December 1, 2015 (Vol. 35, No. 21)

Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications

Method Combines Sensitivity of Fluorescence with the Homogeneous Nature of FRET

At the 6th Cisbio HTRF symposium, “Charting the Course of Drug Discovery,” held recently in Brewster, MA, investigators described how homogeneous time-resolved fluorescence (HTRF®) continues to expand and improve upon the repertoire of available bioassay formats for basic research and drug discovery. Participants described applications of these assays as integral components in studies ranging from identification of allosteric modulators as potential drugs to determination of critical components in protein-modifying biochemical pathways as new drug targets.

A form of time-resolved fluorescence energy transfer (TR-FRET) technology, HTRF brings together the sensitivity of fluorescence with the homogeneous nature of FRET and the low background of time resolution. As in other FRET systems, HTRF uses two fluorophores—a donor and an acceptor that transfer energy when in close proximity to each other. Excitation of the donor molecule by an energy source such as a laser causes the emission of light waves at donor-specific wave lengths.

The HTRF assay can be miniaturized into 384- and 1536-well plate formats, which proponents say can contain reagent costs and reduce quantities of limited target and compound material used in the assay. This assay technology has been applied to many antibody-based assays, including assays for GPCR signaling (cAMP and IP-One); assays for kinases, cytokines, biomarkers, and bioprocess indicators of antibody and protein production; as well as assays for protein-protein, protein-peptide, and protein-DNA/RNA interactions.

Discovering and Characterizing Allosteric Modulators

Introduced to the market in 2010 for the treatment of secondary hyperparathyroidism in adult patients with chronic kidney disease on dialysis, Cinacalcet, a positive allosteric modulator (PAM), activates the calcium-sensing receptor that functions as the principal regulator of parathyroid hormone secretion. Cinacalcet is the first clinically administered allosteric modulator acting on a GPCR, and provided a proof-of-concept for future development of allosteric modulators on other GPCR drug targets.

Hayley Jones, Ph.D., and Jeff Jerman, Ph.D., both of Medical Research Council Technology (MRCT) in the U.K., talked about the characterization of novel PAMs for the dopamine 1 receptor. Dr. Jones noted that although preclinical and clinical data have validated this receptor as a target for drugs to improve cognitive impairment in schizophrenia, attempts to clinically develop agonists have failed.

She and her colleagues have approached this problem by targeting D1R via PAMs. They found that in contrast to “direct” orthosteric D1R agonists, PAMs potentially offer advantages, including physiological spatiotemporal control of dopamine function by enhancing the effect of its endogenous ligand and avoiding over stimulation by self-limiting effects.

The investigators said they had configured a cell-based HTRF assay to screen a subset of an MRCT compound library using CHO cells that transiently express the human receptor. Inclusion of a submaximal concentration of dopamine in the assays facilitated simultaneous detection of both PAMs and agonists, allowing them to identify novel D1R activators.

Michelle Arkin, Ph.D., of the University of California, San Francisco, focuses her research on developing small molecule modulators of allosterically regulated enzymes and protein complexes as potential drug leads. Neurodegenerative diseases such as Alzheimer’s and other “taopathies” are characterized by formation of intracellular tangles comprising aggregated tau proteins. Previous studies have shown that the protein actyltransferase p300 acetylates tau at several sites, competing with ubiquitination and thereby inhibiting tau degradation.

Dr. Arkin and colleagues developed a high-throughput screen using HTRF to identify p300 inhibitors, designing a suite of counter screens and secondary assays to validate hits. Based on previous findings that the protease caspase-6 clips tau at specific sites and that truncated tau forms are associated with disease progression, the investigators developed selective caspase-6 inhibitors.

HTRF assays demonstrated, she said, that small molecule compounds inhibit caspase-6-mediated cleavage of tau in cell lysates, suggesting that the combination of HTRF enzymatic and biophysical assay formats allows characterization of inhibitors of proteins that may be involved in tauopathy progression.

Researchers are working on novel adaptations of HTRF-based assays, as well as their combination with other types of assays, to characterize complex disease pathways that may present multiple drug targets for disease therapy. [iStock/ponsulak]

Lack of Suitable Assays

At Pfizer, Richard Frisbee, a scientist in the hit discovery and lead profiling (HDLP) department, and colleagues have focused on the development of HTS whole-blood assays using HTRF, particularly to monitor anti-inflammatory drug potency. They noted that traditional whole-blood formats such as ELISAs for detecting cytokines require multiple assay plate manipulations, including wash steps and incubation steps, have limited throughput, and are relatively time consuming.

They reported that they had developed a sandwich immunoassay protocol that measures cytokine production in human whole blood in a 384-well format. They described key elements of the assay, including nanoliter spotting of test compounds, miniaturized blood/reagent transfer, and optimized assay incubations. Development of a relatively convenient assay to monitor compound potency in whole blood can facilitate, they said, the prediction of compound doses required for therapeutic efficacy.

Inhibiting the enzyme gamma-secretase, which converts amyloid precursor protein to β-amyloid , thus preventing its accumulation in the brain, has been a goal of drug developers.

Most recently, Bristol-Myers Squibb (BMS) elected to discontinue development of its inhibitor candidate avagacestat into Phase III trials after disappointing Phase II results. BMS remains in the hunt for drugs to treat Alzheimer’s disease. Despite clinical failures of its and other companies’ other gamma-secretase inhibitors, researchers continue to search for next-generation compounds they believe may succeed.

At BMS, Dave Harden, Ph.D., principal scientist and team leader, biochemical screening in the leads discovery and optimization group, has developed novel assays to identify molecules that inhibit secretase by measuring multiple amyloid beta species in cell supernatant. He and his team have capitalized on terbium cryptate’s properties as a donor fluorophore in HTRF. This compound has different photophysical properties compared to the donor fluor europium.

Terbium cryptate’s properties afford the opportunity to measure more than one interaction within a well due to the multiple emission spectra observed upon excitation. Terbium cryptate can therefore serve as a donor fluorophore to green-emitting fluors because it has multiple emission peaks including one at 490 nm as well as the typically used 665 nm (red) emission.

Dr. Harden and colleagues, in order to “enhance” their screening practices by expanding well information content, enabled two-color multiplexed HTRF in multiple settings in large (>1 MM well) screening campaigns. This approach, they reported, successfully identified mechanistically distinct gamma secretase inhibitors by measuring multiple amyloid beta peptide species in cell supernatants. This, and several other examples, the presenters said, demonstrated the power of multiplexed HTRF in maximizing screening outcomes.

Early Detection of HIV

For researchers measuring difficult-to-detect biomarkers, Meso Scale Discovery (MSD), a division of Meso Scale Diagnostics, has developed S-Plex™ sample testing services to provide specific fg/mL detection for a growing list of analytes. The use of an S-Plex immunoassay for the early detection of HIV infection was described in two papers presented at this year’s annual meeting of the American Association of Clinical Chemistry. (The papers, both attributed to Stengelin et al., were entitled “Multi-Array Assay to Discriminate Recent from Long-Standing HIV Infection,” and “HIV p24 Immunoassay with the Sensitivity of PCR Methods.”)

According to the presentations, patients recently infected with HIV contribute disproportionately to the spread of the disease. Therefore, early detection of acute HIV infection is of great importance for public health. Current PCR tests employed for early detection of infection are complex and expensive, and they are unsuitable for all settings.

The detection limit for MSD’s S-Plex HIV p24 immunoassay is approximately 1 fg/mL—10,000× more sensitive than current p24 immunoassays, according to the company. This sensitivity corresponds to less than one virus particle per 25 µL of sample volume.

In the two seroconversion panels tested, the MSD p24 assay result was negative for all PCR-negative samples and positive for all PCR-positive samples, and infection was detected well before conventional p24 immunoassays. Therefore, the S-PLEX p24 immunoassay is comparable in sensitivity to PCR assays and opens up the potential of using next-generation ultrasensitive immunoassays for the detection of biomarkers, explained a company spokesperson.

MSD’s Multi-Array® technology reportedly utilizes electrochemiluminescence detection that delivers high sensitivity, reproducibility, and a wide dynamic range. MSD offers a range of immunoassay platforms for measuring biomarkers. The company’s U-Plex® platform allows for development of multiplex assays, and its V-Plex® products are validated according to the FDA’s analytical validation guidelines.

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