PHERAstar FSX

Residues of human pharmaceuticals, such as β-blockers are increasingly found in effluent wastewater of treatment plants (WWTP) and represent a potential environmental threat. Beta-blockers antagonize β-adrenergic receptors and thereby control hypertension and cardiac arrhythmias. The amino acid sequence of the target, the β1-adrenergic receptor, is evolutionarily highly conserved among vertebrates. Thus, organisms may physiologically respond to β-blockers. For environmental risk assessment one has to know the extent to which aquatic organisms are exposed to β-blockers and metabolites with the same mode of action (MOA).

The mode of action-based β-blocker assay in living cells measures total β-blocker activities in complex mixtures such as WWTP effluents as equivalents of the lead substance metoprolol (MetEQ). The assay is suitable as a standard method in large-scale monitoring of WWTP effluents and the aquatic environment they are discharged into. The CLARIOstar^® microplate reader provides highly stable and reliable results as well as the required robustness for continuous use.

To survive as sessile organisms, plants need to constantly adapt their metabolism to their environment. Flooding of plants is widespread and has severe metabolic consequences as it limits the cellular supply with oxygen to drive respiration, and thus production of ATP. Submergence acclimation typically involves drastic alterations in metabolism to circumvent anoxia and to maintain primary metabolism and ATP supply. We have recently established the use of a genetically encoded protein sensor (ATeam1.03-nD/nA) to assess MgATP dynamics in the model plant Arabidopsis thaliana.

This FRET-system read on a CLARIOstar^® allows for reliable analyses of changes in MgATP in vivo and in real-time and provides a new handle to investigate the energetic consequences of low oxygen for the plant cell. The microplate reader’s orbital averaging function acquires the signal in multiple points of the well to obtain a meaningful result of the non-homogenously distributed Arabidopsis seedlings.

Receptor tyrosine kinases (RTK) are transmembrane receptors that translate stimulation by growth factors into regulation of cell growth, proliferation, cell death and differentiation. Consequently, aberrant RTK-signaling is implicated in cancer, making it a popular anti-cancer drug target. Development of pharmaceutical RTK-inhibitors requires assays that characterize receptor-ligand interaction and that identify inhibitors thereof.

A novel method labeled the vascular endothelial growth factor (VEGF) at a single site with the fluorophore TMR. The labeled ligand was used in combination with HEK293 cells expressing the RTK VEGF-receptor 2 (VEGFR2) fused to the NanoLuc^® luciferase. Upon interaction of ligand and receptor, bioluminescence resonance energy transfer (BRET) between luciferase and fluorophore takes place.

The signal of luciferase and fluorophore were acquired simultaneously with the PHERAstar microplate reader. This way, not only the pKi of a VEGF-inhibitor was determined, but also the time-course of binding and inhibition was monitored.

GPCRs are important drug targets requiring receptor-protein interaction and trafficking studies to reveal how they function. Bioluminescence resonance energy transfer (BRET) is a versatile tool to study such interactions and trafficking. Hitherto, it is limited by the ectopic expression of labelled interaction partners. CRISPR/Cas9 genome editing overcomes the limitation by enabling endogenous expression of luciferase-labelled proteins.

CRISPR/Cas9-edited cells endogenously expressing a CXCR4/NanoLuciferase fusion protein were used in conjunction with β-Arrestin/Venus to monitor receptor activation. Employing two fluorophores fused to a membrane and endosome standing CXCR4-interacting protein, respectively, allowed for monitoring of receptor trafficking.

The novel CRISPR/Cas9 technique successfully fused the Nluc BRET donor to endogenously-expressed CXCR4. The resulting protein levels were sufficient to monitor receptor interactions as well as internalization. The internalization assay depends on two acceptor fluorophores whose selective detection was rendered possible by the CLARIOstar’s monochromator.

Schistosomiasis is a parasitic disease that affects over 200 million people in tropical, developing nations, causing severe morbidity and over 300,000 deaths annually. Schistosomiasis is treated with a single drug and no vaccine is available.

We selected three Schistosoma surface-associated enzymes that are indispensable to parasitic survival: alkaline phosphatase; phosphodiesterase SmNPP-5 and an acetylcholinesterase. The activity of these molecules on the surface of live and intact larval and adult Schistosoma can be assayed in real-time of cultured parasites, providing a tool to assess the efficacy of drugs or vaccines targeting these enzymes. The colorimetric assay was read on a FLUOstar^® Omega microplate reader.

The versatile instrument supports development of assays and drugs. Apart from its absorbance spectrometer, the FLUOstar Omega is equipped with fluorescence and luminescence detection capabilities allowing fast and reliable endpoint and kinetic measurements in all detection modes.