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DIANA assay for in vitro diagnostics and drug discovery

Enzymes are important targets in diagnostics and drug discovery due to their causative involvement in most human diseases. However, methods currently used for enzyme detection and for inhibitor screening suffer from low sensitivity, low reliability and narrow range. Therefore, we developed the DIANA assay, which overcomes all listed limitations.

The DIANA assay is a multiwell-plate based assay suitable for enzyme detection and inhibitor screening. DIANA uses a straightforward protocol, in which the target enzyme is captured by an immobilized antibody, probed with the detection probe consisting of a small-molecule active site ligand attached to a reporter DNA, and subsequently detected by qPCR. We developed this assay for detecting two human cancer markers (PSMA and CAIX) in various biological matrices. We have also shown the ability to screen inhibitors of five human enzymes (PSMA, GCPIII, CAVII, CAIX and FAP), two viral enzymes (HIV protease and influenza neuraminidase) and even one human receptor (Fc receptor). Using DIANA, we also discovered novel scaffolds inhibiting PSMA suitable for further development.

Enzyme detection (1) sensitivity: up to zeptomolar (10-21 M) amounts are detected (2) selectivity: target proteins are selectively quantified in various biological matrices (3) broad linear range of up to six-logs (4) only active form of the enzyme is detected Screening of enzyme inhibitors (1) sensitivity: low enzyme amounts needed (typically picograms) (2) selectivity: no need for recombinant and/or purified proteins (3) inhibition potency can be determined from a single tested concentration (4) sensitive in hit discovery, low false positive and false negative rates (5) ability to screen inhibitors in pooled compound libraries

Automated protocol in 384-well plate Developed for 8 targets, >10 in development Pilot compound library screens

DNA-linked Inhibitor Antibody Assay (DIANA) for sensitive and selective enzyme detection and inhibitor screening. Navrátil V, Schimer J, Tykvart J, Knedlík T, Vik V, Majer P, Konvalinka J and Šácha P. Nucleic Acids Res. 2017 Jan 25;45(2):e10. doi: 10.1093/nar/gkw853.

Patent pending (PCT phase).

Institute of Organic Chemistry and Biochemistry AS CR Flemingovo n. 2, 16610 Prague 6 Czech republic