Drug profiling service procedure

1. Compound derivatization and toxicity test

The compound under investigation is synthesized as a derivative linked to an anchor moiety. The anchor moiety serves to couple the derivatized compound to the bait scaffold and a flexible linker ensures that no sterical hindrance occurs.
The compound synthesis can be performed by Dualsystems Biotech or by the customer.

The yeast reporter strain is grown in the presence of the derivatized compound in different concentrations.
Comparison of growth curves indicates the optimal compound concentration for the subsequent library screening.
As shown in the example on the right, a compound concentration of 1 µM has no negative effect on yeast growth and is therefore the optimal compound concentration.

2. Library transformation and compound-dependency test

The yeast reporter strain expressing a scaffold bait is grown in the presence of the derivatized compound.
The compound is internalized and immobilized on the scaffold via its anchor moiety.
The scaffold-immobilized compound is screened against a cDNA library of choice.
Upon interaction of the compound with a particular prey expressed from the cDNA library, the two yeast reporter genes (HIS3/ADE2) are activated, resulting in growth of the yeast cells under selection.
With our interaction technology, we are able to screen your compound of choice against millions of human proteins or protein fragments simultaneously.

Elimination of false positives (compound-independent interactions). As shown in the figure on the right, all positive clones from the library screen are tested for compound-dependent growth stimulation.
Only the clones above a predefined cut-off (red line) are considered to be compound-dependent interactors and are further analyzed.

The cDNA sequence encoding the compound-dependent interactor is rescued from yeast and the interacting protein is identified by 5' sequencing and BLAST analysis.
All interesting target candidates are retransformed into newly grown yeast and compound-dependent interactions are confirmed by a growth assay.

In order to further characterize the compound-dependent interactions of selected target proteins, different follow up experiments can be performed, such as competition assays, in vitro binding assays (figure on the right) and mammalian three-hybrid assays.