/L). Furthermore, no interferences from other proteins have been documented. The majority of commercially available immunoassays that use monoclonal anti-CK-MB antibodies are the same as those listed in Table 5-2 for cardiac troponin assays. Excellent concordance has been shown between mass concentration and activity assays. A primary reference material is commercially available to assist in harmonization. If used for assay standardization, then this material allows... 

The same compound collection was screened by Stockwell et al. (24) on a primary FCG assay directed towards activators of a regulatory reporter gene (p3TPLux)... 

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