Bioactivity screening technologies

BMA has a long track record on combining liquid chromatographic (LC) separation with bioassays and parallel MS, the so-called high-resolution screening (HRS) approach, enabling the simultaneous determination of bioactivity and chemical composition of compounds in complex mixtures. More recently, we introduced the nanofractionation approach in which high-resolution LC fractionation allows use of at-line plate-reader-based bioassays. Advantages are straightforward implementation of standard assay formats and the ability to use any bioassay of choice.

Venoms potentially are an important source of new lead compounds in drug discovery. We developed new analytical workflows based on on-line screening or high-resolution nanofractionation for identification of bioactive venom peptides. In the on-line approach, post-column infusion of eluting peptides into a continuous-flow fluorescence bioassay is performed. On-line screening by mixing homogeneous flows is often not possible, and therefore we have also developed a nanofractionation strategy in which LC separation is coupled to high-resolution fractionation onto (multiple) microtiter plates with 96 up to 1536 wells allowing post-column assays. In both approaches, MS data on bioactives is collected in parallel using a post-column split. Snake and cone snail venom screening was carried out for the drug targets angiotensin converting enzyme, Factor 10a, thrombin, and the nicotinic acetylcholine receptor. Re-analysis of crude venoms using orthogonal separation techniques, confirms presence of bioactives (Figure). Using a proteomics approach, nano-LC-MS/MS on earlier pinpointed bioactives - optionally preceded by in-solution tryptic digestion - facilitated their unambiguous identification.

Biomolecular Analysis 1

Drug-drug interactions caused by inhibition or induction of drug metabolizing enzymes, such as Cytochrome P450 (CYP), can lead to adverse reactions in humans. Bioaffinity profiling of drug metabolites at an early stage in drug discovery therefore is essential. We developed LC-nanofractionation methodologies with parallel MS for the identification of CYP1A2 inhibitors present in metabolic mixtures. Applying a fluorescence bioassay to the fractions, metabolites with affinity towards CYP1A2 are detected as a negative peak in the bioactivity chromatogram. Phase I metabolic mixtures of the drug ellipticine were screened. The observed bioaffinity peaks were in a good correspondence with the LC-MS data, and the main metabolites, including structural isomers, were separated and identified.

The nanofraction platform was fully developed as a new tool for high-throughput effect directed analysis (HTEDA) in the field of environmental safety profiling. Hyphenation of a human gene reporter assay (ER-luc) to LC was accomplished for the identification of estrogens in surface and drinking water. Both estrogenic/anti-estrogenic and androgenic/anti-androgenic compounds in the environment could be detected rapidly. Moreover, a novel method was developed for high-resolution fractionation after gas chromatography and its feasibility for EDA was demonstrated.

Another line of research involves the label-free, real-time detection of analyte biochemical interactions by surface plasmon resonance spectroscopy (SPR). An estrogen receptor (ER) assay was developed by immobilizing animated estradiol on the sensor surface. The ligand binding domains of the ER alpha and beta showed strong binding. By incubation of ER in presence of estradiol the binding was fully diminished with almost no bulk effect. We developed a bench top SPR multiplexing instrument with a camera pixel algorithm and software implemented. After obtaining successful results with multiple channel flow cells, the same approach was performed with a one-channel flow cell for measurement of binding events of antibodies to 16 spotted proteins on different areas on the gold sensor surface. The multiplexing can theoretically be extended to approximately maximally 100 spots on the sensor surface considering the total sensor surface length. We also developed a variable wavelength (350-890 nm) and angle scanning (35-78 degree) SPR setup.