The DIANA assay is a novel multiwell-plate based assay suitable for enzyme detection and inhibitor screening. DIANA uses a straightforward protocol, in which the target enzyme is captured, probed with the detection probe consisting of a small-molecule active site ligand attached to a reporter DNA, and subsequently detected by qPCR. It can be implemented using standard laboratory equipment and be fully automatized.
DIANA provides multiple unique advantages over the existing assays (such as ELISA, immuno-PCR or proximity ligation/extension) making it suitable for two areas of commercial application: 1) Clinical in vitro diagnostics 2) Screening for enzyme inhibitors in drug discovery.
In clinical diagnostics, it allows for precise and specific detection of miniscule amounts of target protein (e.g. onco-markers) in blood or other clinical samples. In addition to the use in R&D setting, CE IVD detection kits are being developed.
In screening application, DIANA is used in a modified setup where screened compounds compete with the probe for target binding. It offers sensitive hit discovery (ultra-low false-positive and false-negative rate) while providing direct quantification of the compound’s inhibition potential and allowing screening in highly efficient compound pooling setup leading to reduced screening cost and time. This makes it suitable for both large pharma HTS facilities and smaller-scale projects at academic institutions (in-house libraries of ~10k compounds can be screened in just few 96-well plates).
The assay has been developed, validated and automated in 96- and 384-well plates for multiple targets, including two human cancer markers (PSMA and CA-IX) and Fibroblast activating protein (FAP). In addition, DIANA assays are straightforward to develop for majority of relevant target proteins.
IP is owned by the Institute of Organic Chemistry and Biochemistry, the Czech Academy of Sciences, Prague.
The technology is available for licensing, co-development and/or distribution partnerships.
For more info: https://www.dianabiotech.com/
Methods currently used for enzyme detection and for inhibitor screening suffer from low sensitivity, low reliability and narrow range. DIANA assay overcomes these limitations and provides clear advantages in its both areas of application.
In diagnostics, the major benefits are:
• Ultra-high sensitivity - up to zeptomolar (10-21 M) amounts can be detected
• Very broad dynamic range - up to six-logs
• Very low background - lack of antibody interference and non-specific binding
• High selectivity for active form of the target - validation by titration with free inhibitor possible
• Robustness - proteins can be quantified in various biological samples (incl. blood serum)
• Easy protocol - suitable for detection kit format
• High level of reproducibility - suitable for CE IVD
For inhibitor screening, its major benefits are:
• Extremely high signal to noise ratio (Z’ > 0.9; CV < 5%)
• Quantitative - compound potency directly measured from a single well, hits ranked by inhibition potency, easy to screen for specificity in protein family
• Sensitivity - sensitive hit discovery, ultra-low false-positive and false-negative rate
• Robustness - no need for recombinant and/or purified proteins, no interference or non-specific binding
Cost-efficiency - very low compound and target protein consumption (typically picograms of protein needed) and ability to screen inhibitors in pooled compound libraries
The assay is currently developed only for ~10 target proteins (another >10 in development). The development for new targets is relatively straightforward, but it needs existing ligand (small molecule or peptide) that specifically binds to the target. It must be also possible to modify this ligand for attachment to the DNA oligo without significantly compromising its binding affinity. If such ligand is not available, then it may be complicated to develop the DIANA assay for that target.
The protocol, while suitable for full automatization, requires washing steps and detection using qPCR, which may not be compatible with existing protocols at some HTS or clinical diagnostic facilities.
DIANA has two major areas of commercial application.
1) In diagnostics, it is used for a sensitive detection of active-form of protein (e.g. onco-marker) in blood or other clinical samples. For this purpose, detection kits are being developed, which will be composed of antibody coated 96- well plates, detection probe, target protein standard and buffers. Together with standardized experimental setting such kits could be CE IVD certified to allow use in clinical diagnostic facilities.
DIANA-based detection kits can be also used by R&D labs in experiments where other assays do not exist or are not sensitive enough to quantify the target proteins.
2) For drug discovery application, DIANA is used to screen libraries of compound to identify new inhibitors / ligands of target protein. This can be implemented at large pharma screening facilities utilizing the licensing agreements, but also in smaller-scale at research and academic labs using ready-made DIANA screening kits.
We are currently searching for developmental and distribution partnerships for delivering full potential of DIANA assay to the market.