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. 2012 Feb;17(2):163-76.
doi: 10.1177/1087057111423417. Epub 2011 Oct 27.

TR-FRET-based high-throughput screening assay for identification of UBC13 inhibitors

Charitha Madiraju  1 , Kate Welsh, Michael P Cuddy, Paulo H Godoi, Ian Pass, Tram Ngo, Stefan Vasile, Eduard A Sergienko, Paul Diaz, Shu-Ichi Matsuzawa, John C Reed
Affiliations

TR-FRET-based high-throughput screening assay for identification of UBC13 inhibitors

Charitha Madiraju et al. J Biomol Screen. 2012 Feb.

Abstract

UBC13 is a noncanonical ubiquitin conjugating enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of lysine 63-linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair and thus a potential radiosensitizer and chemosensitizer target for oncology. The authors developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)-conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)-conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. The authors defined conditions for optimized performance of the TR-FRET assay in both 384- and 1536-well formats. Chemical library screens (total 456 865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z' scores (typically >0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13.

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Figures

Figure 1
Figure 1. Schematic representation of TR-FRET-based assay for UBC13-UEV1A-mediated ubiquitination
Diagram shows use of terbium-ubiquitin and fluorescein-ubiquitin to generate a FRET reaction. In the presence of Mg2+ and ATP, labeled ubiquitin attaches to ubiquitin activating enzyme (E1) followed by transfer to ubiquitin conjugating enzyme complex (E2, UBC13-UEV1A). This event triggers ubiquitin chain build up, which is monitored by TR-FRET that occurs when terbium-ubiquitin and fluorescein-ubiquitin are in close proximity to each other. Terbium is excited at ~360 nm light emitting at a wavelength (~480 nm) suitable for excitation of fluorescein, which in turn emits at ~520 nm. The TR-FRET signal is measured as an emission ratio (520 nm: 480 nm).
Figure 2
Figure 2. Demonstration of TR-FRET reaction for UBC13-UEV1A reactions
(A) Ubiquitination reactions were performed to monitor ubiquitin chain assembly by TR-FRET methodology. Complete reaction mixture consisting of Fl-Ub (300 nM), Tb-Ub (12.5 nM), E1 (12.5 nM), UBC13-UEV1A heterodimer complex (500 nM), and ATP regenerating system (1X) was compared with reactions lacking some components. In (A), the emission ratio (520 nm: 480 nm) was determined at 1 hr. Data are expressed as mean ± SEM (n=3). Abscissa (x-axis): Reaction components; Ordinate (y-axis): TR-FRET signal expressed as emission ratio (Fl-520 nm/Tb-480 nm). Similar data were obtained at 3 and 5 hrs (not shown). (B) SDS-PAGE analysis of reactions was performed. Reaction aliquots were resolved by 15% SDS-PAGE and imaged. Molecular weight (mw) markers are indicated in kiloDaltons. Polyubiquitin conjugates are indicated by bracket and mono-Fl-ubiquitin by arrowhead.
Figure 3
Figure 3. Assay optimization
(A) Determination of optimal Fl-Ub: Tb-Ub ratio. The acceptor fluorophore, FI-Ub, was titrated against donor Tb-Ub to determine ratio for optimal signal:noise (S/N) results in the UBC13-UEV1A-mediated TR-FRET reaction. Complete ubiquitination reaction mixtures (- • -) consisting of E1 (12.5 nM), UBC13-UEV1A complex (500 nM), Tb-Ub (10 nM), and Mg2+/ATP were incubated with increasing molar ratio of FI-Ub: Tb-Ub, ranging from 0–40. Ubiquitination reaction mixtures lacking UBC13-UEV1A complex (- ◆ -) were similarly incubated with increasing molar concentrations of FI-Ub. Graphical analysis of TR-FRET measurements shows S/N > 6.0 with 15: 1 molar ratio of [FI-Ub]: [Tb-Ub]. TR-FRET data were plotted as emission ratio (y-axis) versus molar ratio of [Fl-Ub]: [Tb-Ub] (x-axis). Data are represented as mean ± SEM (n=2) and represent 1 hr measurements. Note that because the total amount of ubiquitin also changed, the optimal Fl-Ub:Tb-Ub ratio may be different as total ubiquitin varies. (B)Effects of temperature and time on TR-FRET-based ubiquitination assay using UBC13-UEV1A. Time-dependent ubiquitination reactions were performed at RT. Reaction components are indicated. Data are represented as mean ± SEM (n=3). Note that signal:noise ratio remains acceptable for ~8 hr at RT but not at 37 °C.
Figure 4
Figure 4. Cofactor dependence of UBC13-catalyzed poly-ubiquitination
TR-FRET reactions were performed without or with various amounts of cofactor (A) UEV1A or (B) MMS2. Molar ratios of co-factors relative to UBC13 are indicated. Basal reactions contained Fl-Ub (150 nM), Tb-Ub (10 nM), E1 (12.5 nM), UBC13 (250 nM), and ATP regenerating system (1X). TR-FRET reactions were measured at 3 hrs. Data are represented as mean ± SEM (n=3).
Figure 5
Figure 5. HTS implementation of TR-FRET based ubiquitination assay
(A) Z' factor determination of TR-FRET-based UBC13-UEV1A ubiquitination assays. Multiple replicate reactions were performed at RT, taking TR-FRET measurements after 1 hr incubation for samples containing (+) or lacking (−) UBC13 and UEV1A. The Z' score was calculated as 0.71 as described in supplemental information. (B)DMSO tolerance of TR-FRET-based ubiquitination assay. UBC13-UEV1A TR-FRET reactions were performed in the presence or absence of 0.5–2% DMSO (final). Reaction components are indicated. Data are represented as mean ± SEM (n=60). Abscissa (x-axis): percent DMSO in the reaction mixture (0–5%); Ordinate (y-axis): TR-FRET signal represented as emission ratio (Fl-520 nm/Tb-480 nm). (C) Histogram representation of LOPAC library screen. Full inhibition controls include reaction system lacking UBC13-UEV1A and are without any compound added to them. These controls are located at 98% on x-axis (% Inhibition). No inhibition controls include complete reaction system and are without any compound added to them. These controls are located at −4.0% on x-axis (% Inhibition). The majority of non-hits are located at ≈20% on x-axis (% Inhibition) reflecting the effect of “aged” DMSO. Compounds having < 50% inhibition are considered non-hits. x-axis: % Inhibition; y-axis: number of compounds. (D) 3D Scattergram representation of LOPAC library screen. formula image : Full inhibition controls include reaction system lacking UBC13-UEV1A without compound added. formula image : No inhibition controls include complete reaction system without compound added. formula image : LOPAC compounds; x-axis: Well #; y-axis: % Inhibition; z-axis: Plate ID.
Figure 6
Figure 6. Sanford-Burnham internal chemical library screen data
(A) Primary HTS assay performed in a 384-well format using internal chemical library screen [N ≈ 125,000 compounds (378 plates)]. The average Z' factor observed was 0.76. (B) Chemical `hits' results obtained from primary HTS screen represented for each individual plate ID are shown.
Figure 7
Figure 7. Comparison profile of the screening hit in 384 vs. 1536-well formats
Percent inhibition profiles obtained for one of the inhibitors derived from chemical library screen represented. Data obtained from TR-FRET-based HTS assay performed in a 384-well format (A) versus 1536-well format (B) is shown. IC50 values are shown and were determined from log [concentration]-percent inhibition curves constructed for each inhibitor. Data are expressed as mean±SEM (N=3).
Figure 8
Figure 8. NIH chemical library HTS screen data
(A) Shown are Z' values per plate from the primary HTS assay performed in a 1536-well format using the NIH small molecule library [N ≈ 330,000 compounds]. The x-axis represents the assay plate number vs. the y-axis showing the Z'. The average Z' factor observed was 0.78. (B) The primary HTS screen is represented in Histogram format. The x-axis represents % activity vs. the y-axis, which corresponds to the number of compounds. For inhibitors, a hit cutoff of >= 45 % activity (compared to the controls) was chosen, representing two standard deviations from the mean. (C) The primary HTS screen is also represented in Scatterplot format. The x-axis represents SourcePlate ID (each plate representing a subset of chemical compounds) vs the y-axis, which corresponds to the % Activity. For inhibitors, a hit cutoff of ≥ 45 % activity (compared to the controls) was chosen, representing two standard deviations from the mean.
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