Screening Improvements

In order to enhance the quality of screening data generated from highly valuable natural products, the nature of the extracts can be improved. There are a number of approaches to this problem, but the common goal has been to simplify the materials that are tested. When the offending materials are well understood, such as the presence of tannins in plant extracts, pretreatment of the crude extracts may suffice to remove the tannins from the screening samples. A broader approach that is applicable to samples derived from a variety of sources is diagrammed in Figure 6.8. This HPLC-based method seeks to remove offending nonspecific materials, as well as simplifying the actual screening samples. As diagrammed in Figure 6.8, the natural products are concentrated, often in the form of a solvent extract, and then subjected to a separation by reversed phase HPLC. The compounds are separated by employing gradient elution and only the material contained in the shaded area is retained for testing. This area contains components with polarities consistent with drug-like properties, having eliminated the early-eluting highly polar compounds such as saccharides and amino acids and the highly retained lipophilic materials that elute at the end of the run. The area is divided into 10 fractions that are concentrated and plated for use in high-throughput screening.

Fractionated screening samples derived from chromatographic separations as indicated in Figure 6.8 offer several advantages over crude extracts. The major one is the ability to independently evaluate diverse components produced by a particular source organism. In cases where one component in an extract is toxic, its adverse effect on the test organism in a phenotypic assay may obscure the positive response of a second component. Actinomycetes are particularly notable in their ability to produce numerous families of compounds. For example, it has been well documented that Streptomyces species that produce the milbemycin class of macrolides, such as the previously mentioned nemadectin, generally also produce oligomycins. The oligomycin macrolides are known

Concentrate

Microbial broth

Microbial broth t

Reversed phase HPLC

Plating for HTS

Reversed phase HPLC

2 6 10 14 18 22 26 30 34

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