SAMPLE SCREENING

Crude extracts from ground up samples are used for biological and chemical assays aimed at detecting novel compounds with discernable biological activity such as the ability to kill cancer cells in culture.

Microorganism samples are also used to produce extracts used in these evaluations. To accomplish this, purified colonies from petri dishes are fermented, or grown, in flasks of various liquid growth media. The liquid acts as an extract for compounds produced by the microorganisms and can be used in assays.

One method for discovering natural products with therapeutic potential is known as a random approach. In this process, a large number of extracts are tested in various biochemical and biological tests, called "screens," which are designed to identify natural products with high potential for treating a particular disease.

For example, drug screening to identify a possible treatment for lung cancer could entail applying the extract to living cultures of the commercial A549 human lung tumor cell line. If cell proliferation or growth is inhibited by a particular extract in such a treatment, relative to an untreated control cell culture, the cell screen has then successfully identified an extract with some potential to fight lung cancer. Researchers then begin what can be a long process of separating out the various chemicals in an extract (more on this below) to home in on the one that actually has the therapeutic effect on the lung tumor cells.

Another drug discovery method is target-directed screening. In this approach, specific molecules such as enzymes, structural cell components, or chemical receptor sites are selected as targets based on their involvement in a particular disease. Screens are designed to identify those natural products that specifically interact with these targets. For example, certain enzymes are known to be necessary for activating the T-cells of the immune system. Natural products that inhibit the activity of this enzyme would therefore be expected to suppress the immune system, an important goal in preventing rejection of transplanted organs.

In many cases, target-directed screening and cell culture screening can be complementary. For instance, if marine chemical extracts screened using molecular-target based assays show bioactivity against the intended targets, then follow-up screens using cultured cell lines might be used to confirm the suspected bioactivity within intact living systems.

Those products that continue to show very promising biomedical potential are selected for further research and testing. Eventually a compound is promoted to the next level of screening involving whole animal testing, in which purified compounds are tested in appropriate animal models. These models could include mice or zebrafish with afflictions similar to those for which treatments are sought, such as cancer. Because the animal screening approach is very expensive, time consuming, and requires large amounts of purified natural product, it is used for only the select few products that show the most potential as disease treatments.

But animal testing, as well as any detailed study of a compound requires that it first be purified, and its structure identified. This is accomplished through Natural Products Chemistry.

Natural Products Chemistry

A crude extract derived from a marine organism typically contains a large number of compounds, but usually only one of these will be responsible for a particular bioactivity exhibited by the extract during laboratory screening.

Teasing out the compound of interest from a crude extract is a complex process. Active compounds are separated and purified in steps using a wide variety of methods. Initially, compounds in the extract might be separated into groups by adding solvents that only dissolve certain types of compounds. For instance, polar solvents will only separate the polar solute fraction from the crude extract. Compounds can also be purified from extracts using a combination of chromatographic methods, including thin layer chromatography (TLC), open column chromatography, vacuum column chromatography (VCC), high performance liquid chromatography (HPLC), and centrifugal counter current chromatography (CCC).

Purified compounds are used in further testing of compound bioactivity, for animal testing, and if all goes well, ultimately for human testing. Teams will often have to return to collection sites to gather more of a sample to produce compound needed for studies. Eventually the need for a compound may exceed the amount that can be generated from samples collected, and researchers have to determine a sustainable method for producing a compound.

Once a compound is purified, determining its structure is critical so that scientists can check chemical databases to ensure that a compound is in fact novel and not something whose biomedical properties have already been determined. Knowing the compound's structure also helps researchers determine why a compound has a particular biological effect such as killing tumor cells, so that its activity in humans can be predicted.

Biologically active compounds display a range of complexity and structural types. In order to determine the structure of purified compounds, researchers use several spectral methods, including nuclear magnetic resonance (NMR), as well as infrared (IR), ultraviolet (UV), and mass spectroscopy (MS). If a suitable crystal of the parent compound or a derivative can be obtained, structures are determined by X-ray crystallography.

The data obtained using these tools allows chemists to define key structural elements and functional groups of natural products, and finally to determine a molecular formula and structure.