Fluorescence lifetime plate reader: Resolution and precision meet high-throughput

Petersen, Karl J.; Peterson, Kurt C.; Muretta, Joseph M.; Higgins, Sutton E.; Gillispie, Gregory D.; Thomas, David D.

doi:10.1063/1.4900727

Cited by 40 publications

(67 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our initial screen of a 400-compound library resulted in four verified hits. Although the rate of false-positive compounds identified after the initial round of screening is significantly lower than in a previously reported PfHT inhibitor assay (15), further improvements can be made by selecting a higher threshold of FRET ratio reduction, especially when screening a larger library or using different detection methods like fluorescence lifetime (25,26). The hits identified in this screen include three compounds (MMV009085, MMV020548, and MMV665879) that were previously identified and characterized by Ortiz et al (15) to selectively inhibit glucose uptake, but not proline transport, in PfHT-overexpressing Leishmania mexicana.…”

Section: Discussionmentioning

confidence: 99%

A Novel Fluorescence Resonance Energy Transfer-Based Screen in High-Throughput Format To Identify Inhibitors of Malarial and Human Glucose Transporters

Kraft

Heitmeier

Putanko

et al. 2016

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z= factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counterscreen PfHT hits against the human orthologues GLUT1, -2, -3, and -4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum. Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC 50 ) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and orthologue selectivity, thereby successfully validating our assay for antimalarial screening. Malaria is a major threat to the human population in large areas of the world, affecting over 200 million people per year (1, 2). Beyond the effects of this disease on human life, malaria also cripples economic development and burdens the health care systems of countries where malaria is endemic (3). The emergence of parasites with resistance to even the most potent existing antimalarial drugs, such as the artemisinins (4), has made paramount the development of novel drugs that target essential pathways for parasite survival. Blood stage parasites utilize glucose for both biomass production and ATP synthesis (5). The malarial hexose transporter, Plasmodium falciparum hexose transporter (PfHT), first cloned by Woodrow et al. in 1999 (6), mediates parasite transport of glucose, fructose, mannose, and galactose (7). Since PfHT is essential for parasite survival (8), this protein is a highly promising molecular target for antimalarial drug development. This is supported by the ability of compound 3361, a glucose analogue that inhibits PfHT with high selectivity over the human orthologue GLUT1, to inhibit asexual intraerythrocytic growth in culture (9). Compound 3361 is also active against Plasmodium berghei liver and transmission stage parasites in infected mice (10), suggesting that PfHT is a promising target for full life cycle activity. However, while compound 3361 validates efforts to target PfHT, this compound is not itself considered drug-like and is therefore not a valid candidate for lead development (11). We have recently extended these earlier find...

show abstract

Section: Discussionmentioning

confidence: 99%

A Novel Fluorescence Resonance Energy Transfer-Based Screen in High-Throughput Format To Identify Inhibitors of Malarial and Human Glucose Transporters

Kraft

Heitmeier

Putanko

et al. 2016

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

show abstract

“…13 This set the stage for a proof-of-principle structure-based small-molecule screen, using a prototype fluorescence lifetime (FLT) microplate reader. 2,14 This seminal study proved that a genetically-encoded FRET sensor could be stably expressed in human embryonic kidney (HEK293) cells and utilized for HTS in a microplate format.…”

Section: Introductionmentioning

confidence: 99%

High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA

et al. 2017

Self Cite

View full text Add to dashboard Cite

A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green (GFP, donor) and red (RFP, acceptor) fluorescent proteins fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA’s distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA’s ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS.

show abstract

“…Changes in FRET due to an RyR modulator reflect changes in FKBP and/or CaM binding, and/or in RyR structure, as shown in Figure 1C. We integrated this FRET-based molecular toolkit that targets RyRs with the use of an FLT plate-reader (FLT-PR) (19,20) to undertake a duplicate screen of the 727-compound NIH clinical collection (NCC). Using this technology, a high-precision (S/N>100) TR-fluorescence decay, averaged from 200 fluorescence waveforms is read in 200 ms, and a whole 384-well plate is scanned within 3 min.…”

Section: Introductionmentioning

confidence: 99%

High-Throughput Screens to Discover Small-Molecule Modulators of Ryanodine Receptor Calcium Release Channels

et al. 2017

Self Cite

View full text Add to dashboard Cite

Using time-resolved fluorescence resonance energy transfer (FRET), we have developed and validated the first high-throughput screening (HTS) method to discover compounds that modulate an intracellular Ca2+ channel, the ryanodine receptor (RyR), for therapeutic applications. Intracellular Ca2+ regulation is critical for striated muscle function, and RyR is a central player. At resting [Ca2+], increased propensity of channel opening due to RyR dysregulation is associated with severe cardiac and skeletal myopathies, diabetes and neurological disorders. This leaky state of the RyR is an attractive target for pharmacological agents to treat such pathologies. Our FRET-based HTS detects RyR binding of accessory proteins calmodulin or FKBP12.6. Under conditions that mimic a pathological state, we carried out a screen of the 727-compound NIH Clinical Collection, which yielded six compounds that reproducibly changed FRET by >3SD. Dose-response of FRET and [3H]ryanodine binding readouts reveal that five hits reproducibly alter RyR1 structure and activity. One compound increased FRET and inhibited RyR1, which was only significant at nM [Ca2+], and accentuated without CaM present. These properties characterize a compound that could mitigate RyR1 leak. An excellent z′-factor and the tight correlation between structural and functional readouts validate this first HTS method to identify RyR modulators.

show abstract

Fluorescence lifetime plate reader: Resolution and precision meet high-throughput

Cited by 40 publications

References 29 publications

A Novel Fluorescence Resonance Energy Transfer-Based Screen in High-Throughput Format To Identify Inhibitors of Malarial and Human Glucose Transporters

A Novel Fluorescence Resonance Energy Transfer-Based Screen in High-Throughput Format To Identify Inhibitors of Malarial and Human Glucose Transporters

High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA

High-Throughput Screens to Discover Small-Molecule Modulators of Ryanodine Receptor Calcium Release Channels

Contact Info

Product

Resources

About