Whole cell assays

Oldfield s work confirmed the complete pathway and identified all the intermediates via an exhaustive experimental scheme, which included whole cell assays, cell-free extracts of IGTS8 as well as extracts from clones containing individual genes expressed in Escherichia coli. The need for NADH was clearly demonstrated in cell-free assays by amendment of NADH. The experimental evidence for involvement of FMN in the pathway was demonstrated [53,66,67],... 

The specificity of several of the enzymes identified in the 4S pathway of different organisms has been studied. In case of the DBT desulfurizing enzymes, little difference is expected in the specificity of the enzymes, say DszA, from different Rhodococcus strains found to date. This is essentially because the DNA sequence for the enzymes investigated so far has been the same. The difference in the specificity observed with whole cell assays is essentially due to the differences in substrate intake via the cell membrane and not necessarily due to a difference in the intrinsic enzyme specificity. It has been found that while isolated enzyme DszC (from KA2-5-1) can desulfurize up to 4,6 dipropyl DBT, whole cells cannot, indicating substrate transport as limiting factor. 

Analog ent-(+)-8 is 20-fold more potent than racemic acifran and is threefold more potent than niacin in the cAMP whole-cell assay [55]. The selectivity was also improved with respect to acifran with 10-fold selectivity for GPR109A over GPR109B. The importance of the absolute configuration of ent-(+)-8, which is tentatively assigned S, is demonstrated by the 70-fold better cAMP whole-cell activity of the eutomer compared to that of the distomer. 

Functional in vitro assays can be whole-cell assays or animal tissue assays. For in vitro cell-based assays, recombinant molecular targets can be expressed in cell lines and linked to second-messenger systems to measure functional responses. Examples of functional... 

Whole-cell assays (allowing measurement of phosphoinositide hydrolysis, adenylate cyclase activity, etc.)... 

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. 

Schwaneberg, U., Otey, C., Cirino, P.C., Farinos, E. and Arnold, F.H., Cost-effective whole-cell assay for laboratory evolution of hydroxylases in Escherichia coli. J. Biomol. Screen., 2001, 6, 111-117. 

A number of approaches have been used to discover new antibiotics in past decades. One approach is empirical screening, in which a whole cell assay is used to find antibacterial activities first, followed by the... 

Fig. 1. Empiric observation that more than approx 20 compounds from any scaffold need to be tested in order to be sure that an active will be found. (A) This shows the results from a whole cell assay in which the compounds have been classified using the Level 1 Ring System of the Structural Browsing Index (SBI) also known as molecular equivalence numbers or meqnums, which is the most-detailed level. The most-populated SBI containing only inactives is labeled 1, it contains 16 compounds. The most populated SBI containing actives is labeled 2. (B) Looking at only the compounds present in the most-populated SBI (2 above) and arranging the compounds in this SBI randomly, the smallest set of compounds in which an active (indicated by +) can be found is approx 30 compounds.

Berg, J., Christoph, T., Widerna, M., Bodenteich, A. Isoenzyme-specific cyclooxygenase inhibitors a whole cell assay system using the human erythroleukemic cell line HEL and the human monocytic cell line Mono Mac 6, J. Pharmacol. Toxicol. Methods 1997, 37, 179-186. 

Preliminary pharmacokinetic behavior can be tested through a number of whole cell assays. Most commercially successful drugs are administered orally, meaning the drug must be able to enter the bloodstream by crossing membranes in the intestines. The most common membrane permeability assay is performed by monitoring the absorption and secretion of a compound by colon carcinoma cells (Caco-2). Diffusion across Caco-2 cell membranes is considered to be a valid model for molecular transport in the small intestines.16... 

Ten compounds were ultimately selected, and five compounds showed MIC < 12.5 pg/mL in whole-cell assays with no cytotoxicity, while the binding of these compounds to enzyme remained to be demonstrated... 

Whole-Cell Assays with Transfected Chinese Hamster (CHO) Cells Expressing COX-1 and COX-2... 

Whole-Cell Assays with Osteosarcoma Cells (COX-2) and U937 Cells (C0X-1)... 

Whole cell growth inhibition screens combined with subsequent target identification using molecular methods have proven viable approaches to the discovery of novel antibacterial inhibitors. Andries and colleagues (2005) at Johnson Johnson employed whole cell assays to discover a series of antimycobacterial diarylquinolines (DARQs). Chemical optimization of a lead compound led to DARQ derivatives exhibiting potent in vitro activities against several mycobacteria including Mycobacterium tuberculosis (Andries et al., 2005 Ji et al., 2006), with MICs below 0.5 pg/mL. Antimycobacterial efficacy in vivo was confirmed for three of the derivatives. 

A PROKARYOTIC WHOLE CELL ASSAY TO IDENTIFY SIGNAL TRANSDUCTION INHIBITORS... 

---