What is the current value of fluorescence polarization assays in small molecule screening?

KEYWORDS:

• Fluorescence polarization
• high-throughput screening
• lead generation
• small molecule screening

1. Fluorescence polarization (FP)-based high-throughput screening (HTS) assays

In the lead generation phase of drug development, the screening and characterization of new bioactive compounds, pharmacological hits, are of crucial importance [1]. The quality of these research activities affects the number and origin of compounds that reach preclinical in-vivo studies as drug candidates. In effect, FP-based assays have an internal quality control parameter defined as the ratio of the emission intensities of the bound and free probes. Because FP-based assays are frequently utilized in the drug discovery industry, an ‘Assay guidance manual’ has been published and updated since 2004 by Eli Lilly & Company and the National Center for Advancing Translational Sciences [2]. The manual, last updated on July 8th, 2019, suggests that FP-based assays are actively used by companies in their screening campaigns.

In the last decade of the 20th century, an active shift in technique from radioactive screening assays to optical assays occurred in both biotechnology and pharmaceutical companies as well as in academic research institutions [3]. Optical assays have the advantages of no radioactive waste, safety for technicians, and cost-effectiveness. This rapid technological transfer was supported by wider access to new optical screening technologies based on surface plasmon resonance (SPR) and fluorescence phenomena. Around 1995, HTS capability was introduced to fluorescence measurements when 8 cm × 12 cm multi-well microplates and sensitive multi-mode fluorescence plate readers became widely accessible for compound testing [3]. By means of this equipment, testing methods were made amenable to automation as pipetting devices and stackers could be combined with sensitive and fast multi-mode fluorescence plate readers for HTS.

Since the first use of plate readers for HTS, FP-based detection methods have played a leading role. FP-based assays can be performed in homogeneous solutions since no physical separation of the bound and free ligands is required, resulting in fewer handling steps. Thus, FP-based assays are faster than the alternative filtration assays, which require physical separation and thus additional handling steps. Furthermore, alternative scintillation proximity assays are expensive to perform [2,p.105]. Several useful properties of the FP phenomenon support the success of FP-based assays in HTS experiments. Namely, ratiometric FP techniques are tolerant to small fluctuations in instrument variance and fluorescence intensity. FP-based assays are also rapid and precise; several instruments have measurement standard deviations of less than 1 mP at a tracer concentration of 1 nM and a reading time of less than 1 min for a 384-well plate. Often, quality factor Z′ values [4] greater than 0.7 have been achieved for FP assays, where assays are considered suitable for HTS at Z′ values of 0.5–1.0. Glickman, in a comparison between two types of FP-based assays (Polar Screen™ and IMAP®) and alternative assays used in HTS [2,p.88], concluded that the major drawback of Polar Screen and IMAP is a susceptibility to compound fluorescence interference.

FP measurements function based on depolarization of the emission intensity of a fluorophore when excited with plane-polarized light. The degree of depolarization is a function of molecular properties, specifically Brownian molecular rotation, and hence can serve as an effective sensor of molecular complex size [3–6]. The terms ‘FP’ and ‘fluorescence anisotropy (FA)’ contain the same information, and their values are interrelated. The construction of tracers is straightforward, and ligands labeled with typical fluorescent dyes (fluorescence lifetimes in the range 1–5 ns) inevitably result in a substantial increase in FP upon binding to a biopolymer (e.g., target protein, antibody, nucleic acid, aptamer) with MW > 10 kD. Therefore, FP is an effective means for measuring the affinity of a compound under evaluation via concurrent displacement of the fluorescent ligand (fluorescent tracer) from the complex with the target biopolymer for identifying small molecule modulators of the activity of various enzymes or identifying proteins (based on substrate-free enzymatic reactions or reactions with substrate ± immunodetection) [2–4].

A search of the PubChem BioAssay database for ‘fluorescence polarization’ and ‘fluorescence anisotropy’ on 23 October 2019, identified 3,140 FP- and FA-based assays. Of these, 423 were annotated according to their implementation in HTS campaigns. While 34% of these HTS assays were used for primary screening, 66% were implemented in counter or orthogonal screening to identify false positives and confirm activity against the targets. The targets, which included kinases, other enzymes, and nuclear receptors, represent more than 50% of targets interrogated by FP-based assays in drug discovery. The Transcreener® ADP2 FP, recently introduced by Bellbrook Labs, is a competitive FP assay based on ADP detection, which renders it compatible with any enzyme class that produces ADP; this includes protein, lipid, and carbohydrate kinases, ATPases, DNA helicases, carboxylases, and glutamine synthetase.

One success story of FP-based HTS is the discovery of INCA-6, an anti-inflammatory agent, from a library of 16,320 compounds. Its action mechanism was confirmed by T2-filtered NMR titration experiments [7]. Furthermore, GW0742, a reversible inhibitor of the interaction between the vitamin D receptor and steroid receptor coactivator 2, was discovered in a follow-up HTS campaign to eliminate irreversible inhibitors of the latter interaction. GW0742, which is also a highly selective agonist for peroxisome proliferator-activated receptor δ, is now in preclinical studies [4].

2. Expert opinion

FP-based assays will continue to be in the forefront of biochemical HTS technologies. Biological interactions are better described when the interacting partners are considered to be in their ‘natural’ milieu. Thus, FP has the advantage of resisting some significant in situ optical interferences [2,p.813,4]. Although the development of suitable fluorescent tracers may be complicated in special cases, their use pays off quickly because of the simplicity and reliability of FP-based binding assays. Furthermore, malfunctioning of the G-protein-coupled galanin receptor type 2, melanocortin 1 receptor, and formyl peptide receptor 2 is correlated with epilepsy, obesity, and Alzheimer’s disease, respectively. Many enzymes and G-protein-coupled receptors (GPCRs) are associated with severe diseases and therefore necessitate new and improved drugs. Thus, improved screening assays are an important component of the discovery of novel compounds. Alternatively, SPR has been used to screen 6,369 compounds as potential GPCR ligands (neurotensin receptor 1) [8].

Classical FP-based assays have at least two considerable restrictions. First, FP-based measurements require excess target protein compared with the fluorescent tracer, which limits the application of FP-based assays to the screening of GPCR ligands. GPCRs represent a major domain of pharmaceutical discovery because of the wide variety of human physiological processes that they regulate without toxic effects to normal cells. In recent years, success has been achieved in the field through overexpression GPCRs in host cell membranes on the surface of budded baculovirus particles or other vesicles together with the application of bright tracers with low nonspecific binding [9]. Second, in the classical assay format, it is impossible to characterize binders whose affinity toward the target protein is higher than that of the tracer [10]. However, in other detection formats (e.g., FI measurement with FRET-based probes), measurable K_D values may be substantially shifted by the application of a significant excess of probes, compared with the target protein, that emit a negligible signal in their free form [11].

The number of kits that are commercially available for FP-based assays is increasing [4], rendering the application of FP methods more affordable. The ease of constructing new tracers depends on the availability of high-affinity ligands that can be conjugated with a fluorescent dye such that the binding characteristics of the ligand are retained by the tracer. Brighter probes with high affinities have been used in the HTS of GPCRs, and spectral interference has been avoided by using a red-shifted dye [12], illustrating that probes still have room for improvement. For testing by biochemical assays with purified proteins, even nonselective inhibitors with known binding modes to a group of proteins have great potential because a single tracer constructed from such inhibitors can be used to screen inhibitors toward this group. To avoid false positive hits in FP-based screening, it is reasonable to use alternative photoluminescence detection methods to confirm the results or even an assay procedure based on another physical phenomenon (e.g., SPR, radioactivity).

The success of fabricating bright, high-affinity tracers with low nonspecific binding to receptors and equipment plastics, along with efficient protein expression systems, has enabled the implementation of FP-based assays for the screening of GPCR ligands (agonists and antagonists). Although testing with FP technology for the analysis of cells remains under evaluation [13], the results of current studies do not predict the applicability of these procedures for high-content screening of ligands in the near future. Besides, microfluidic multiplexing FP-based assays could gain importance for future studies on protein-ligand interactions owing to the large number of picoliter droplets that can be analyzed in a miniaturized and automatic multiplexing format.

Several new tracers for FP measurements are currently commercially available. Important changes have occurred in FP measurements upon introducing the technology to compare ligands [14] according to their residence times [15]. This opens a new path to screening and characterizing ligands according to their protein binding kinetics, an important characteristic for several types of drugs. Most importantly, however, FP-based HTS assays will continue to be a reliable, simple, quick, and cheap method for the primary screening of hit compounds and subsequent characterization of ligands at every stage of drug development.

Declaration of interest

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer Disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

The authors are supported by the Estonian Research Council via grant PRG454.