Protocols screening tests

This chapter presents methods and protocols suitable for the identification and characterization of inhibitors of the prokaryotic and/or eukaryotic translational apparatus as a whole or targeting specific, underexploited targets of the bacterial protein synthetic machinery such as translation initiation and amino-acylation. Some of the methods described have been used successfully for the high-throughput screening of libraries of natural or synthetic compounds and make use of model universal mRNAs that can be translated with similar efficiency by cellfree extracts of bacterial, yeast, and HeLa cells. Other methods presented here are suitable for secondary screening tests aimed at identifying a ... 

In the following section, we describe protocols for tests aimed at screening for compounds capable of interfering with some of the main activities of this factor, such as (a) recognition and binding of initiator tRNA (b) codon-dependent ribosomal binding of fMet-tRNA leading to the formation of a 30S or 70S initiation complex (c) ribosome-dependent hydrolysis of GTP and (d) accommodation of fMet-tRNA in the ribosomal P-site and formation of the first peptide bond (initiation dipeptide formation). 

Rather than assess all attributes for each prototype, a subset of properties was selected for use as a screening process. Protocols for screening tests that are broadly equivalent to those implemented by the Unilever product scientists during the development of Dove have appeared in the patent literature, and we note these references in Table 9.3-1. The tests were relatively easy to perform, and served to rapidly eliminate unsuitable compositions. Only if a prototype passed all screening tests would it be subject to the remaining property assessments, listed in Table 9.3-2. 

Frog embryo teratogenesis assay on Xenopus (FETAX) has been routinely used in oiu laboratory for the last 12 years as a routine developmental toxicity screening test for pharmaceutical candidate compoimds. To date, out of more than 400 candidates tested in FETAX, around 60 have also been evaluated in mammalian embryotoxicity studies according to standard ICH protocols. 

Frequently, results are highly dependent upon the test protocol. This is especially true for screening tests. For example, many screening tests do not employ an acclimation step prior to the test start and/or may not run long enough to allow for acclimation during the test. In addition, the reproducibility of individual tests often is poor, especially between laboratories, and in some cases even within the same laboratory. 

How general are such conclusions Do they apply to more complex proteins Are there more elusive features that influence turn preferences Selection for catalytic activity, an extremely sensitive probe of structural integrity, has the potential to reveal more subtle sequence preferences than simple screening protocols. We tested this premise using the EcCM enzyme (Fig. 3.4B) as a template [85]. The two identical polypeptides that make up this helical bundle protein consist of three helices joined by two loop segments (Fig. 3.12A) [33], The L2 loop connecting the H2 and H3 helices lies farthest from the active site and was targeted for analysis. 

The Bitrex (Denatonium benzoate) solution aerosol QLFT protocol uses the published saccharin test protocol because that protocol is widely accepted. Bitrex is routinely used as a taste aversion agent in household liquids which children should not be drinking and is endorsed by the American Medical Association, the National Safety Council, and the American Association of Poison Control Centers. The entire screening and testing procedure shall be explained to the test subject prior to the conduct of the screening test. 

Once phage or scFv which specifically bind the antigen of interest have been identified it may be necessary to cany out further screening tests to assess which antibodies fiom the positive population have the highest affinities. There are a variety of wtq of achieving this including surface plasmon resonance (BlAcore) screening, fluorescence quench measmement, and competition ELISAs. Descriptions of these protocols are beyond the scope of this chapter. 

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