Objective

Many diseases are treated by inhibitors of disease-related enzymes. To find those inhibitors, small-molecule libraries are commonly screened. However, currently used methods suffer from low sensitivity and low reliability and require purified enzyme.
We developed a novel assay suitable for both ultrasensitive enzyme quantification and screening of enzyme inhibitors, which overcomes the current limitations. In the DNA-linked Inhibitor ANtibody Assay (DIANA), the target enzyme is captured by an immobilized antibody, probed with the detection probe consisting of a small-molecule inhibitor attached to a reporter DNA, and subsequently detected by qPCR.
DIANA enables quantitative screening of enzyme inhibitors by using just microliters of blood sera containing picogram quantities of endogenous target enzyme. Furthermore, it permits determination of an inhibition constant from a single well measurement using a single concentration of the inhibitor. Moreover, the high signal-to-background ratio, low false positive rate and low false negative rate enables screening for inhibitors in pooled libraries or compound mixtures. In this manner, we were able to discover novel and potent inhibitory scaffolds against two clinically relevant enzymes (PSMA and CAIX).
Thanks to the straightforward protocol, DIANA has potential to become a method routinely used in many biochemical laboratories around the world for both disease detection and drug discovery.