Method for detecting ADP-ribosyl groups or their attachment to polymers and kits for carrying out said method

Abstract

The present invention is directed to methods, kits, systems and fusion proteins for detecting binding to an ADP-ribosyl group or polymer thereof, said group or polymer being conjugated to a peptide or protein, said methods comprising the steps of: i) providing a first entity comprising a first label or tag, said entity comprising an amino acid sequence comprising a cysteine ​​residue to which at least one ADP-ribosyl group or analogue thereof is conjugated via an S-glycosidic bond; ii) contacting said first entity with a second entity in an assay, said second entity being capable of binding or believed to be capable of binding to an ADP-ribosyl group or polymer thereof conjugated to a peptide or protein; and iii) measuring a signal derived from said first label or localized by said tag, wherein a signal detected when said second entity is bound to said at least one ADP-ribosyl group of said first entity is different or differently localized than a signal detected when no binding interaction between said second entity and said ADP-ribosyl group occurs. The kits of the invention provide means for carrying out the methods of the invention.

Description

[Technical field]

[0001] The present invention relates to an assay technique for detecting interactions between biomolecules. In particular, the present invention relates to the identification of proteins that interact with ADP-ribosylation in cancer-related pathways and viral infections. The objective of the present invention is to provide a robust assay technique suitable for high-throughput screening of a wide range of hydrolyzable and non-hydrolyzable ADP-ribose binders. [Background technology]

[0002] ADP-ribosylation is a post-translational modification involved in the regulation of many diverse processes in cells. Despite its physiological importance, the complex interplay of ADP-ribose transfer, detection and removal is poorly understood at the molecular level.

[0003] The human genome encodes many different conjugates, ADP-ribosyl-modifying proteins ("readers"), as well as proteins capable of hydrolyzing and removing the ADP-ribosyl group ("erasers") (Teloni & Altmayer, 2016). Macrodomains are one of the largest classes of ADP-ribose conjugates known in humans. Many of them are encoded as part of other multidomain proteins, such as ADP-ribosyltransferases (PARP9, PARP14, PARP15) or histones (macroH2A variants). Other macrodomains, such as MDO1, MDO2, PARG or TARG1, have hydrolytic activity and are essential actors in the ADP-ribose signaling pathway.

[0004] There are several other domains that are primarily involved in poly ADP-ribose binding: examples of these are the PAR-binding zinc finger (PBZ) domain of APLF, the WWE domains of several ADP-ribosyltransferases and E3 ubiquitin ligases, and the PAR-binding motif (PBM) of XRCC1 (Teloni & Altmayer, 2016).

[0005] Pathogenic viral macrodomains are another class of ADP-ribose binders. Viruses such as coronaviruses or togaviruses are known to possess macrodomains capable of removing ADP-ribose from proteins in the host cell. These viral macrodomains have been suggested to weaken the host's viral defense mechanisms by interfering with the host's ADP-ribosylation signaling machinery and have been shown to be necessary for viral replication and pathogenesis. Viruses with these macrodomains include Chikungunya virus, MERS-CoV (camel influenza) and SARS-CoV-2 (COVID-19).

[0006] Although the development of inhibitors against ADP-ribosyltransferases has been studied for decades, mainly in the context of cancer therapeutics, the development of inhibitors against ADP-ribose conjugates or hydrolysates has only gained momentum in recent years. Inhibitors of ADP-ribose binding proteins will be valuable tools to help decipher the complex ADP-ribose signaling mechanisms in cells. These inhibitors may also show therapeutic potential. Furthermore, inhibition of viral macrodomains to combat diseases caused by these viruses is currently being investigated.

[0007] Previous assays measuring hydrolysis of ADP-ribosylated substrate proteins are not directly applicable to non-hydrolyzable conjugates of ADP-ribose (Wazir et al., 2021). There remains a need for improvement in this field, as known assays for ADP-ribose binding are complex, expensive, or not amenable to high-throughput processes. Summary of the Invention [Problem to be solved by the invention]

[0008] The aim of the present invention was to design a system that would allow easy development of assays suitable for the detection of a wide variety of ADP-ribosyl conjugates and -hydrolases. The inventors reasoned that by using a non-hydrolyzable ADP-ribose probe, it would be possible to measure the binding of both hydrolyzable and non-hydrolyzable ADP-ribose conjugates.

[0009] In proteins, many different residues can act as acceptors for ADP-ribose. Residues such as serine, aspartic acid or glutamic acid form O-glycosidic bonds, while others such as lysine, arginine or asparagine form N-glycosidic bonds with ADP-ribose. In addition, a less common modification is the chemically stable S-glycosidic bond that can be formed with cysteine ​​residues. Many different ADP-ribosylhydrolases exist and can remove ADP-ribose from O- or N-glycosidic bonds, but to date no enzymes capable of restoring the S-glycosidic bond have been reported in humans. [Means for solving the problem]

[0010] Pertussis toxin from the bacterium Bordetella pertussis is known to efficiently catalyze the transfer of ADP-ribose units to specific C-terminal cysteine ​​residues of the αi subunit of heterotrimeric G proteins (Gαi). As an example of how the present invention works, we reduced Gαi to an 8-mer peptide and recombinantly fused it to the C-terminus of other proteins, again observing efficient modification, allowing the site-specific addition of ADP-ribose units to any protein with an accessible C-terminus. This was exploited to generate MAR-ylated YFP proteins with stable S-glycosidic bonds that can bind to ADP-ribose leaders or erasers. We further extended this system by finding conditions for extending MARs to PARs, allowing the binding of MAR and PAR conjugates to be probed as well.

[0011] Accordingly, the present invention relates to a method for detecting the binding of an ADP-ribosyl group or a polymer thereof, said group or polymer being conjugated to a peptide or protein and comprising the steps of: i) providing a first entity comprising a first label or tag, said entity comprising an amino acid sequence comprising a cysteine ​​residue to which at least one ADP-ribosyl group or analogue thereof is conjugated via an S-glycosidic bond; ii) contacting said first entity with a second entity in an assay, said second entity being capable or believed to be capable of binding to an ADP-ribosyl group or polymer thereof conjugated to a peptide or protein; and iii) measuring a signal derived from or localized by said first label, wherein a signal detected when said second entity is bound to at least one ADP-ribosyl group of said first entity is different or differently localized than a signal detected when no binding interaction between said second entity and said ADP-ribosyl group occurs.

[0012] In another related aspect, the invention provides a kit for detecting the binding of an ADP-ribosyl group or a polymer thereof, said group or polymer being conjugated to a peptide or protein, - in a first container: a first entity comprising a first label or tag, said entity comprising an amino acid sequence comprising a cysteine ​​residue to which at least one ADP-ribosyl group is conjugated via an S-glycosidic bond; - in a second container: a second entity conjugated to a second label or tag, the second entity being capable of binding to an ADP-ribosyl group or polymer thereof conjugated to the peptide or protein.

[0013] In another related aspect, the present invention provides a fusion protein comprising a first domain and a second domain, wherein said second domain comprises an amino acid sequence corresponding to the C-terminal sequence of a Gα subunit of a G protein, preferably SEQ ID NO:4, or having at least 75% sequence identity with the C-terminal sequence of a Gα subunit of a G protein, wherein said amino acid sequence comprises a cysteine ​​residue to which at least one ADP-ribosyl group or an analog thereof is conjugated via an S-glycosidic bond.

[0014] In further related aspects, the present invention provides i) a fusion protein comprising a first domain and a second domain, the second domain comprising an amino acid sequence comprising a cysteine ​​residue to which at least one ADP-ribosyl group or an analogue thereof is conjugated via an S-glycosidic bond; ii) a biopolymer, preferably selected from the group consisting of a macrodomain, an ARH family protein, a BRCT domain, a PAR-binding zinc motif, and a WWE domain, which specifically recognizes said at least one ADP-ribosyl group or an analog thereof of said fusion protein; In the system, the biopolymer binds to the at least one ADP-ribosyl group or analogue thereof of the fusion protein such that the biomolecule and the fusion protein are linked together to form a coupled entity. [Brief description of the drawings]

[0015] [Figure 1]Figures 1a-d are diagrams of a molecular toolbox for in vitro interaction studies and assay development of ADP-ribosyl binding proteins. Figure 1a shows that site-specific ADP-ribosylation of a C-terminal Gαi-based 10-mer peptide (GAP tag) by pertussis toxin subunit S1 (PtxS1) can generate a single S-glycosidically linked mono-ADP-ribosyl (MAR) group. Figure 1b shows that the MAR group of the GAP tag is extended to a poly-ADP-ribosyl (PAR) group by PARP2. This system can be used to measure the binding of proteins interacting with mono- or poly-ADP-ribosyl groups by FRET or other conjugation techniques. Figure 1c shows that the GAP tag can be used for site-specific labeling with NAD+ analogues. Figure 1d shows that the high-affinity ADP-ribosyl conjugate fused to nanoluciferase (Nluc) can be used as a luminescent probe to rapidly, sensitively and selectively detect mono- and poly-ADP-ribosylated proteins using a blot-based method. [Diagram 2]Figure 2a-d are diagrams of the initial development of the toolkit components. Figure 2a is a diagram of the testing of ADP-ribosylation by PtxS1 with different Gαi constructs. We tested full-length Gαi constructs, unlabeled or fused to YFP, and GAP-tagged YFP as cysteine-ADPr acceptors when treated with 50 nM (+) or 250 nM (++) PtxS1. As controls, we used buffer or YFP-GAP, in which the cysteine ​​of the acceptor was mutated to alanine. Reactions were blotted onto nitrocellulose membranes and detected with Nluc-eAf1521. Figure 2b shows that the MAR group of the GAP tag can be extended to PAR by PARP2. 10 μM YFP-GAP or YFP-GAP(MAR) were mixed with 1 mM NAD+ and 400 nM (+) or 4 μM (++) PARP2 or buffer as a control. Reactions were visualized by SDS-PAGE with Coomassie Blue or by Western blotting with detection with Nluc-eAf1521 or Nluc-ALC1. Figure 2c shows the detection of MAR and PAR by Nluc-eAf1521. Figure 2d shows the selective detection of PAR by Nluc-ALC1. A dilution series of YFP-GAP (±MAR) or TNKS1 (±PAR) was blotted onto nitrocellulose membrane. [Diagram 3]Figures 3a-b show that the Gαi-tag can be used to introduce site-specific modifications with NAD+ analogues. Figure 3a shows site-specific biotinylation of the GAP tag. GAP-tagged YFP was mixed with NAD+ or 6-biotin-17-NAD+ in the absence or presence of PtxS1. Reactions were blotted onto nitrocellulose membranes and biotin was detected using streptavidin-HRP. Figure 3b shows that YFP-GAP was MARed by PtxS1 with NAD+ or 6-propargyl adenine-NAD+ containing an alkyne group. The resulting proteins YFP-GAP(MAR) or YFP-GAP(MAR-alkyne) or buffer were mixed with Cy3-azide or Cy5-azide, and copper(I)-catalyzed alkyne-azide cycloaddition reaction was carried out by adding 5 mM sodium ascorbate, 300 μM CuSO4, and 600 μM L-histidine. The samples were incubated at room temperature for 3 h, blotted onto nitrocellulose membranes, and color images were taken. Unreacted Cy3-azide or Cy5-azide was removed by washing the membrane with TBS-T, and color and fluorescent images were taken. [Figure 4]Figure 4a-e shows the test of interaction of YFP-GAP with reported and potential leaders and erasers. Figure 4a shows the interaction of MAR-ylated YFP-GAP with potential and confirmed ADP-ribosyl conjugates fused to CFP. 1 μM of CFP fusion protein was mixed with 5 μM of YFP-GAP or 5 μM of YFP-GAP(MAR) in the absence or presence of 200 μM of ADP-ribose. Ratiometric FRET signals were measured. Figure 4b shows the test of ADP-ribosyl removal from the GAP tag. 10 μM of YFP-GAP(MAR) was prepared in the absence (-) or presence (+) of 1 μM of CFP fusion protein or 0.01 μM to 1 μM of snake venom phosphodiesterase I (SVP). Samples were incubated at room temperature for 24 h and blotted onto nitrocellulose membranes. Detection was performed with Nluc-eAf1521. Figure 4c shows the interaction of poly ADP-ribosyl conjugates with PARylated YFP-GAP. 250 nM of CFP fusion protein was mixed with 500 nM YFP or 500 nM YFP-GAP(PAR) in the absence or presence of 100 μM ADP-ribose or 2.5 μM automodified PARP2. Ratiometric FRET signals were measured. Figure 4d shows a representative dose-response curve for the competition of 1 μM CFP-SARS-CoV nsp3 with 5 μM YFP-GAP(MAR) ADP-ribose. Figure 4e shows a representative dose-response curve for the competition of 250 nM CFP-ALC1 with 500 nM YFP-GAP(PAR) with PARylated PARP2. [Diagram 5]Figures 5a-d show that various assay techniques are readily available to detect ADPr binding. Figure 5a shows the measurement of interaction by FRET. Ratiometric FRET signal of CFP-MDO2 and YFP-Gαi (MAR) shown in Figure 4a in the absence (control) or presence of 200 μM ADP-ribose. Figure 5b shows the measurement of interaction by BRET. Ratiometric BRET signal of Nluc-MDO2 and YFP-Gαi (MAR) in the absence (control) or presence of 200 μM ADP-ribose. Figure 5c shows the measurement of interaction by AlphaScreen. Biotinylated MDO2 and His-tagged MAR-Gαi were mixed with streptavidin donor beads and chelating acceptor beads in the absence (control) or presence of ADP-ribose. Luminescence signal was detected by excitation of the donor beads. Figure 5d shows the measurement of interaction by Biolayer Interferometry. His-tagged YFP-GAP(MAR) was bound to the optical sensor surface, and the signal change was measured following association (0 s) or dissociation (120 s) of unlabeled MDO2 protein in the absence or presence of 3.16 μM ADP-ribose. [Figure 6]6a-f are diagrams of the development of a screening assay based on YFP-GAP(MAR) probes using CFP-tagged SARS-CoV-2 nsp3 macrodomain. Fig. 6a is a diagram of signal validation of the screening assay using CFP-SARS-CoV-2. 1 μM SARS-CoV-2 was mixed with 5 μM YFP-GAP(MAR) in the absence or presence of 200 μM ADP-ribose, and a Z'-factor of 0.7 was calculated. Fig. 6b is a diagram of the screening of the ENZO FDA approved drug library. One compound showed more than 30% inhibition and was moved to further validation. Fig. 6c is a diagram of the structure of the hit compound suramin. Fig. 6d is a diagram of the dose-response curve with suramin showing an IC50 of 8.7 μM against the FRET-based SARS-CoV-2 nsp3 macrodomain. Fig. 5e is a diagram of suramin showing stabilization of the SARS-CoV-2 nsp3 macrodomain by DSF. Figure 5f shows the inhibition profile of suramin against the human and viral ADP-ribosyl conjugates used in this study. The inhibition percentage was calculated based on the ratiometric FRET signal of CFP fusion conjugates mixed with YFP-GAP(MAR) or YFP-GAP(PAR). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] "ADP-ribosylation," which includes both "MARylation" and "PARylation," is catalyzed by enzymes such as ADP-ribosyltransferases, including poly(ADP-ribose) polymerase (PARP), arginine-specific ectoenzymes such as ARTC1-6, and many bacterial toxins. Examples of ADP-ribosyltransferases include PARP in humans and the bacterial toxin DarT in bacteria.

[0017] "MARylation" refers to the transfer of a single mono(ADP-ribose) (MAR) group onto a protein or nucleic acid by ADP-ribosylation.

[0018] "PARylation" refers to the transfer of multiple ADP-ribose (ADPr) groups onto a protein or nucleic acid by ADP-ribosylation.

[0019] The term "PARP family enzymes" or "PARP" refers to poly(ADP-ribose) polymerases (PARPs), a family of related enzymes that share the ability to catalyze the transfer of ADP-ribose to target proteins. PARPs play important roles in a variety of cellular processes, including regulation of chromatin structure, transcription, replication, recombination, and DNA repair.

[0020] As used herein, the term "Gα subunit" or "Gαi" refers to one of the three subunits of G proteins, which are membrane-bound heterotrimeric G proteins (i.e., alpha (α), beta (β), and gamma (γ) subunits). G proteins, also known as guanine nucleotide-binding proteins, are a family of proteins that act as molecular switches within cells and are involved in the transmission of signals from various extracellular stimuli to the interior of the cell. An exemplary sequence of a human Gα subunit is shown in SEQ ID NO:4.

[0021] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers as well as to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acids. Polypeptides can be modified, for example, by the addition of carbohydrate residues to form glycoproteins. The terms "polypeptide", "peptide" and "protein" include glycoproteins and non-glycoproteins.

[0022] The term "signal" as used herein refers to any physical or chemical effect. A signal may be, for example, a fluorescent, luminescent, colorimetric or electroluminescent signal, although this list is not limiting.

[0023] The term "luminescent protein" or "luminescent label" refers to an entity or domain that has the property of releasing part of the absorbed energy upon excitation in the form of photons with energy of non-thermal origin, thus involving the deactivation of the excited molecule to a lower energy state. In other words, a luminescent molecule is a molecule that can act on a suitable substance to generate luminescence. Advantageously, a luminescent protein or label has the property of emitting blue, yellow or green light. The luminescent protein can be selected from among those known to the person skilled in the art.

[0024] A "fluorescent label" refers to a molecule that has the property of absorbing light energy (excitation light) and then rapidly recovering it in the form of fluorescent light by very quickly releasing a photon (emission light). Upon absorbing the photon of energy, the molecule generally becomes electronically energized. In other words, it may be a fluorophore or a fluorescent dye.

[0025] The term "FRET" refers to the process of fluorescence resonance energy transfer that occurs between two chromophores. As used herein, chromophores include, for example, fluorescent, luminescent, and other non-fluorescent moieties.

[0026] The term "BRET" refers to resonance energy transfer (RET) between a bioluminescent donor moiety (ie, a BRET energy donor) and a fluorescent acceptor moiety (ie, a BRET energy acceptor).

[0027] The term "tag" as used herein is meant to be understood in its broadest sense and includes, but is not limited to, any suitable enzymatic, fluorescent, or radioactive label and suitable epitope, including, but not limited to, biotin tag, HA tag, Myc tag, T7, His tag, FLAG tag, calmodulin-binding protein, glutathione-S-transferase, strep tag, KT3 epitope, EEF epitope, green fluorescent protein and variants thereof. A "tag" can also be any means of binding and / or immobilizing a protein, such as the MARylated or PARylated protein of the present invention, to a surface.

[0028] The term "domain" may be construed herein to encompass functional amino acid sequences in a polypeptide, e.g., sequences for binding or target sites for post-translational modification, that retain their function when incorporated into a fusion protein. The present invention provides a system for in vitro studies that allows simple and efficient setup of binding assays for ADP-ribosyl leaders and erasers based on site-specific cysteine ​​ADP-ribosylation. We have extended this system to incorporate peptide-tagged C-terminal proteins into chemically modified NADs. + We demonstrated that the ELISA kit can be modified with analogs. This method paves the way for the development of various in-vitro assay systems (Figure 1). To demonstrate its potential for screening, we set up a binding assay against the macrodomain of SARS-CoV-2 nonstructural protein 3 and identified the FDA-approved drug suramin as a moderate inhibitor.

[0029] Specifically, the present invention is directed to a method for detecting the attachment of an ADP-ribosyl group or a polymer thereof, said group or polymer being conjugated to a peptide or protein, said method comprising the steps of: i) providing a first entity comprising a first label or tag, said entity comprising an amino acid sequence comprising a cysteine ​​residue to which at least one ADP-ribosyl group or analogue thereof is conjugated via an S-glycosidic bond; ii) contacting said first entity with a second entity in an assay, said second entity being capable or believed to be capable of binding to an ADP-ribosyl group or polymer thereof conjugated to a peptide or protein; and iii) measuring a signal derived from said first label or localized by said tag, wherein said assay is configured such that a signal detected when said second entity is bound to at least one ADP-ribosyl group of said first entity differs from a signal detected when no binding interaction between said second entity and said ADP-ribosyl group occurs.

[0030] In a preferred embodiment, the amino acid sequence corresponds to the C-terminal sequence of a Gα subunit of a G protein, preferably SEQ ID NO:4, or has at least 75% sequence identity with the C-terminal sequence of said Gα subunit, preferably said amino acid sequence corresponds to the C-terminal sequence of said Gα subunit or has at least 75% sequence identity with the C-terminal sequence of a heterotrimeric G protein, is a sequence or peptide of at least 4, more preferably up to 50, 60, 70, 80, 90, 100, 150, 200, 250 or 300 amino acids in length.

[0031] In another preferred embodiment, said S-glycosidic conjugation of said at least one ADP-ribosyl group to said cysteine ​​residue is catalyzed by pertussis toxin and, in the case of a polymer, is preferably extended by a PARP family enzyme.

[0032] In a further preferred embodiment, the first entity comprises a C-terminal sequence of at least 4 amino acids in length, CGLF (SEQ ID NO:1) or CGLY (SEQ ID NO:2), corresponding to the C-terminal sequence of the Gα subunit of a G protein, wherein at least one ADP-ribosyl group is conjugated to a cysteine ​​(C) of SEQ ID NO:1 or SEQ ID NO:2 via an S-glycosidic bond.

[0033] In certain preferred embodiments, the first entity has the C-terminal sequence KX 1 NLKX 2 CGLX 3 (SEQ ID NO:3), wherein X 1 is E or N, and X 2 is E or D, and X 3 is F or Y.

[0034] In a further preferred embodiment, said first entity is a fusion protein, preferably including but not limited to a luminescent or fluorescent protein domain or entity.

[0035] In a further preferred embodiment, said first entity is a fusion protein comprising a binding or enzymatic tag, such as, but not limited to, a GST tag or a digoxigenin tag.

[0036] In a particularly preferred embodiment, the second entity comprises a biopolymer capable of binding to the ADP-ribosyl group conjugated to a peptide or protein or a polymer thereof.

[0037] In a further preferred embodiment, the biopolymer is selected from the group consisting of, but not limited to, a macrodomain, an ADP-ribosyl acceptor hydrolase (ARH) family protein, a BRCA1 C-terminal (BRCT) domain, a poly(ADP-ribose)-binding zinc finger motif, and a conserved globular WWE domain of a poly ADP-ribose polymerase homolog.

[0038] In certain preferred embodiments, said second entity comprises a second label and in step iii) signals originating from the first and second labels are measured, wherein a signal detected when said second entity is bound to said at least one ADP-ribosyl group of said first entity is different or differently localized than a signal detected when no binding interaction between said second entity and said ADP-ribosyl group occurs.

[0039] In another preferred embodiment, said first and second labels are distinct luminescent or fluorescent labels.

[0040] In certain preferred embodiments, a candidate inhibitor compound is also added to the assay in step ii), said candidate inhibitor compound being known or suspected to inhibit the binding interaction between said second entity and said ADP-ribosyl group or polymer thereof bound to said first entity.

[0041] In another embodiment, the candidate inhibitor compound is found to be an inhibitor of ADP-ribosyl binding if the binding interaction is inhibited in an assay in the presence of the candidate inhibitor compound, but not inhibited in an assay in the absence of the candidate inhibitor compound.

[0042] In a particular preferred embodiment, the method of the present invention comprises the steps of: i) providing a first luminescent or fluorescent fusion protein comprising an amino acid sequence that includes a cysteine ​​residue to which at least one ADP-ribosyl group is conjugated via an S-glycosidic bond; ii) contacting the first luminescent or fluorescent fusion protein with a second luminescent or fluorescent fusion protein capable of binding an ADP-ribosyl group and a candidate inhibitor compound in an assay; and iii) measuring a luminescence or fluorescence signal from the assay, wherein a signal detected when the second fusion protein is bound to at least one ADP-ribosyl group of the first fusion protein differs from a signal detected when an interaction between the second fusion protein and the ADP-ribosyl group is inhibited by the presence of the candidate inhibitor compound, and wherein the candidate inhibitor compound is found to be an inhibitor of ADP-ribosyl binding if the binding interaction is inhibited in the assay in the presence of the candidate inhibitor but not in the assay in the absence of the candidate inhibitor.

[0043] In certain preferred embodiments, the first and second fusion proteins comprise a fluorescent protein selected from the group consisting of the following: GFP ("green fluorescent protein"), YFP ("yellow fluorescent protein"), CFP ("cyan fluorescent protein"), eYFP ("enhanced yellow fluorescent protein"), eCFP ("enhanced cyan fluorescent protein"), derivatives and variants thereof, such that the fluorescent protein of the first fusion protein is preferably different from the fluorescent protein of the second fusion protein.

[0044] In a further preferred embodiment, the method of the present invention comprises the steps of: - an initial step of providing an entity conjugated to said first label or tag, said entity comprising an amino acid sequence of at least 4 amino acids in length, CGLF (SEQ ID NO:1) or CGLY (SEQ ID NO:2), which corresponds to the C-terminal sequence of the Gα subunit of a G protein; - Pertussis toxin is used to convert the cysteine ​​residues of SEQ ID NO: 1 or 2 to NAD + or an analog thereof to obtain said first entity; - optionally an initial step of extending said ADP-ribosyl group conjugated to said first entity into a polymer using a PARP enzyme.

[0045] The present invention also provides a kit for detecting the attachment of an ADP-ribosyl group or a polymer thereof, said group or polymer being conjugated to a peptide or protein, said kit comprising: - in a first container: a first entity comprising a first label or tag, said entity comprising an amino acid sequence comprising a cysteine ​​residue to which at least one ADP-ribosyl group is conjugated via an S-glycosidic bond; - in a second container: a second entity conjugated to a second label or tag, said second entity being capable of binding to an ADP-ribosyl group or polymer thereof conjugated to a peptide or protein.

[0046] In a preferred embodiment of the kit, said amino acid sequence preferably corresponds to the C-terminal sequence of a Gα subunit of a G protein, preferably of SEQ ID NO.4 or has at least 75% sequence identity with the C-terminal sequence of a Gα subunit of a G protein, more preferably said C-terminal sequence corresponds to the C-terminal sequence of a Gα subunit of a G protein or has at least 75% sequence identity with the C-terminal sequence of a Gα subunit of a G protein, most preferably said C-terminal sequence is a sequence or peptide of at least 4, more preferably up to 50, 60, 70, 80, 90, 100, 150, 200, 250 or 300 amino acids in length of the C-terminal sequence of SEQ ID NO:4 or a sequence having at least 75% sequence identity thereto.

[0047] In another preferred embodiment, said first and second labels may be, but are not limited to, distinct luminescent or fluorescent labels.

[0048] In a particularly preferred embodiment, said first entity is a fusion protein comprising a binding or enzymatic tag, such as a GST tag or a digoxigenin tag.

[0049] In a particularly preferred embodiment, said first and second entities are fusion proteins including, but not limited to, green fluorescent protein (GFP) or a derivative or variant thereof.

[0050] In certain preferred embodiments, the first entity comprises an amino acid sequence of at least 4 amino acids in length, CGLF (SEQ ID NO:1) or CGLY (SEQ ID NO:2), corresponding to the C-terminal sequence of the Gα subunit of a G protein, wherein at least one ADP-ribosyl group is conjugated to a cysteine ​​(C) of SEQ ID NO:1 or SEQ ID NO:2 via an S-glycosidic bond.

[0051] In another preferred embodiment, the first entity has the C-terminal sequence KX 1NLKX 2 CGLX 3 (SEQ ID NO:3), wherein X 1 is E or N, and X 2 is E or D, and X 3 is F or Y.

[0052] In a particularly preferred embodiment, the second entity comprises a biopolymer capable of binding to the ADP-ribosyl group conjugated to a peptide or protein or a polymer thereof.

[0053] In certain preferred embodiments, the biopolymer is selected from the group consisting of, but not limited to, a macrodomain, an ARH family protein, a BRCT domain, a PAR-binding zinc motif, and a WWE domain.

[0054] The present invention also provides a fusion protein comprising a first domain and a second domain, wherein said second domain comprises an amino acid sequence corresponding to the C-terminal sequence of a Gα subunit of a G protein, preferably SEQ ID NO:4, or having at least 75% sequence identity with the C-terminal sequence of a Gα subunit of a G protein, wherein said amino acid sequence comprises a cysteine ​​residue to which at least one ADP-ribosyl group or an analogue thereof is conjugated via an S-glycosidic bond.

[0055] In a preferred embodiment of said fusion protein, said C-terminal sequence corresponds to the C-terminal sequence of the Gα subunit of a G protein, preferably SEQ ID NO:4, or has at least 75% sequence identity with the C-terminal sequence of the Gα subunit of a G protein, and is a sequence or peptide at least 4 amino acids in length.

[0056] In another preferred embodiment, the amino acid sequence of the second domain comprises the amino acid sequence CGLF (SEQ ID NO:1) or CGLY (SEQ ID NO:2) corresponding to the C-terminal sequence of the Gα subunit of a G protein, wherein at least one ADP-ribosyl group is conjugated to a cysteine ​​(C) of SEQ ID NO:1 or SEQ ID NO:2 via an S-glycosidic bond.

[0057] In another preferred embodiment, the amino acid sequence of the second domain comprises the C-terminal sequence KX 1 NLKX 2 CGLX 3 (SEQ ID NO:3), wherein X 1 is E or N, and X 2 is E or D, and X 3 is F or Y.

[0058] In another preferred embodiment of the fusion protein, the first domain is preferably a fluorescent protein selected from the group consisting of: GFP ("green fluorescent protein"), YFP ("yellow fluorescent protein"), CFP ("cyan fluorescent protein"), eYFP ("enhanced yellow fluorescent protein"), eCFP ("enhanced cyan fluorescent protein"), derivatives and variants thereof.

[0059] The present invention further comprises the following i) a fusion protein comprising a first domain and a second domain, the second domain comprising an amino acid sequence comprising a cysteine ​​residue to which at least one ADP-ribosyl group or analogue thereof is attached via an S-glycosidic bond; ii) a biopolymer, preferably selected from the group consisting of a macrodomain, an ARH family protein, a BRCT domain, a PAR-binding zinc motif, and a WWE domain, said biopolymer specifically recognizing said at least one ADP-ribosyl group or an analog thereof of said fusion protein; The system is directed to a system in which the biopolymer binds to the at least one ADP-ribosyl group or analogue thereof of the fusion protein such that the biomolecule and the fusion protein are linked together to form a conjugate.

[0060] In a preferred embodiment of the system, the second domain comprises an amino acid sequence that corresponds to the C-terminal sequence of a Gα subunit of a G protein, preferably SEQ ID NO:4, or has at least 75% sequence identity to the C-terminal sequence of a Gα subunit of a G protein.

[0061] In a preferred embodiment, the system comprises means for detecting the presence of the conjugate within the system, which may preferably be selected from the group of luminescent and fluorescent labels and devices capable of receiving signals from these labels.

[0062] The following examples illustrate the principles of the present invention in one or more specific applications, but it will be apparent to those skilled in the art that numerous changes in form, use and details of implementation can be made without the exercise of inventive faculty and without departing from the principles and concepts of the present invention. Accordingly, it is not intended that the present invention be limited, except as by the scope of the claims which follow.

[0063] In this document, the verbs "comprise" and "include" are used as open limitations that do not exclude or require the presence of any unrecited features. Features recited in the claims may be freely combined with each other, unless otherwise expressly stated. Furthermore, it is to be understood that the use of "a" or "an" throughout this document, i.e., the singular, does not exclude the plural.

[0064] Throughout this specification, a reference to an embodiment or an embodiment means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, although the phrases "in one embodiment" or "in an embodiment" appear in various places throughout this specification, they do not necessarily all refer to the same embodiment. For example, when a numerical value is referred to using a term such as about or substantially, the exact numerical value is also disclosed. EXAMPLES

[0065] Materials and Methods Cloning Expression constructs of CFP or nanoluciferase fusion proteins were cloned into pNIC28-CFP or pNH-Nluc by sequence- and ligation-independent cloning. pNIC28-CFP and pNH-Nluc were prepared by inserting the sequence of CFP or nanoluciferase between the His6-tag and the TEV protease cleavage site of pNIC28-Bsa4 or pNH-Trxt. Other protein constructs were cloned into pNIC28-Bsa or pNIC-MBP vectors.

[0066] Protein expression Plasmids were transformed into E. coli BL21(DE3) or E. coli Rosetta2 cells. Autoinducing medium with trace elements (Formedium, Hunstanton, Norfolk, UK) supplemented with 8 g / L glycerol and antibiotics was inoculated 1:100 with an overnight preculture grown in LB. Flasks were incubated at 37°C with shaking until an OD600 of approximately 1 was reached. The temperature was set to 18°C ​​and incubation continued overnight. Cells were harvested by centrifugation at 4,200 x g for 30 min at 4°C. The pellet was suspended in lysis buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 15 mM imidazole). Resuspended cells were stored at -20°C until purification.

[0067] Protein purification All constructs were first purified by immobilized metal affinity chromatography (IMAC). Cells were thawed and lysed by sonication. The lysates were centrifuged, the supernatants were filtered, and equilibrated with lysis buffer and Ni 2+ The eluate was loaded onto a 5 mL HiTrap HP column charged with 0.1% ethanol. The column was washed with 2-10 column volumes of lysis buffer and 2-10 column volumes of lysis buffer containing 10-50 mM imidazole. Elution was performed with elution buffer containing 100-500 mM imidazole. After IMAC, most of the proteins were further purified by size exclusion. Finally, the proteins were concentrated to concentrations of 0.04-3 mM, then aliquoted, flash frozen in liquid nitrogen, and stored at -70 °C.

[0068] Preparation of mono- and poly ADP-ribosylated Gαi proteins YFP with a C-terminal Gαi-peptide tag was purified by IMAC and dialyzed against 20 mM HEPES pH 7.5, 350 mM NaCl. YFP-Gαi was diluted to 100 μM in 50 mM sodium phosphate buffer pH 7.0 and mixed with 1.5 μM pertussis toxin catalytic S1 domain and 150 μM sodium β-nicotinamide adenine dinucleotide. The reaction was incubated for 1 h at room temperature. A second aliquot of 150 μM was added to the reaction to ensure the reaction was complete. The incubation was continued for 1 h at room temperature. The reaction mixture was loaded onto an IMAC column to remove pertussis toxin, hydrolysis products and unreacted NAD. + The protein was then flash frozen in liquid nitrogen and stored at -70°C. IMAC was performed as described in the purification procedure above. The buffer was exchanged to 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TCEP, and the MAR-ylated YFP-Gαi was subsequently concentrated to approximately 1 mM concentration using Amicon Ultra-15 centrifugal filter units (MWCO: 10 kDa). The protein was flash frozen in liquid nitrogen and stored at -70°C.

[0069] PAR-ylated YFP-Gαi for FRET experiments was prepared from MAR-ylated YFP-Gαi. 10 μM MAR-ylated YFP-Gαi was mixed with 400 nM PARP2 (residues 90-583) and 1 mM NAD + The reaction mixture was incubated for 2 h at room temperature in a buffer containing 50 mM Tris [pH 8.0] and 5 mM MgCl2 in the presence of 100 mM glycerol. The reacted samples were then purified by IMAC as described above to remove PARP2. The sample buffer was exchanged into 30 mM HEPES [pH 7.5], 150 mM NaCl, 10% glycerol, 0.5 mM TCEP using Amicon Ultra-15 centrifugal filter units (MWCO: 10 kDa). Proteins were aliquoted, flash frozen in liquid nitrogen, and stored at -70 °C.

[0070] To produce PAR-YFP-Gαi of Tankyrase1, MAR-YFP-Gαi was incubated with 200 nM Tankyrase1 SAM catalytic domain dimer, 1 and 10 mM NAD + The mixture was incubated with 10 mM bis-Trispropane [pH 7.0] and 0.01% Triton X-100 in a buffer. The reaction was carried out at room temperature for 16 hours.

[0071] Blot-based detection of mono- and poly ADP-ribosylation For dot blot experiments, the sample solution was transferred to a dry nitrocellulose membrane at 0.5 μL per spot using an Echo650. All the following steps were performed at room temperature. After drying of the spots, the membrane was blocked with 15 mL of 5% (w / v) nonfat dry milk in TBS-T for 10 min on a shaker. The blocking solution was discarded and the membrane was incubated with 15 mL of mpNanoluc-eAf1521 at 0.1 μg / mL in 1% (w / v) nonfat dry milk in TBS-T on a shaker. After discarding the Nanoluc-eAf1521 solution, the membrane was rinsed with 15 mL of TBS-T and incubated with 15 mL of TBS-T for 15 min on a shaker. After a final rinse with 15 mL of TBS-T, the membrane was imaged with 500 μL of 1:1000 NanoGlo substrate (Promega) diluted in 10 mM sodium phosphate buffer pH 7.0.

[0072] For Western blot, 10 μL of sample was first subjected to SDS-PAGE (Mini-Protean TGX 4-20% gradient gel, BioRad). Proteins were then transferred to nitrocellulose membranes (Trans-Blot Turbo, BioRad) using the TransBlot semi-dry system (BioRad). After transfer, the membranes were treated with the same procedure as for dot blots above. Nanoluc-eAF1521 and Nanoluc-ALC1 were used at 0.1 μg / mL.

[0073] Testing the sensitivity and selectivity of Nluc-eAf1521 and Nluc-ALC1 To generate autoPARylated TNKS1, 10 μM of the TNKS1 construct was dissolved in 50 mM Bis-Trispropane pH 7.0, 0.01% TritonX-100, 0.5 mM TCEP and 1 mM NAD. + To generate a control containing TNKS1 without PAR, the TNKS1 construct was mixed with 2 μM snake venom phosphodiesterase I to eliminate the partial auto-PARylation that occurred during recombinant expression in E. coli.

[0074] 6-Biotin-17-NAD + Modification of YFP-Gαi by 10 μM YFP-Gαi or YFP-Gαi (mutants) in 1 μM NAD + or 6-biotin-17-NAD + (Biolog). Reactions were prepared in the absence or presence of 0.5 μM PtxS1 and incubated at room temperature for 1 h before blotting onto dry nitrocellulose membranes (0.5 μL of sample solution per spot on dry nitrocellulose membrane, using Echo650). After drying the membranes were blocked in 15 mL of blocking buffer (1% casein in TBS, BioRad) for 10 min on a shaker. The blocking solution was discarded and incubated with 15 mL of 1:5000 streptavidin-HRP in blocking buffer for 1 h on a shaker. After discarding the streptavidin-HRP solution, the membranes were rinsed with 15 mL of TBS-T and incubated with 15 mL of TBS-T for 15 min on a shaker. After a final rinse with 15 mL of TBS-T, the membranes were imaged using ECL solution (BioRad).

[0075] 6-Parg-NAD + Modification of YFP-Gαi with added Cy3 and Cy5 azides and by CuAAC YFP-Gαi(6-PARG-MAR) was transfected with NAD + Instead of 6-PARG-NAD +The reaction was prepared as described above for YFP-Gαi using the CuAAC. To test the addition of Cy3 / Cy5 to YFP-Gαi(6-PARG-MAR) by CuAAC, reactions were prepared in 25 mM HEPES pH 7.5 by mixing 15 μM YFP-Gαi(6-PARG-MAR) or YFP-Gαi(MAR) with 10 mM sodium ascorbate, 50 μM Cy3-azide or Cy5-azide, and premixed 300 μM CuSO4 and 600 μM L-histidine. In addition, a no protein control was also prepared. The reaction was incubated at room temperature for 3 h and then blotted (5 μL per spot) onto a nitrocellulose membrane. The membrane was washed with 15 mL TBS-T for 30 min and imaged. Fluorescence imaging was performed using an Azure600 imaging system (Azure Biosystems) using Cy3 or Cy5 filter settings, respectively.

[0076] FRET measurements Samples were excited at 410 nM and emissions at wavelengths of 477 nM and 527 nM were measured. Ratiometric FRET values ​​(rFRET) were calculated by dividing the fluorescence intensity at 527 nm by the fluorescence intensity at 477 nm. Experiments were performed in assay buffer (10 mM Bis-Tris-propane pH 7.0, 3% (w / v) PEG 20,000, 0.01% (v / v) Triton X-100 and 0.5 mM TCEP) in a volume of 10 μL per well unless otherwise noted.

[0077] BRET measurement Reactions were performed in 384-well white OptiPlates (PerkinElmer). Reaction volumes were 40 μL per well. 50 nM Nluc-MDO2 was mixed with 1 μM MAR-ylated YFP-Gαi. Reactions were initiated by adding 1:4000 NanoGlo substrate (Promega, Cat# N1110). Reactions were incubated for 5 min and emission was measured at wavelengths 445-470 nM and 520-545 nM using a Tecan Spak multimode plate reader with luminescence readout, set time 10 ms, and integration time 100 ms). Ratiometric BRET values ​​(rBRET) were calculated by dividing the emission intensity at 520-545 nM by the emission intensity at 445-470 nM. Experiments were performed in assay buffer (10 mM Bis-Tris-propane pH 7.0, 3% (w / v) PEG 20,000, 0.01% (v / v) Triton X-100, 0.5 mM TCEP).

[0078] Alpha Screen Reactions were performed in a total volume of 25 μL using 384-well flat grey Alphaplates (PerkinElmer). Reactions consisted of 300 nM His-tagged Gαi(MAR) mixed with 300 nM Bio-MDO2 in a buffer containing (25 mM HEPES pH 7.5, 100 mM NaCl and 0.1 mg / mL BSA). The plate was sealed and incubated for 80 min at RT with constant shaking at 300 rpm. Finally, 5 μg / mL of nickel chelate acceptor and streptavidin donor beads were added to the plate and incubated for an additional 3 h. The plate included blank wells (assay buffer and AlphaScreen beads only), control 1 (Bio-MDO2, His-Gαi) and control 2 (Bio-MDO2, modified His-Gαi, ADPr). Luminescence was read using a Tecan infinite M1000Pro plate reader with an AlphaScreen detection module.

[0079] Biolayer Interferometry Biolayer interferometry (BLI) assays were performed in a buffer containing 10 mM bis-trispropane [pH 7.0], 150 mM NaCl, 1% BSA, and 0.02% TritonX-100 at 30°C with shaking at 1500 rpm in an Octet Red system (Forte Bio). 2+ -NTA-coated sensors were loaded with 1,000 ng / mL DMSO, followed by a washing step in buffer. Association to MDO2 was measured by immersing the sensor in a solution containing 0–2 μM MDO2 for 120 s, and the dissociation step involved immersing the sensor in buffer for 120 s.

[0080] In ADP-ribose competition experiments, 10 μg / mL of YFP-Gα i or MAR-YFP-Gα i was loaded onto Ni2+-NTA-coated sensors. For association, the sensors were immersed in 100 nM MDO2 mixed with a half-log dilution series of ADPr (10 nM to 10 μM) for 120 s and then transferred to buffer for the dissociation step.

[0081] Cysteine-ADP-ribosyl hydrolysis assay All samples were incubated for 24 hours at room temperature before blotting. 1 μM CFP fusion protein or 0.01, 0.1, or 1 μM SVP were mixed with 10 μM MARylated YFP-Gαi in 10 mM HEPES pH 7.5, 25 mM NaCl, 0.5 mM TCEP. After incubation, 0.5 μL of the reaction mixture per spot was blotted next to a 0.5 μL spot of 10 μM MARylated YFP-Gαi. As a control, 10 μM non-MARylated YFP-Gαi was blotted next to 10 μM MARylated YFP-Gαi.

[0082] Concomitant screening of FDA-approved drug libraries For screening, 40 nL of 10 mM compound stock dissolved in DMSO from an FDA-approved drug library (Enzo Life Sciences) was transferred to a 384-well black low-volume polypropylene plate (Fisherbrand). Sample mixtures containing 1 μM CFP-SARS-CoV-2 nsp3 macrodomain and 5 μM MARylated YFP-Gαi were prepared in assay buffer (10 mM Bis-Trispropane pH 7.0, 3% (w / v) PEG 20,000, 0.01% (v / v) TritonX-100, and 0.5 mM TCEP) and dispensed 20 μL per well using Mantis (Formulatrix). After 5 min of incubation, the rFRET signal was measured. Sample mixtures with or without 200 μM ADP-ribose were used as positive and negative controls, respectively.

[0083] Verification of suramin binding by DSF Untagged SARS-CoV-2 nsp3 macrodomain was diluted to 5 μM in 10 mM HEPES pH 7.5, 25 mM NaCl, 0.5 mM TCEP buffer and mixed with 5x SYPRO Orange. Samples were prepared with 10 μM, 50 μM, 100 μM or 1 mM suramin. Samples with or without 1 mM ADP-ribose were used as controls. Samples were transferred to a 96-well qPCR plate. Measurements were performed in a BioRad C1000 CFX96 thermal cycler. Melting curve data points were recorded at 1 min intervals from 20-95 °C, with the temperature increasing at 1 °C / min. Data were analyzed in GraphPad Prism 7 using nonlinear regression analysis (Boltzmann sigmoid equation) of normalized data.

[0084] Example 1. Initial preparation of protein for toolbox studies All proteins used in this study were recombinantly produced in E. coli. As a tool to detect ADP-ribosylation in a blot-based manner, we produced the recently reported ADP-ribosyl superconjugate eAf1521 (Nowak et al., 2020) as a nanoluciferase-tethered fusion protein. We found that this protein functions in a simple and rapid one-step protocol for sensitive detection of mono- and poly-ADP-ribosylated proteins (Figures 2c and 2d).

[0085] We reasoned that mono-ADP-ribosylation of Gαi by pertussis toxin would be a suitable probe to investigate ADP-ribosylation because the modification is well-defined at one residue and uses a cysteine ​​to generate a stable S-glycosidic bond. To generate a mono-ADP-ribosylated Gαi probe, we tested the ability of pertussis toxin to modify only the 10 amino acid C-terminal peptide of Gαi fused to YFP, in comparison with a full-length Gαi mutant (Figure 2a). The results showed that it could effectively modify the fusion peptide.

[0086] Next, we aimed to introduce poly ADP-ribosyl chains into Gαi peptides. We hypothesized that the mono ADP-ribosyl group of the modified Gαi peptides could be the starting point for elongation to poly ADP-ribose by the PARP enzyme. After optimizing the reaction conditions, we found that PARP2 could generate poly ADP-ribosylated Gαi constructs (Fig. 2b).

[0087] Example 2. NAD + G.ALPHA.i peptides as tags for site-specific labeling with analogues. The Gαi peptide tag allows for site-specific labeling of proteins with accessible C-termini with ADP-ribose, but this system can be extended to incorporate chemical NAD at the C-terminus of proteins. + We thought we could introduce analogues. Many NAD + Analogs already exist and are biotinylated, fluorescent and click chemistry compatible. + Analogs and other analogs are commercially available.+ Modification of YFP-Gαi-peptide tagged proteins with the analogs was tested. Site-specific modification was detected using dot blots with streptavidin-conjugated horseradish peroxidase (Figure 3a).

[0088] In addition, 6-propargyl adenine-NAD, which allows the copper(I)-catalyzed alkyne-azide cycloaddition of azide groups, was synthesized. + We examined the modification of the YFP-fused Gαi peptide tag with 6-propargyl adenine-NAD by dot blot analysis (Figure 3b). + The proteins containing the modified Gαi peptide could be tagged with azide groups containing the fluorescent dyes Cy3 and Cy5, but not with the canonical NAD. + It was not possible to add Cy3 or Cy5 to the Gαi peptide modified with .

[0089] Example 3. Evaluation of binding to confirmed and potential ADP-ribosyl conjugates A set of 27 proteins was selected to test binding to MARylated Gαi. These proteins were recombinantly produced in E. coli as fusions with CFP and tested for ratiometric FRET signal upon binding to MARylated YFP-Gαi constructs (Figure 4a). To compete the interaction, we used a non-MARylated YFP-Gαi construct as a control, as well as MARylated YFP-Gαi with 200 μM ADP-ribose. Higher FRET signal correlates with higher occupancy of the binding partner and therefore higher binding affinity, but is also influenced by the distance and orientation of the fluorophore. We found that proteins previously reported to bind ADP-ribose showed higher FRET signal compared to the control, indicating binding to the MARylated probe. ARH1 is known to bind ADP-ribose with low affinity, which may be the reason why we were unable to measure the FRET signal of this construct. Although all three histone macrodomains, macroH2A1.1, macroH2A1.2 and macroH2A2, have very similar sequence identity, only macroH2A1.1 has the ability to bind ADP-ribose, which is consistent with our observation that only macroH2A1.1 exhibits a higher FRET signal than the control.

[0090] It is expected that binding to cysteine-ADP-ribosyl groups will not be of physiological significance for some or even most of the ADP-ribosyl-binding proteins tested. It is surprising that no binder appears to be highly specific for either O- or N-glycosidically linked ADP-ribose and that cysteine ​​sulfur does not inhibit binding in any of the identified ADP-ribosyl-binding proteins.

[0091] Although cysteine-ADP-glycosyl hydrolase activity has been detected in human erythrocytes and mitochondria, a specific enzyme in humans capable of hydrolyzing the S-glycosidic bond of cysteine-ADP-ribose has not been identified so far. The above CFP fusion constructs were mixed with mono-ADP-ribosylated YFP-Gαi and tested for ADP-ribose hydrolysis after 24 h of incubation using a dot blot (Figure 4b). Although controls containing snake venom phosphodiesterase I showed loss of signal due to cleavage of the ADP-ribose diphosphate bond, none of the proteins tested were able to hydrolyze the S-glycosidic bond under the conditions tested. These findings highlight the versatility of this system to measure ADP-ribosyl bonds in proteins capable of hydrolyzing O- or N-glycosidic bonds such as MDO1, TARG1, ARH3 or SARS-CoV-2 nsp3. We next tested the ratiometric FRET signal of the CFP-fused polyADP-ribosyl conjugates when mixed with YFP-Gαi containing polyADP-ribosyl groups (Fig. 2b). The polyADP-ribosyl conjugates showed an increased ratiometric FRET signal with PAR-ylated YFP-Gαi compared to the control, suggesting binding to PAR rather than MAR groups (Fig. 4c). Representative dose-response curves of mono- or polyADP conjugates show good signal quality (Fig. 4d, e).

[0092] Although we used FRET to test ADP-ribosyl binding above, we showed that binding to MAR-tagged Gαi can also be measured using a different binding technique. For this, we used MDO2 as an example protein. Similar to the binding of CFP-fused MDO2 to MAR-tagged YFP-Gαi for FRET measurements (Fig. 5a), we used Nanoluc-fused MDO2 to generate a BRET signal upon MAR-tagged YFP-Gαi interaction (Fig. 5b).

[0093] Although AlphaScreen protocols exist for ADP-ribosyl leaders or erasers, our system adapted for AlphaScreen technology allows one to directly probe the binding of potentially any leader or eraser to MARylated Gαi, as shown for example with the otherwise hydrolyzing MDO2 domain (Fig. 5c).

[0094] We further demonstrated binding using biolayer interferometry (BLI, Figure 5d), a method that has recently become popular as a screening method for small molecule compounds, and which also allows for easy quantification of kinetic binding parameters such as the dissociation constant.

[0095] Example 4. Screening of inhibitors against SARS-CoV-2 nsp3 macrodomain Given the current situation regarding the COVID-19 pandemic, we decided to demonstrate the applicability of this binding system to screen small molecule inhibitors against the macrodomain of SARS-CoV-2 nsp3. Currently, researchers around the world are working to find inhibitors against this macrodomain. The coronavirus nsp3 macrodomain has been shown to be important for viral replication, and small molecule inhibitors may be promising therapeutic agents against infections caused by SARS-CoV-2 (COVID-19) and other viruses.

[0096] We assessed the quality of the FRET signals of alternating positive and negative controls by mixing the CFP-fused SARS-CoV-2 nsp3 macrodomain with MAR-ylated YFP-Gαi in the absence and presence of 200 μM ADP-ribose in a 384-well plate (Figure 6a). Although the SARS-CoV-2 macrodomain has been reported to have a relatively low binding affinity with ADP-ribose (Kd = 17 μM), we still found that the signals showed sufficient separation between the positive and negative controls. The Z'-factor was calculated to be 0.7, indicating that this assay is suitable for high-throughput screening.

[0097] The compound was screened against the ENZO FDA-approved drug library, which contains 640 small molecule compounds, at a compound concentration of 20 μM (Figure 6b). From the screen, only one compound, suramin, was identified as a hit with an inhibition rate of 82% and was tested in a FRET-based assay with an IC 50 The binding activity of this compound to the SARS-CoV-2 nsp3 macrodomain was 8.7 μM (Fig. 6c, 6d). To confirm the binding of this compound to the SARS-CoV-2 nsp3 macrodomain, DSF analysis was performed, which showed a concentration-dependent stabilization of the protein (Fig. 6e). Interestingly, suramin has been used as a broad-spectrum antiviral and antiparasitic drug and has been reported to inhibit SARS-CoV2 infection in cell culture-based models. A review of the literature related to suramin revealed that it has been reported to inhibit many target proteins, from DNA and RNA polymerases to sirtuins, ATPases and G protein-coupled receptors, indicating low target specificity. We next tested suramin for inhibition of the viral and human ADP-ribose readers and erasers produced in this study (Fig. 6f). Not surprisingly, suramin showed strong inhibition even at 10 μM against many of the proteins tested, confirming the low target specificity of this compound.

[0098] [References] Nowak K, Rosenthal F, Karlberg T, Butepage M, Thorsell AG, Dreier B, Grossmann J, Sobek J, Imhof R, Luescher B, Schuler H, Pluckthun A, Leslie Pedriol i DM, Hottiger MO. (2020) Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins. Nat. Commun. 11:5199, https: / / doi.org / 10.1038 / s41467-020-18981-w TeloniF and Altmeyer M.(2016)Readers of poly(ADP-ribose): designed to be fit for purpose. Nucleic Acids Res. 44:993-1006、doi: 10.1093 / nar / gkv1383 Wazir S、MaksimainenmM、Alanen HI、Galera-Prat A、Lehtioe L.(2021)Activity-Based Screening Assay for Mono-ADP-Ribosylhydrolases. SLAS Discovery 26:67-76、doi: 10.1177 / 2472555220928911

Claims

1. 1. A method for detecting the binding of an ADP-ribosyl group or a polymer thereof, said group or polymer being conjugated to a peptide or protein, comprising the steps of: i) providing a first entity comprising a first label or tag, said entity comprising an amino acid sequence comprising a cysteine ​​residue to which at least one ADP-ribosyl group or analogue thereof is conjugated via an S-glycosidic bond; ii) contacting said first entity with a second entity in an assay, said second entity being capable or believed to be capable of binding to an ADP-ribosyl group or polymer thereof conjugated to a peptide or protein; and iii) measuring a signal derived from said first label or localized by said tag, wherein a signal detected when said second entity is bound to at least one ADP-ribosyl group of said first entity is different or differently localized than a signal detected when no binding interaction between said second entity and said ADP-ribosyl group occurs.

2. 2. The method of claim 1, wherein the amino acid sequence corresponds to the C-terminal sequence of a Gα subunit of a G protein or has at least 75% sequence identity with the C-terminal sequence of the Gα subunit, preferably the amino acid sequence corresponds to the C-terminal sequence of the Gα subunit or has at least 75% sequence identity with the C-terminal sequence of a heterotrimeric G protein is a sequence or peptide at least 4 amino acids in length.

3. 2. The method of claim 1, wherein the conjugation of the at least one ADP-ribosyl group to the cysteine ​​residue via the S-glycosidic bond is catalyzed by pertussis toxin, and in the case of the polymer, the ADP-ribosyl group conjugated to the cysteine ​​residue is preferably extended by a PARP family enzyme.

4. 2. The method of claim 1, wherein the first entity comprises a C-terminal sequence of at least 4 amino acids in length, CGLF (SEQ ID NO:1) or CGLY (SEQ ID NO:2), corresponding to the C-terminal sequence of the Gα subunit of a G protein, and at least one ADP-ribosyl group is conjugated to the cysteine ​​(C) of SEQ ID NO:1 or SEQ ID NO:2 via an S-glycosidic bond.

5. The first entity has the C-terminal sequence KX １ NLKX ２ CGLX ３ (SEQ ID NO: 3), wherein X １ is E or N, and X ２ is E or D, and X ３ The method of claim 4 , wherein is F or Y.

6. The method of claim 1 , wherein the first entity is a fusion protein, preferably comprising a luminescent or fluorescent protein domain or entity.

7. The method of claim 1 , wherein the first entity is a fusion protein comprising a binding or enzyme tag, such as a GST tag or a digoxigenin tag.

8. The method of claim 1 , wherein the second entity comprises a biopolymer capable of binding to the ADP-ribosyl group conjugated to a peptide or protein or a polymer thereof.

9. The method of claim 8, wherein the biopolymer is selected from the group consisting of a macrodomain, an ARH family protein, a BRCT domain, a PAR-binding zinc motif, and a WWE domain.

10. 2. The method of claim 1, wherein the second entity comprises a second label, and in step iii) signals from the first and second labels are measured, and a signal detected when the second entity is bound to at least one ADP-ribosyl group of the first entity is different or differently localized than a signal detected when no binding interaction between the second entity and the ADP-ribosyl group occurs.

11. The method of claim 10, wherein the first and second labels are distinct luminescent or fluorescent labels.

12. 2. The method of claim 1, wherein in step ii) a candidate inhibitor compound is also added to the assay, said candidate inhibitor being known or suspected to inhibit the binding interaction between said second entity and said ADP-ribosyl group or polymer thereof conjugated to said first entity.

13. The method of claim 12, wherein the candidate inhibitor compound is found to be an inhibitor of ADP-ribosyl binding if the binding interaction is inhibited in an assay in the presence of the candidate inhibitor but not in an assay in the absence of the candidate inhibitor.

14. i) providing a first luminescent or fluorescent fusion protein comprising an amino acid sequence that includes a cysteine ​​residue to which at least one ADP-ribosyl group is conjugated via an S-glycosidic bond; ii) contacting said first luminescent or fluorescent fusion protein with a second luminescent or fluorescent fusion protein capable of binding an ADP-ribosyl group and a candidate inhibitor compound in an assay; iii) measuring a luminescence or fluorescence signal from the assay, wherein a signal detected when the second fusion protein is bound to at least one ADP-ribosyl group of the first fusion protein differs from a signal detected when an interaction between the second fusion protein and the ADP-ribosyl group is inhibited by the presence of the candidate inhibitor compound, and wherein the candidate inhibitor compound is found to be an inhibitor of ADP-ribosyl binding if the binding interaction is inhibited in an assay in the presence of the candidate inhibitor but not in an assay in the absence of the candidate inhibitor.

15. 15. The method of claim 14, wherein the first and second fusion proteins comprise green fluorescent protein (GFP) or a derivative thereof.

16. - an initial step of providing an entity conjugated to said first label or tag, said entity comprising an amino acid sequence of at least 4 amino acids in length CGLF (SEQ ID NO: 1) or CGLY (SEQ ID NO: 2) corresponding to the C-terminal sequence of the Gα subunit of a G protein; - Pertussis toxin is used to ligate the cysteine ​​residues of SEQ ID NO: 1 or 2 to NAD ＋ or an analog thereof to obtain said first entity; - optionally an initial step of extending the ADP-ribosyl group conjugated to said first entity into a polymer using a PARP enzyme.

17. 1. A kit for detecting the binding of an ADP-ribosyl group or a polymer thereof, said group or polymer being conjugated to a peptide or protein, - in a first container: a first entity comprising a first label or tag, said entity comprising an amino acid sequence comprising a cysteine ​​residue to which at least one ADP-ribosyl group is conjugated via an S-glycosidic bond; - in a second container: a second entity conjugated to a second label or tag, said second entity being capable of binding to an ADP-ribosyl group or polymer thereof conjugated to a peptide or protein.

18. 18. The kit of claim 17, wherein the amino acid sequence corresponds to or has at least 75% sequence identity with the C-terminal sequence of a Gα subunit of a G protein, preferably the C-terminal sequence corresponds to or has at least 75% sequence identity with the C-terminal sequence of a Gα subunit of a G protein and is a sequence or peptide at least 4 amino acids in length.

19. 20. The kit of claim 18, wherein the first and second labels are distinct luminescent or fluorescent labels.

20. 18. The kit of claim 17, wherein the first entity is a fusion protein comprising a binding or enzyme tag, such as a GST tag or a digoxigenin tag.

21. 20. The kit of claim 18, wherein the first and second entities are fusion proteins comprising green fluorescent protein (GFP) or a derivative or mutant thereof.

22. 18. The kit of claim 17, wherein the first entity comprises an amino acid sequence of at least 4 amino acids in length CGLF (SEQ ID NO:1) or CGLY (SEQ ID NO:2) corresponding to the C-terminal sequence of the Gα subunit of a G protein, and at least one ADP-ribosyl group is conjugated to the cysteine ​​(C) of SEQ ID NO:1 or SEQ ID NO:2 via an S-glycosidic bond.

23. The first entity has the C-terminal sequence KX １ NLKX ２ CGLX ３ (SEQ ID NO: 3), wherein X １ is E or N, and X ２ is E or D, and X ３ is F or Y.

24. 18. The kit of claim 17, wherein the second entity comprises a biopolymer capable of binding to the ADP-ribosyl group conjugated to a peptide or protein or a polymer thereof.

25. 25. The kit of claim 24, wherein the biopolymer is selected from the group consisting of a macrodomain, an ARH family protein, a BRCT domain, a PAR-binding zinc motif, and a WWE domain.

26. A fusion protein comprising a first domain and a second domain, said second domain comprising an amino acid sequence corresponding to or having at least 75% sequence identity with the C-terminal sequence of a Gα subunit of a G protein, said amino acid sequence comprising a cysteine ​​residue to which at least one ADP-ribosyl group or an analog thereof is coupled via an S-glycosidic bond.

27. 27. The fusion protein of claim 26, wherein the C-terminal sequence corresponds to or has at least 75% sequence identity with the C-terminal sequence of a Gα subunit of a G protein and is a sequence or peptide at least 4 amino acids in length.

28. 27. The fusion protein of claim 26, wherein the amino acid sequence comprises the amino acid sequence CGLF (SEQ ID NO: 1) or CGLY (SEQ ID NO: 2) corresponding to the C-terminal sequence of the Gα subunit of a G protein, and at least one ADP-ribosyl group is conjugated to the cysteine ​​(C) of SEQ ID NO: 1 or SEQ ID NO: 2 via an S-glycosidic bond.

29. The amino acid sequence is the C-terminal sequence KX １ NLKX ２ CGLX ３ (SEQ ID NO: 3), wherein X １ is E or N, and X ２ is E or D, and X ３ is F or Y.

30. 27. The fusion protein of claim 26, wherein the first entity is preferably selected from the group consisting of GFP ("green fluorescent protein"), YFP ("yellow fluorescent protein"), CFP ("cyan fluorescent protein"), eYFP ("enhanced yellow fluorescent protein"), eCFP ("enhanced cyan fluorescent protein"), derivatives and mutants thereof.

31. The analogue may be a biotinylated, fluorescent or click chemistry-enabled NAD ＋ 27. The fusion protein of claim 26 which is derived from an analog.

32. 27. The fusion protein of claim 26, wherein the first domain comprises a luminescent label, a fluorescent label, a binding tag, or an enzymatic tag, such as a GST tag or a digoxigenin tag.

33. 27. The fusion protein of claim 26, wherein the first domain is a fluorescent protein preferably selected from the group consisting of GFP ("green fluorescent protein"), YFP ("yellow fluorescent protein"), CFP ("cyan fluorescent protein"), eYFP ("enhanced yellow fluorescent protein"), eCFP ("enhanced cyan fluorescent protein"), derivatives and mutants thereof.

34. i) a fusion protein comprising a first domain and a second domain, said second domain comprising an amino acid sequence comprising a cysteine ​​residue to which at least one ADP-ribosyl group or analogue thereof is conjugated via an S-glycosidic bond; ii) a biopolymer, preferably selected from the group consisting of a macrodomain, an ARH family protein, a BRCT domain, a PAR-binding zinc motif, and a WWE domain, which specifically recognizes said at least one ADP-ribosyl group or analog thereof of said fusion protein; In the system, the biopolymer binds to the at least one ADP-ribosyl group or analogue thereof of the fusion protein such that the biomolecule and the fusion protein are linked together to form a conjugate.

35. 35. The system of claim 34, wherein the second domain comprises an amino acid sequence that corresponds to or has at least 75% sequence identity with the C-terminal sequence of a Gα subunit of a G protein.

36. 35. The system of claim 34, comprising a means for detecting the presence of the conjugate in the system.

37. 36. The system of claim 35, wherein the means comprises biolayer interferometry technology.

38. 34. The system of claim 33, wherein the first domain comprises a luminescent label, a fluorescent label, a binding tag, or an enzymatic tag, such as a GST tag or a digoxigenin tag.

39. 34. The system of claim 33, wherein the first domain is a fluorescent protein preferably selected from the group consisting of GFP ("green fluorescent protein"), YFP ("yellow fluorescent protein"), CFP ("cyan fluorescent protein"), eYFP ("enhanced yellow fluorescent protein"), eCFP ("enhanced cyan fluorescent protein"), derivatives and mutants thereof.

Images