Design strategies for water-soluble small molecular chromogenic and fluorogenic probes.

Protein-fragment complementation assays (PCAs) are widely used for investigating protein interactions. However, the fragments used are structurally compromised and have not been optimized nor thoroughly characterized for accurately assessing these interactions. We took advantage of the small size and bright luminescence of NanoLuc to engineer a new complementation reporter (NanoBiT). By design, the NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly associate so that their assembly into a luminescent complex is dictated by the interaction characteristics of the target proteins onto which they are appended. To ascertain their general suitability for measuring interaction affinities and kinetics, we determined that their intrinsic affinity (KD = 190 μM) and association constants (kon = 500 M–1 s–1, koff = 0.2 s–1) are outside of the ranges typical for protein interactions. The accuracy of NanoBiT was verified under defined biochemical conditions using the previously characterized interaction...

http://pubs.acs.org/doi/pdfplus/10.1021/acschembio.5b00753

NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells

3D tumor spheroids: an overview on the tools and techniques used for their analysis.

Although all of these attempts were successfulin principle, there were significant limitations associated withthe use of high-intensity UV or visible light in the photo-activation process. Excessive exposure to UV light can causephotoreactions in nucleic acids and result in cellular damage.Furthermore, short-wavelength UV or visible light does notpenetrate into tissue very far, which limits its utility for deep-tissue imaging by photoactivation of the caged compounds.Alternatively, multiphoton photolysis with long-wavelengthexcitation has been used to enable deep-tissue imaging and totarget gene expression

/pdf/in-vitro-and-in-vivo-uncaging-and-bioluminescence-imaging-by-qyyyge7f6g.pdf

In Vitro and In Vivo Uncaging and Bioluminescence Imaging by Using Photocaged Upconversion Nanoparticles

In the development of high throughput screening (HTS) as a central paradigm of drug discovery, fluorescence has generally been adopted as the favored methodology. Nevertheless, luminescence has maintained a prominent position among certain assay formats, most notably genetic reporters. Recently, there has been growing partiality for luminescent assays across a wider range of applications due to their sensitivity, broad linearity, and robustness to library compounds and complex biological samples. This trend has been fostered by the development of several new assay designs for diverse targets such as kinases, cytochrome p450s, proteases, apoptosis, and cytotoxicity. This review addresses recent progress made in the use of bioluminescent assays for HTS, highlighting new detection capabilities brought about by engineering luciferase genes, enzymes, and substrates. In genetic reporter applications, modifications to the luciferase genes have improved assay sensitivity by substantially increasing expr...

https://www.urmc.rochester.edu/MediaLibraries/URMCMedia/hts/documents/luciferase.pdf

Bioluminescent Assays for High-Throughput Screening

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ∼2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ∼150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly conf...

http://pubs.acs.org/doi/pdf/10.1021/cb3002478

Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.

Novel bioluminogenic substrates were designed for probing monoamine oxidase (MAO) activity based on a simple and effective β-elimination strategy. By modifying the amino group and the central core of luciferin derivatives, we have developed a series of substrates useful for assays of MAO A or B, or both. One of these substrates, exhibiting low Km values and high signal-to-background ratios with both isozymes, was shown to accurately measure the Ki values of known MAO inhibitors. This substrate is a key component in the development of a highly sensitive homogeneous MAO assay for high-throughput screening (HTS) of compounds in drug discovery and for monitoring MAO activity in complex biological systems. This design strategy should be applicable to fluorogenic MAO substrates and could broaden the structural requirements of substrates for other enzyme assays.

New bioluminogenic substrates for monoamine oxidase assays.

A bioluminescent assay for monoamine oxidase activity.

Bioluminescent assays couple a limiting component of a luciferase-catalyzed photon-emitting reaction to a variable parameter of interest, while holding the other components constant or non-limiting. In this way light output varies with the parameter of interest. This review describes three bioluminescent assay types that use firefly luciferase to measure properties of drugs and other xenobiotics which affect their absorption, distribution, metabolism, elimination and toxicity. First, levels of the luciferase enzyme itself are measured in gene reporter assays that place a luciferase cDNA under the control of regulatory sequences from ADMET-related genes. This approach identifies activators of nuclear receptors that regulate expression of genes encoding drug-metabolizing enzymes and drug transporters. Second, drug effects on enzyme activities are monitored with luminogenic probe substrates that are inactive derivatives of the luciferase substrate luciferin. The enzymes of interest convert the substrates to free luciferin, which is detected in a second reaction with luciferase. This approach is used with the drug-metabolizing CYP and monoamine oxidase enzymes, apoptosis-associated caspase proteases, a marker protease for non-viable cells and with glutathione-S-transferase to measure glutathione levels in cell lysates. Third, ATP concentration is monitored as a marker of cell viability or cell death and as a way of identifying substrates for the ATP-dependent drug transporter, P-glycoprotein. Luciferase activity is measured in the presence of a sample that supplies the requisite luciferase substrate, ATP, so that light output varies with ATP concentration. The bioluminescent ADMET assays are rapid and sensitive, amenable to automated high-throughput applications and offer significant advantages over alternative methods.

Bioluminescent assays for ADMET.

A method to detect the presence or amount of at least one molecule in a sample which employs a derivative of luciferin or a derivative of a fluorophore is provided.

Michael P. Valley

Papers

Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.

New bioluminogenic substrates for monoamine oxidase assays.

A bioluminescent assay for monoamine oxidase activity.

Bioluminescent assays for ADMET.

Luminogenic and fluorogenic compounds and methods to detect molecules or conditions