Biomedical Engineering Reference
In-Depth Information
linked to a biological assay. A positive response in the assay (a “hit”) is determined by the intrinsic
potency of a given compound and its concentration in the screening sample. The sources of natu-
ral products used in the screening process can be quite diverse, ranging from bacterial products
to higher plants and animals, however the processes involved are similar. Once a sample, which
is typically an extract of an organism, or a part of an organism (e.g., fermentation broth, fruiting
body of a mushroom, leaves, or roots of plants, etc.) has shown a positive response in a given assay
the process of “bioassay-guided fractionation” begins. This process is shown as a loop diagram in
Figure 6.6. Resolution of the active principle(s) in these materials is a highly experimental process.
The ease of resolution is dependent upon such parameters as the concentration of the active
compound in an extract, the overall constitution of the extract, in terms of interferences (e.g., tan-
nins, fatty acids and other lipids, and complex carbohydrates), as well as the chemical properties
of the compound of interest. Trial and error is the operational mode of these processes and is
highly dependent on the preferences and experience of the individual investigator. As indicated in
Figure 6.6 the original crude material is initially split into fractions, by a rough process such as
differential solubility in solvents with different polarities, or by liquid-liquid partitioning between
immiscible phases, usually aqueous versus organic. Subsequent steps are generally of higher reso-
lution often with different forms of chromatography, perhaps using a normal phase high-capacity
technique like silica gel chromatography in organic mobile phases i rst, followed by a reversed
phase system with a hydrophobic stationary phase eluted with an aqueous-organic mixture. It is usu-
ally the case that a suite of structurally related compounds is isolated in this process, each having
some activity in the bioassay of interest. Subtle differences in the potency or selectivity shown by
these congeners form the basis for the “natural structure-activity relationship” (SAR) of the series
that may be useful in designing improved compounds by synthetic or biosynthetic methods during
subsequent optimization of the lead. Once a compound is shown to have the activity of interest
and passes a criterion of purity, it can proceed for resolution of its chemical structure and further
biological evaluation.
Crude extract
Fractionate (HPLC, Partition, HP20, etc.)
Fractions
~2 to 3
cycles
Test fractions
Active
?
Active
?
Active
?
Active
?
Active
?
No
Ye s
Ye s
Ye s
Ye s
HPLC or HPLC/MS
No
Pure
?
Pure
?
Pure
?
Ye s
Ye s
Pure compound
Determine structure
assess potency, etc.
FIGURE 6.6
Bioassay-guided fractionation of natural products.
6.4.2 W
HOLE
O
RGANISM
S
CREENING
Often referred to as “phenotypic screening,” as well, the screening of natural products in whole
organisms was historically the original mode, and one that still has a great deal of potential. One of
the most robust examples of this approach is screening for antimicrobial activity by inhibiting the
