Development of a Real-Time Homogeneous TR-FRET Conjugation and Deconjugation Assay Platform By South Bay Bio, 5941 Optical Court, Suite 229, San Jose CA, 95138 Contact [email protected], Tel. (415) 935-3226

Introduction TR-FRET Tech

Ubiquitin, a highly conserved 76 amino acid , is an important post- Rare earth cryptates are macrocyclical structures encasing a rare earth lanthanide translational modifier responsible for regulating a wide variety of cellular functions atom. Lanthanide ions don’t exhibit suitable fluorescence properties on their own, including but not limited to protein degradation and recycling, control, and and require incorporation with organic moieties functioning as “light-harvesting” DNA damage repair. Ubiquitination, the modification of by the attachment of antennas to collect and transfer energy by intramolecular non-radiative processes. ubiquitin or polyubiquitin chains, is catalyzed by a three step process in which ubiquitin Our cryptate is composed of a macrocycle in a cage-shaped assembly composed of 3+ 3+ is activated by an ATP-dependent E1 activating , transferred to an E2 three bipyridine arms, which complex a Eu ion (Eu TBP) as shown in the structure conjugating enzyme, then finally conjugated to a target substrate by an E3 ubiquitin below. Unlike other common flavors of rare earth complexes like chelates, cryptates ligase. The process is reversed by ubiquitin deconjugating (DUBs.) Thus far, are extremely robust and stable structures that show no sensitivity to assay development within the ubiquitin field has suffered a lack of tools for probing the photobleaching. This is largely due to cryptates having no dissociation between the activity of the conjugation pathway. Herein, we report the development of a novel complexed ion and the macrocycle, contrary to chelates, which often exhibit assay platform capable of measuring ubiquitin conjugation and deconjugation in real uncoordinated bonds with solvent. These properties allow cryptates to perform in time in a homogenous, single step assay, utilizing TR-FRET technology. stringent conditions, e.g. in presence of strong chelators, of divalent cations (e.g. Mn2+, Mg2+), extremes of pH, organic solvents, and high TR-FRET (Time Resolved Fluorescence Resonance Energy Transfer) uses the extended temperatures (PCR or other thermoregulated fluorescence emission decay lifetimes typical of rare-earth lanthanides to impart a processes). short time-delay between FRET donor excitation and emission. This delay provides a means to separate “true” signal from short-lived background fluorescence and reduce interference from compound fluorescence and other assay artifacts. Using ubiquitin labeled with either Europium-Cryptate (donor) or Cyanine5 (acceptor), we demonstrate ubiquitin conjugation and deconjugation measured homogenously in real time, with assays commonly exhibiting Z’ ≥ 0.8. We show examples of auto-ubiquitination kinetics of several human recombinant E3 ligases of significant interest: , Parkin, ITCH, NEDD4, and XIAP. Additionally, we are able to show the reversal of this process by deubiquitinating ubiquitinated MDM2 with the DUB USP2, and compare USP2’s kinetics with this substrate versus Ubiquitin-Rhodamine 110. Activity of All Types of E3 Ubiquitin Ligases can be Measured in a Real-Time Homogeneous TR-FRET Assay RING HECT RBR

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E E E 3 .1 2 5 n M 0 .0 0 .0 0 .0 0 .0 0 .0 1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 1 2 0 1 3 5 1 5 0 1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 1 2 0 1 3 5 1 5 0 1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 1 2 0 1 3 5 1 5 0 1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 1 2 0 1 3 5 1 5 0 1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 1 2 0 1 3 5 1 5 0 tim e (m in ) tim e (m in ) tim e (m in ) tim e (m in ) tim e (m in ) TR-FRET MDM2 Autoubiquitination: Serial dilutions TR-FRET XIAP Autoubiquitination: Serial dilutions of TR-FRET ITCH Autoubiquitination: Serial dilutions of TR-FRET NEDD4 Autoubiquitination: Serial dilutions TR-FRET ParkinW403A Autoubiquitination: Serial of MDM2 from 200nM to 12.5nM mixed with UBA1, XIAP from 350nM to 43.75nM mixed with UBA1, ITCH from 130nM to 8.125nM mixed with UBA1, of NEDD4 from 50nM to 3.125nM mixed with UBA1, dilutions of ParkinW403A from 50nM to 6.25nM mixed UBE2D3, trf-Ub mix. Reaction was initiated with UBE2D2, trf-Ub mix. Reaction was initiated with UBE2L3, trf-Ub mix. Reaction was initiated with UBE2L3, trf-Ub mix. Reaction was initiated with with UBA1, UBE2D3, trf-Ub mix. Reaction was addition of Mg-ATP, excited at 304 nm, and emission addition of Mg-ATP, excited at 304 nm, and emission addition of Mg-ATP, excited at 304 nm, and emission addition of Mg-ATP, excited at 304 nm, and emission initiated with addition of Mg-ATP, excited at 304 nm, detected at 620 nm, and 665 nm. detected at 620 nm, and 665 nm. detected at 620 nm, and 665 nm. detected at 620 nm, and 665 nm. and emission detected at 620 nm, and 665 nm. 300nM wt-Parkin shown for comparison. E3 Ligase Autoubiquitination Measured in an Endpoint TR-FRET Assay Deconjugation Deconjugating enzymes (DUBs) catalyze the removal of ubiquitin from substrate

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TR-FRET MDM2-Ubn Digests with USP2CD & Measures Ki Values Summary References

U b -A ld e h y d e T itra tio n & 2 5 0 n M U S P 2 U b iq u itin a te d M D M 2 v s . U S P 2 C D T itra tio n C D In this report, we show the capabilities of a novel Amerik, A. Y., & Hochstrasser, M. (2004). Mechanism and

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z IC = 0 .1 7 5  M simple assays tailored for the ubiquitin

i 5 0 Kimura, M., ... & Endo, T. (2014). Ubiquitin is

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a proteasome system. Unlike existing technologies, phosphorylated by PINK1 to activate parkin. Nature,

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o our platform does not rely on secondary 510(7503), 162. -1 0 0 N 0 .0 detection methods such as antibodies. We 0 2 0 4 0 6 0 8 0 1 0 0 0 .0 0 1 0 .0 1 0 .1 1 1 0 demonstrate this platform’s robustness with Magennis, S. W., Parsons, S., Pikramenou, Z., Corval, A., U S P 2 C D (n M ) U b iq u itin -A ld e h y d e ( M ) assays commonly exhibiting Z’ ≥0.8, and show its & Woollins, J. D. (1999). Imidodiphosphinate ligands as antenna units in luminescent lanthanide TR-FRET MDM2-Ubn Chains Titrated with USP2CD: 50nM TR-FRET MDM2-Ubn Chains & USP2CD Ub-Aldehyde Dose- adaptability to miniaturized formats, making it MDM2 was conjugated with trf-Ub chains using UBA1, Response: 50nM MDM2 pre-conjugated with trf-Ub chains ideal for high-throughput screening experiments. complexes. Chemical communications, (1), 61-62. was digested with 250nM USP2 along with serial dilutions UBE2D3, and Mg-ATP for 3 hours. TR-FRET MDM2-Ubn CD We further provide a proof-of-concept for the chains were then digested using titrating amounts of USP2 , of Ub-Aldehyde. Initial rates of each inhibitor concentration Zheng, N., & Shabek, N. (2017). Ubiquitin Ligases: CD system’s utility and simplicity in making K from 93nM to 11nM. Initial velocities (delta (sec-1)) are were calculated and normalized relative to the no-inhibition i Structure, Function, and Regulation. Annual Review of determinations, and demonstrate that the shown plotted as a function of USP2CD concentration. control. Normalized activity was plotted against the log of Biochemistry, (0). Increasing the DUB concentration increases the reaction rate inhibitor concentration, and fit with a 4th parameter agonist platform holds its weight against a common DUB substrate, Ubiquitin Rhodamine 110. linearly. curve yielding an IC50 of 152nM. A similar dose-response using 500nM Ubiquitin-Rhodamine 110 substituted for trf-

MDM2-Ubn yields similar results.