Cell-based reporters for ubiquitin ligases

Abstract

A reporter for an intramolecular protein-fragment complementation assay, wherein the reporter is a fused protein comprising a first fragment, a second fragment and an E3 ligase sequence section, wherein the first fragment and the second fragment are derived from different sections of the same split protein, preferably luciferase split protein, and fragments of the split protein are configured for providing a signal indicating an assembly of the first fragment and the second fragment, wherein the E3 ligase sequence section intervenes between the first fragment and the second fragment, wherein E3 ligase sequence section is derived from an E3 ligase reference sequence, as well as a polynucleotide, a cell and methods for conducting such an assay.

Description

[0001] CELL-BASED REPORTERS FOR UBIQUITIN LIGASES

[0002] The invention relates to an E3 ligase reporter for a protein-fragment complementation assay, a polynucleotide and a cell for a protein-fragment complementation assay as well as methods of conducting such an assay to monitor for example target engagement of candidate molecules.

[0003] BACKGROUND OF THE INVENTION

[0004] Targeted protein degradation has emerged as a promising new strategy, especially for the treatment of cancers, and enables molecular targeting of hitherto “undruggable” proteins [Samarasinghe et al., 2021, Dale et al., 2021], In these approaches, the cellular ubiquitin- proteasome system (UPS) is harnessed to specifically degrade the respective disease driver protein. Here, a molecular interaction between the protein of interest (POI) and an E3 ligase enzyme is artificially induced to promote ubiquitylation and subsequent proteasome-mediated degradation of the target protein [Zhao et al., 2022], Among several strategies that have been developed to induce the interaction between the POI and the E3 ligase, the application of proteolysis-targeting chimeras (PROTACs) is considered to hold a high therapeutic potential [Samarasinghe et al., 2021], PROTACs are bifunctional small molecules, consisting of an E3 ligase-recruiting part of the small molecule which is linked to the POI-binding second ‘warhead’.

[0005] Although more than 600 distinct E3 ligases are encoded in the human genome [Zheng et al., 2017], the vast majority of PROTACS developed so far function through recruitment of the endogenously present proteins cereblon (CRBN) or Von-Hippel-Lindau (VHL) [Liu et al., 2023], Both of these represent subunits of multi-protein E3 ligase complexes and served as workhorses for early PROTAC developments. Their popularity is grounded on their ubiquitous presence in most tissues, the availability of highly specific recruitment agents, coupled with a high degree of molecular flexibility when recruited to the POI. In contrast to CRBN and VHL, which represent the substrate recruiting subunit in a multi-protein complex [Bekes et al., 2022], many E3 ligases are functional as an individual protein. The different types can be subclassified based on their sequence homology and Fig. 1 shows phylogenetic trees of human proteins annotated as substrate receptors of multi-subunit ubiquitin E3 ligases (Fig. 1 A) or as autonomous ubiquitin E3 ligases (Fig. IB).

[0006] To promote research especially on the not yet thoroughly investigated autonomous E3 ligases, it will be critical to identify molecular glue lead structures that facilitate artificial substrate recruitment while retaining catalytic capability of the enzyme. Thus, readouts need to be developed to determine the activity state of E3 ligases upon molecular interaction with a bioactive small molecule or prospective glue compound.

[0007] WO 2023 / 235313 A2 discloses a method of identifying a substrate of an E3 ligase, the method comprising: contacting a ubiquitinated substrate of an E3 ligase with a biotin ligase fused to the E3 ligase, wherein the E3 ligase substrate is ubiquitinated with one or more ubiquitin or ubiquitin-like molecules, each of which is fused to a biotin ligase peptide substrate comprising a biotinylation site of the biotin ligase, wherein the biotin ligase fused to the E3 ligase biotinylates the tagged ubiquitinated E3 ligase substrate when in proximity to the tag; and identifying the substrate of the E3 ligase by detecting and / or selecting the biotinylated ubiquitinated E3 ligase substrate. Pulldown assays and proximity-based detection assays respective luminescent assays may be used. Further, mutated E3 ubiquitin ligases are foreseen.

[0008] WO 2022 / 256623 Al discloses a system for detecting modulator-dependent proximity-based interactions between an E3 ligase and a target protein, the system comprising: a) cell(s) expressing one or more fusion proteins, each fusion protein comprising an E3 ligase substrate receptor and a proximity labeling enzyme; and b) an E3 ligase binding modulator.

[0009] WO 2020 / 079687 discloses a method relying on a bacterial cell system comprising a first and a second polypeptide, wherein the bacterial cell outputs a detectable or selectable signal which correlates with the non-direct binding of the first polypeptide to the second polypeptide. Said method makes use of recruitment of ubiquitin transfer and recruitment of ubiquitin E3 ligase. CN115786373A discloses a recombinant nucleic acid molecule, wherein the comprised sequences include a linker sequence encoding for a cleavable linker, so that it is ensured that the recombinant polypeptides separated from each other in the cell and are suitable for an intermolecular protein-fragment complementation assay between a target protein and a polypeptide having ubiquitin ligase activity.

[0010] Due to the increasing importance of ubiquitin ligases, there is and remains a need to provide biosensors that differentiate in their properties in order to study target engagement through recordings of small molecule or activity controlled conformational profiles of ubiquitin ligases upon binding of PROTACs, their E3 interacting small molecule-based war-heads and E3 ligase inhibitors as well as under different regulatory influences.

[0011] SHORT DESCRIPTION OF THE INVENTION

[0012] The present invention provides a reporter for an intramolecular protein-fragment complementation assay, wherein the reporter is a fused protein comprising a first fragment, a second fragment and an E3 ligase sequence section, wherein the first fragment and the second fragment are derived from different sections of the same (luciferase) split protein, and wherein the E3 ligase sequence section intervenes between the first fragment and the second fragment, and the E3 ligase sequence section is derived from an E3 ligase reference sequence.

[0013] The inventors found that the intramolecular E3 ligase reporters based on different E3 ligase reference sequences were suitable to provide a readout signal (measured as change in relative luminescence units (RLU) during conditions suitable for detecting the luciferase signal). Furthermore, the readout signal was found to be sensitive to endogenous and exogeneous conditions known to modulate activity of the respective E3 ligase. Hence, the invention provides a reporter for monitoring the intramolecular conformational changes and for studying engagement of candidate molecules with the E3 ligase as target (target engagement).

[0014] The reporter can detect subtle changes in the E3 ligase protein conformation dynamics in living cells. It can identify even small conformational alterations that may be missed by other techniques. Further, it can be easily adapted to high throughput screening platforms, enabling the analysis of large drug candidate libraries. This scalability allows for efficient screening and identification of compounds that modulate E3 ligase conformation dynamics, thus representing a target engagement setup for drug discovery and development efforts.

[0015] In other aspects, the invention relates to a polynucleotide encoding for a reporter according to the invention and a cell comprising said polynucleotide and expressing a reporter according to the invention.

[0016] In another aspect, the invention relates to a method for measuring an intramolecular interaction within an E3 ligase reporter in a protein-fragment complementation assay comprising the steps of a) providing a reporter according to the invention and b) providing conditions suitable for detecting a signal from the split protein, wherein said signal indicates assembling of the first fragment and the second fragment upon an intramolecular interaction within the reporter.

[0017] Finally, the invention relates to a method for measuring an effect of a candidate compound on the intramolecular interaction within an E3 ligase reporter, wherein the method for measuring an intramolecular interaction within an E3 ligase reporter is conducted in presence of the candidate compound and the effect of the candidate compound on the interaction is determined by comparing the signal as detected in presence of the candidate compound versus the signal in absence of the candidate compound.

[0018] DETAILED DESCRIPTION OF THE INVENTION

[0019] The reporter according to the invention is a fused protein construct which enables an intramolecular protein-fragment complementation assay (PCA). The engineered construct may be encoded by a polynucleotide and expressed in transient as well as stable cell lines, i.e. it is a biosensor.

[0020] Thus, the fused reporter enables conducting a method for quantifying E3 ligase conformational changes in a cell-based bioluminescence assay. The general concept of a PCA is for example described in EP 0 966 685 Bl. According to the PCA strategy a molecular interaction, i.e. the special contact between two proteins can be investigated by fusing the potentially interacting partners to different sections of a so-called split protein. In literature, the split protein, from which protein the fragments are derived, may also be referred to as reporter protein. A reporting signal results from complementation of the fragments in case the contact of the interacting partners is established. If the interaction between the two fragments is established a functional unit is achieved. For example, the fragments may be luciferase fragments, and the assembled functional luciferase enzyme produces a bioluminescence signal upon oxidation of a substrate.

[0021] In the present invention the reporter protein is a reporter for an intramolecular interaction. The concept of an intramolecular PCA system has been used before for assaying conformational changes, e.g. US 8,178,654 B2 describes an estrogen receptor system; in US 11,237,173 the concept is applied on kinases. In such intramolecular PCA systems, both PCA fragments are part of one reporter construct. The intramolecular reporter is a single protein that includes two fragments of a split protein, for example a first and a second fragment of a luciferase protein (luciferase split protein). A readout signal measured in relative luminescence units (RLU) can be detected when the conformation of the reporter allows the two fragments F[l] and F[2] of the reporter arrange to form a functional unit. A change in the RLU signal indicates a conformational change of the reporter protein.

[0022] The reporter according to the present invention is an E3 ligase reporter as it comprises an E3 ligase sequence section derived from an E3 ligase reference sequence. Preferably, the E3 ligase sequence section comprises a sequence with E3 ligase activity (i.e. is capable of catalytically and / or specifically contributing to transfer of ubiquitin to a target protein). Accordingly, in a preferred embodiment, the E3 ligase sequence section comprises at least a substrate recognition site of the E3 ligase reference sequence (i.e. a section or domain considered responsible for substrate binding and recognition).

[0023] The term “E3 ligase reference sequence” refers to a protein sequence as present in and originating from a living being and is characterized by an E3 ligase activity (i.e. is capable of catalytically and / or specifically contributing to transfer of ubiquitin to a target protein). As originating from a living being the term refers to the native full-length protein without limitation to a specific isoform and includes the wild type as well as patient variation sequences. Preferably, the E3 ligase reference sequence originates from a vertebrate, preferably a mammal, more preferably a human. Accordingly, the E3 ligase reference sequence may be selected from a vertebrate, preferably a mammal, and more preferably a human E3 ligase reference sequence.

[0024] Human E3 ligase reference sequences are of special interest for drug development. However, the inventive concept is not limited to human E3 ligase reference sequence and E3 ligases from other organisms may be of interest e.g. in animal models or for veterinary applications.

[0025] In a preferred embodiment, the E3 ligase reference sequence is an amino acid sequence of a full-length, human protein with E3 ligase activity, preferably a protein identified as autonomous ubiquitin E3 ligase or substrate receptor of multi-subunit ubiquitin E3 ligases. Preferably, the E3 ligase reference sequence is selected from the group consisting of (human) autonomous ubiquitin ligases and (human) substrate receptors of a multi-subunit ubiquitin ligase. In exemplary embodiments, the E3 ligase reference sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 27. These sequences represent various different human families of E3 ligases.

[0026] Figure 1 shows phylogenetic trees of known human proteins identified and annotated as substrate receptors of multi-subunit ubiquitin ligases (Fig. 1A) and autonomous ubiquitin ligases (Fig. IB). Protein families of multi-subunit ubiquitin ligases are annotated as BTB (BR-C, ttk and bab domain), APC activators (APC act.), Von Hippel-Lindau (VHL), suppressor of cytokine signaling (SOCS), F-box / WD repeat-containing (FBXW), F-box (FBXO), F-box / Leucine-rich repeats (FBXL), DDB1 and CUL4 associated factors (DCAF). Protein families of autonomous ubiquitin ligases are annotated as Mouse double minute homologs (MDM), Homologous to the E6-AP Carboxyl Terminus (ELECT), Deltex (DTX), Baculovirus IAP Repeat (BIR), ubiquitin-protein ligase E3 component n-recognin (UBR), Membrane-associated RING-CH (MARCH), Mex-3 homolog (MEX), Plant U-box homologs (UBOX), Tripartite motif (TRIM), TNF receptor associated factors (TRAF), or RING finger (RNF). Others are categorized as miscellaneous (Misc). The exemplary E3 ligase reference sequences investigated herein cover e.g. autonomous ubiquitin ligases from the family referred to as RNF (RING finger) or RBR (RING between RING (RBR) family, e g. PARKIN (SEQ ID NO: 1), the family referred to as Mouse double minute homologs, e.g. MDM2 (SEQ ID NO: 2), and the family referred to as BIR (Baculovirus IAP Repeat) domain-containing ubiquitin ligases, e.g. BIRC2 or cIAPl (SEQ ID NO: 27). On the other hand, the exemplarily investigated Cereblon, short CRBN (SEQ ID NO: 3), is a substrate receptor of a multi-subunit ubiquitin ligase, in particular a Cullin-RING E3 Ubiquitin Ligase (CRL).

[0027] The skilled person will understand that the term “E3 ligase reference sequence” includes all naturally occurring isoforms as well as patient variations thereof, i.e. it may be a canonical (wildtype) human E3 ligase reference sequence or a patient variation thereof.

[0028] The term “patient variation” is used to cover any sequence variation including mutation that is detected in living individuals, particular human individuals, i.e. patients. In a preferred embodiment, a patient variation of the reference sequence differs from the wildtype sequence by a disease associated mutation. E3 ligase mutations are associated with various diseases including cancer, immunological disorders, and neurological diseases.

[0029] For example, mutations in the E3 ligases PARK2 have been identified in highly prevalent in Parkinson' s disease, but specific mutations in other E3 ligases are associated with rare neurological disease [George et al., 2018], In the context of targeted protein degradation, patient mutations in the cereblon gene are associated with acquired resistance following tumor therapy [Hanzl et al., 2023]; [Kortum et al., 2016],

[0030] The wording that the E3 ligase sequence section is “derived from the E3 ligase reference sequence” implies a relation between the amino acid sequence of the reporter representing the E3 ligase sequence section and the amino acid sequence of or encoded by said E3 ligase reference sequence. Particularly, the relation is that the amino acid sequence section of the reporter representing the E3 ligase sequence section and the amino acid sequence of said E3 ligase reference sequence are identical or similar to each other over the full length of the E3 ligase reference sequence or over a part of the E3 ligase reference sequence, wherein, typically, said part of the E3 ligase reference sequence comprises a domain considered responsible for substrate recognition. In one embodiment, the E3 ligase sequence section comprises or consists of an amino acid sequence with a sequence identity of at least 95 % to the full-length of the E3 ligase reference sequence or to a part of the E3 ligase reference sequence.

[0031] It is expected that the invention allows a certain sequence variability regarding the sequences comprised in the reporter constructs. In other words, up to 5% of the sequence of the E3 ligase sequence section may differ from the respective (part of the) E3 ligase reference sequence.

[0032] Accordingly, the E3 ligase sequence section may comprise or consist of an amino acid sequence with a sequence identity of 95%, preferably 98%, and more preferably 100%, to the amino acid sequence of the (part of the) E3 ligase reference sequence.

[0033] In a preferred embodiment, the E3 ligase sequence section is derived from the E3 ligase reference sequence by way of truncation of the full-length E3 ligase reference sequence.

[0034] In this preferred embodiment, the E3 ligase sequence section and the amino acid sequence of said E3 ligase reference sequence are similar or identical to each other, preferably identical, over a part of the E3 ligase reference sequence. However, the E3 ligase sequence section does not comprise the full-length amino acid sequence of said E3 ligase reference sequence but only a part of it. The E3 ligase sequence section consists of or comprises a part of the E3 ligase reference sequence, but not the full-length E3 ligase reference sequence as another part is omitted in the reporter construct, i.e. truncated.

[0035] The inventors studied different E3 ligase constructs for an intramolecular protein-fragment complementation assay based on the different human E3 ligase reference sequences and surprisingly found that a sequence truncation, wherein the reference sequence was not incorporated over its full-length in the receptor gave access to reports with a remarkably higher RLU signal. The higher readout signal represents an improvement of the truncated variant reporter in comparison to the full-length reporter because it improves sensitivity of the reporter according to the invention. Further, the inventors found that truncation of the E3 ligase sequence section with respect to the corresponding full-length reference sequence can result in reporters exhibiting also a higher sensitivity towards changes in conditions known to influence the respective E3 ligase conformation. Thus, the truncated E3 ligase derived conformation reporter is a suitable cell-based monitor for measuring target engagement and regulatory protein activities.

[0036] The examples as provided herein show evidence for several E3 ligase reference sequences that truncation of the ligases enables the recordings of conformational arrangements and hence positively influence the signals for intramolecular protein-fragment complementation. On the other hand, the truncations were not destructive to the applicability of the reporter as it was found to be sensitive towards the change in known modulatory conditions, including enzyme- mediated activations (i.e. by phosphorylation of the E3 ligase) and interactions with a collection of small molecules underlying structural integrity of the used E3 ligase reporter hybrid protein.

[0037] Suitable truncations can be derived from an E3 ligase reference sequence for example based on the annotation of the full-length E3 ligase reference sequence indicating specific domains, N- and C-terminal disordered regions or structural motifs. Functional data on mode of action of E3 ligases assist in defining the respective target engagement reporter derived from the E3 ligase sequence. In addition to sequence-based information, structural information on the E3 ligase reference sequence can be considered when available. The removal of domains that sterically hinder access to an interaction site or the special approximation of the terminally added split proteins can guide truncations.

[0038] For example, with respect to the full-length PARKIN (SEQ ID NO: 1), a reference sequence with 465 residues, a C-terminal truncation of residues 378 to 465 was investigated based on truncating the sections encoding for REP (repressor element of Parkin) and RING2 (really interesting-new-gene 2 domain). Typically, appropriate selections can be made by the skilled person with a preference for the removal of terminal domains or terminal sections without functional annotation, and preference of maintaining a native sequence stretch and / or the domains considered responsible for substrate recognition. Exemplarily, RING domains as redundant features in the autonomous E3 ligases, especially if in terminal position of the domain organization, can be considered for truncation. Additionally, it is exemplarily referred to autoinhibitory elements for truncation. Autoinhibitory elements or cis-regulatory elements are involved in controlling E3 ligase activities and described for E3 ligases of the RBR and ELECT family [Sluimer et al., 2018]; [Spratt et al., 2014], For some examples, it is evident, while for others the skilled person can predict that the N- and C-terminal (autoinhibitory) elements sterically hinder that the reporter fragments come together as required for the luciferase activity.

[0039] In the exemplary studies on the CRBN reference sequence (SEQ ID NO: 3), respectively, truncations from the N-terminal and C-terminal terminus were investigated by the inventors. A C-terminal truncation (e.g. residues 426-442) resulted in a reporter with increased sensitivity towards the substrate lenalidomide in comparison with a N-terminal truncation (e.g. residues 1-40, or 1-48). The removed amino acid stretches represent according to alpha fold predictions of non-structured or partially non-structured regions.

[0040] Thus, in a preferred embodiment, the truncation of the E3 ligase reference sequence may be N-terminally or C-terminally.

[0041] Preferably, also when derived by truncation, the E3 ligase sequence section has a high similarity or identity to the amino acid sequence of a part of the E3 ligase reference sequence, i.e. said part remaining after truncation. Hence, in one embodiment, the E3 ligase sequence section is derived from the E3 ligase reference sequence by truncation of the full-length E3 ligase reference sequence, and the E3 ligase sequence section comprises or consists of an amino acid sequence with a sequence identity of at least 95 %, preferably 98%, more preferably 100%, to the amino acid sequence of the remaining (consecutive) part of the E3 ligase reference sequence.

[0042] Advantageously, said E3 ligase sequence section has a high sequence similarity or identity to a remaining and consecutive part of the E3 ligase reference sequence, said part preferably including a biologically relevant section of the E3 ligase reference sequence, more preferably a part comprising a substrate recognition section. In other words, it is preferred that the truncation of the E3 ligase reference sequence is not modified in a way to cut out a domain identified as substrate interaction site. In one embodiment, the E3 ligase sequence section comprises or consists of an amino acid sequence with a sequence identity of at least 95 % to a consecutive part of at least 50, 60, 70, 75, 80, or 85%, preferably 75%, in length of the full-length E3 ligase reference sequence. In this embodiment, the identity or similarity with a (remaining consecutive) part of at least 75 or 70% in length of the full-length E3 ligase reference sequence, limits the length of the section being omitted by the truncation to at most 25 or 30 % of the full-length of the E3 ligase reference sequence.

[0043] Preferably, the truncation omits a section with a length of at most 30, 25, 20, 15, 10 or 5% of the full-length E3 ligase reference sequence, wherein said length may be truncated from the C-terminal side, the N-terminal side, or the sum of truncations from both sides of the sequence. More preferably, the truncation omits a section with about 5 to 25% of the full-length E3 ligase reference sequence from the C-terminal side only or the truncation omits a section with about 5 to 30% of the full-length E3 ligase reference sequence from the N-terminal side only.

[0044] The term “fragment” (used in the present application in the context of the first and second fragment of a split protein) refers to a sequence section derived from a protein suitable to give an appropriate signal for readout. The two fragments comprise sequences from different sections of a split protein. To obtain the fragments the native sequence of a protein that gives a signal for an assay readout is split up. Various proteins can be split into two parts and reconstitute non-covalently. Known split proteins for deriving fragments suitable in PCA are for example P-lactamase, dihydrofolate reductase (DHFR), focal adhesion kinase (FAK), Gal4, GFP (split-GFP), e.g. EGFP (enhanced green fluorescent protein) and IFP (increased fluorescent protein), horseradish peroxidase, infrared fluorescent protein IFP1.4, P-galactosidase (LacZ), luciferase, tobacco etch virus protease (TEV), and ubiquitin. Depending on the split protein, the readout of a signal may be colorimetric or fluorometric. Often the reconstituted split protein is an enzyme catalyzing formation of a detectable product when an appropriate substrate is provided. Alternatively, the complementation might be detected with a labeled ligand binding (e.g. Fluorescein-conjugated methotrexate fMTX as ligand for DHFR). In case of a fluorescent split protein (e.g. split GFP), the reconstituted split protein itself is detectable. This variant is also referred to as bimolecular fluorescence complementation. Alternative to an optical readout, the fragment complementation may be detected by clonal selection, when the complemented split protein provides an essential function for survival of the cells expressing the reporter. This method may be for example applied for a DHFR-based complementation assay in DHFR deficient cells. These cells can only grow on a nucleotide free medium, when the conditions for complementation of the DHFR-PCA fragments are met [Remy, et al., 1999], An overview of PCA techniques and potential split proteins is for example given in the review by Michnick et al. 2007.

[0045] In a reporter according to the invention the first fragment and the second fragment preferably are derived from a luciferase protein. Accordingly, in one embodiment, the reporter according to the invention is a fused protein comprising a first luciferase fragment, a second luciferase fragment and an E3 ligase sequence section, wherein the first and the second luciferase fragments are derived from different sections of the same luciferase, and wherein the E3 ligase sequence section intervenes between the first fragment and the second fragment.

[0046] The term “luciferase fragment” refers to a protein fragment, wherein the sequence of this fragment is derived from a section of full-length luciferase protein. The first and the second luciferase fragments are derived from two different sections of the same full-length luciferase protein. Depending on the fragmentation point, their amino acid sequence may be of different size / length. The Renilla luciferase fragments, i.e. derived from a Renilla luciferase ( / due) sequence, turned out to be valuable reporter protein fragments. In addition to / ue other luciferases may be used to generate a reporter according to the invention. Any luciferase based PCA reporter enzyme might be applicable to generate a reporter according to the invention. Preferably the luciferase fragments are derived from a luciferase selected out of the group consisting of Renilla luciferase, Gaussia luciferase, firefly luciferase, and artificial systems such as NanoLuc, NanoBit, ReBiL (recombinase enhanced bimolecular luciferase). The optimization of / due fragmentation point for another PCA assay has been described before. It is preferred that the fragmentation of the Renilla luciferase sequence applies after residue 110 [Stefan et al., 2007], The fragments are derived from the N-terminal residues 3 to 110 or the C-terminal residues 111 to 311 of the sequence of native luciferase from Renilla reniformis. respectively. SEQ ID NO: 4 comprises 109 residues beginning with an additional alanine residue located N-terminally and SEQ ID NO: 5 comprises a 201 residues sequence from the C-terminal part. The Gaussia luciferase fragments (SEQ ID NO: 6 and SEQ ID NO: 7) are derived from the very small Gaussia luciferase (Glue) originally secreted by the copepod Gaussia princeps. The fragmentation and application of Glue in PCA was described before [Remy et al., 2006],

[0047] In a preferred embodiment, the reporter construct is characterized in that the luciferase fragments are derived from Renilla luciferase or Gaussia luciferase. More preferably, the first luciferase fragment has a sequence identity of at least 95 %, preferably at least 98 %. to SEQ ID NO: 4 or 6 and the second luciferase fragment has a sequence identity of at least 95 %, preferably at least 98 %, to SEQ ID NO: 5 or 7. Mutations in the native sequence of / due may be tolerated or even beneficial for bioluminescence activity of the reassembling fragments. Thus, 95 % sequence identities with the native ue or Glue sequence are sufficient for the fragments.

[0048] Generally, the location of the first fragment and the second fragment within the reporter is open. Two alternative variants may be conceived: for the arrangement of the fragment of the N-terminal part may be located i) N-terminally to the E3 ligase sequence section or ii) C- terminally to the E3 ligase sequence section, with the C-terminal fragment being located at the other side, respectively. However, it is preferred that the first luciferase fragment having a sequence identity of at least 95 % to SEQ ID NO: 4 or 6 is located N-terminally to the E3 ligase sequence section and the second luciferase fragment having a sequence identity of at least 95 % to SEQ ID NO: 5 or 7 is coupled C-terminally to the E3 ligase sequence section. More generally, it may be preferred that the first fragment is derived from an N-terminal section of a split protein and within the reporter said first fragment is located N-terminally to the E3 ligase sequence section and that the second fragment is derived from a C-terminal section of a split protein and within the reporter said second fragment is located C-terminally to the E3 ligase sequence section.

[0049] Typically, the reporter according to the invention is constructed to comprise an N-terminal sequence section comprising a fragment, an E3 ligase sequence section and a C-terminal section comprising another fragment. Additionally, the reporter may preferably comprise one or two linker sequence(s) intervening between the N-terminal fragment and the E3 ligase sequence section and / or between the E3 ligase sequence section and the C-terminal fragment. Suitable linker sequences typically provide an inert linkage between the polypeptide sections without any further biological functional activity. Preferably, they provide structural flexibility, while ensuring the chemical integrity and stability of the reporter, in particular stability with respect to targeted cleavage.

[0050] In one embodiment, further elements may be included, i.e. fused, in the reporter, preferably such elements are located terminally to the arrangement of the E3 ligase section and the two fragments and preferably such elements are included together with an accompanying linker. For example, such further elements could be included to optimize stability or analytical detectability.

[0051] In one embodiment, the reporter according to the invention comprises or essentially consists of the first fragment, the E3 ligase sequence section, the second fragment, and intervening (non-functional) linker sequences. In one embodiment, the reporter according to the invention essentially consists of the first fragment, the E3 ligase sequence section, the second fragment, and two non-functional linker sequences

[0052] Preferably, two linkers enclose the E3 ligase sequence section; e.g. the reporter comprises two interjacent linkers rich in glycine and serine residues. Preferably linker sequence(s) consist(s) of small and flexible residues with no or small side chains such as glycine and / or serine residues or consist(s) of a sequence of 12 residues of glycine and / or serine. Optionally, the glycine rich section can be flanked with one to three additional residues on the N-terminal side, the C-terminal side or both. Linker sequences may consist of for example 5 to 50, 5 to 20, 8 to 16 or 10 to 14 residues. Exemplary linker sequences are disclosed by SEQ ID NO: 8 to 10.

[0053] In a preferred embodiment of the invention, the linker(s) is / are glycine rich linker(s), preferably the linker(s) has / have a sequence according to any one of SEQ ID NO: 8 to 10.

[0054] The term “polynucleotide” is to be understood synonymous to higher oligonucleotide and denotes nucleic acid single-stranded and double-stranded polymers of nucleotide monomers, including 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA). The person skilled in the art may derive the respective RNA or DNA sequence easily from the protein reporter sequence. The general approach for generating a polynucleotide with a luciferase fragment for a PCA assay was described previously [Stefan et al., 2007], In one embodiment the polynucleotide may be a plasmid comprising a DNA sequence section encoding for the reporter. It may be preferred that the plasmid further contains suitable sequence section(s) for expression in eukaryote cells and / or for selection of cells. Such a plasmid is useful as vector for generating a cell expressing the reporter according to the invention.

[0055] Moreover, the invention provides a cell comprising a polynucleotide according to the invention, said cell expressing a reporter according to the invention.

[0056] Thus, a cell according to the invention is capable of expressing the engineered reporter according to the invention. Such a cell or cell line according to the invention is useful for studying and quantifying the influence of mutations (in particular patient variants) and exogenous factors such as drugs on E3 ligases and the intramolecular conformation of the E3 ligase protein. The polynucleotides may be transfected to any modified cancer cell line. In one embodiment the cell line may be a tumor cell line. An exemplary procedure for obtaining a cell according to the invention is given in the examples. Exemplarily, the cell according to the invention may be derived from an established cell line such as a cell line selected out of the group consisting of HEK293, SW480 and U2OS.

[0057] The invention also relates to a method for measuring an intramolecular interaction within the reporter and studying the effect of a candidate molecule’s binding properties.

[0058] The method includes the essential steps for a PCA assay, which allow to detect the conformation / activities of a reporter with a specific E3 ligase sequence section. Thus, the method can be used to study the influence of mutations and external factors (such as drug interactions) on the protein’s conformation / activity. Preferably, the method is performed as a cell-based method, wherein the reporter is expressed in an engineered cell. Suitable cells have been described before. While a cell-based assay may be preferred, the method can also be conducted in an embodiment without cells, e.g. the reporter is provided in an isolated form. The reporter might be generated in vitro, excreted from cells or obtained by breaking up cells and using the lysate.

[0059] Suitable conditions for step b) depend on the fragments and the split protein from which the fragments are derived. Detecting a signal may also refer to detecting viability of cells expressing the reporter under specific conditions (i.e. clonal selection). Preferably, the split protein itself or its catalytic activity is detectable with a non-invasive readout technique in a cellular context, e.g. colorimetric or fluorometric. In some embodiments it may be necessary to provide a substrate of the split protein to detect a signal associated with reassembly of the split protein via its catalytic activity.

[0060] In a preferred embodiment, the fragments in the reporter are derived from a luciferase and step b) includes providing a bioluminescence substrate, preferably a luciferase substrate, and detecting bioluminescence, wherein a bioluminescence signal indicates that the luciferase fragments assemble to exhibit a luciferase activity. Thus, the bioluminescence signal depends on the intramolecular interaction within the reporter.

[0061] The term “luciferase substrate” refers to so called luciferins, which are compounds that are oxidized by an active luciferase to form a light emitting molecule. The luciferase substrate provided in the method (step b) may be for example selected out of the chemical group of coelenterazine compounds, which are also referred to as CTZ or CLZN. Suitable examples may be benzylcoelenterazine (also known as coelenterazine h, 2,8-dibenzyl-6-(4- hydroxyphenyl)imidazo[l,2-a]pyrazin-3(7H)-one, CAS: 50909-86-9). This substrate of Renilla luciferase ( / ue) may be preferred in combination with constructs comprising SEQ ID NO: 4 and 5. Alternatively, native coelenterazine may be used (6-(4-hydroxyphenyl)-2-[(4- hydroxyphenyl)methyl]-8-(phenylmethyl)-7H-imidazo[3,2-a] pyrazin-3-one, CAS: 55779- 48-1). Coelenterazine is a substrate for a Gaussia luciferase (Glue) and may be preferred in combination with constructs comprising SEQ ID NO: 6 and 7. Other luciferins from the coelenterazine class useful according to the invention include e.g. Coelenterazine 400a (Bisdeoxy coelenterazine, 2,8-dibenzyl-6-phenyl-imidazo[l,2A]pyrazin-3-(7H)-l, CAS 70217-82-2), e-Coelenterazine (Coelenterazine-E, Benz[f]imidazol[l,2-a]quinoxalin-3(6H)- one,5,l l-dihydro-8-hydroxy-2-[(4-hydroxyphenyl-methyl]-12-(phenylmethyl), CAS: 114496-02-5), Coelenterazine-Fluoride (Coelenterazine F, 8-benzyl-2-(4-fluorobenzyl)-6-(4- hydroxyphenyl)imidazo[l,2- a]pyrazin-3(7H)-one, CAS: 123437-16-1), e-Coelenterazine-F (Benz[f]imidazol[l,2-a]quinoxalin-3(6H)-one,5,l l-dihydro-8-hydroxy-2-[(4-fluorophenyl- methyl]-12-(phenylmethyl)), v-Coelenterazine (Coelenterazine-v, 16-benzyl-5-hydroxy-13- [(4-hydroxyphenyl)m ethyl]- 11 , 14, 17-triazatetracyclo[8.7.0.0A{2,7} ,0A{ 11,15 }]heptadeca- l(10),2(7),3,5,8,13,15-heptaen-12-one), Coelenterazine hep (2-benzyl-8- (cyclopentylmethyl)-6-(4-hydroxyphenyl)imidazo[l,2-a]pyrazin-3(7H)-one CAS: 123437- 32-1), Coelenterazine cp (8-(cyclopentylmethyl)-2-(4-hydroxybenzyl)-6-(4- hydroxyphenyl)imidazo[l,2-a]pyrazin-3(7H)-one, CAS: 123437-25-2), Coelenterazine fcp (8-(cyclopentylmethyl)-2-(4-fluorobenzyl)-6-(4-hydroxyphenyl)imidazo[l,2-a]pyrazin- 3(7H)-one CAS: 123437-33-2), Coelenterazine ip (8-(isopropylmethyl)-2-(4-hydroxybenzyl)- 6-(4-hydroxyphenyl)imidazo[l,2-a]pyrazin-3(7H)-one). These compounds may be suitable in a method according to the invention as long as they are a substrate of the intact luciferase formed by the fragments. A person skilled in the art can easily verify a substrate as suitable with the respective full-length luciferase.

[0062] The method is also applicable to study the effect of endogenous or exogenous factors on the intramolecular interaction of the E3 ligase as read-out. For example, the co-expression of PINK1, a kinase known to be involved in regulation of the E3 ligase Parkin, was shown to change the RLU readout signal for the truncated Parkin reporter according to the invention (Example 1).

[0063] The method according to the invention was exemplary applied with CRBN reporters and the known substrate lenalidomide, which is used as for E3 ligase binding in PROTAC development (Example 2) and with a MDM2 reporter and its established inhibitor Nutlin3a (Example 3). The results show a dose-dependent change in RLU signals upon presence of the respective E3 ligase interacting small molecule. This indicates the value of this extendable target engagement reporter for investigating small molecule interactions with E3 ligases, to determine their specificity and efficacy in altering conformation and / or activation states. The concept could also be reproduced with a reporter for the experimental drug target cIAPl (Example 4) showing that the dynamics of the truncated reporter variation were sensitive enough to study time dependent effects of the investigational drugs on this target. Hence, the method may be for example used to investigate a candidate compound. A “candidate compound” could be any (macro)molecule for which it is of interest to study the influence on the E3 ligase conformation / activity.

[0064] Thus, the method may be applied to screen for compounds influencing the conformation / activity. In such situation the candidate compound may be any compound, preferably a small organic molecule. Alternatively, the compound may be a compound which is known to affect the E3 ligase conformation or activity, such as specific E3 ligase targeting agents. For example, a known war-head for PROTAC development.

[0065] In this embodiment the method is conducted in presence of the candidate compound and the effect of the candidate compound on the interaction is determined by comparing the signal as detected in presence of the candidate compound versus the signal in absence of the candidate compound. Preferably, the candidate compound is added before the conditions suitable for detecting a signal are provided (before step b)). For example, the candidate compound is added, e.g. several minutes, several hours, such as 0. 5, to 3 hours, but also days before addition of a substrate of the split protein such as a luciferase substrate in case of a reporter with luciferase fragments.

[0066] FIGURES AND EXAMPLES

[0067] The invention will now be described in more detail by the following figures and non-limiting examples.

[0068] The figures show:

[0069] Figure 1: Phylogenetic trees of human proteins annotated as substrate receptors of multisubunit ubiquitin ligases and autonomous ubiquitin ligases. A) Protein families of the substrate receptors of multi-subunit ubiquitin ligases are annotated as BTB (BR-C, ttk and bab domain), APC activators (APC act.), Von Hippel-Lindau (VHL), suppressor of cytokine signaling (SOCS), F-box / WD repeat-containing (FBXW), F-box (FBXO), F-box / Leucine-rich repeats (FBXL), DDB 1 and CUL4 associated factors (DCAF) for multi-subunit ubiquitin ligases. B) Protein families of autonomous ubiquitin ligases are indicated as Mouse double minute homologs (MDM), Homologous to the E6-AP Carboxyl Terminus (ELECT), Deltex (DTX), Baculovirus IAP Repeat (BIR), ubiquitin-protein ligase E3 component n-recognin (UBR), Membrane-associated RING-CH (MARCH), Mex-3 homolog (MEX), Plant U-box homologs (UBOX), Tripartite motif (TRIM), TNF receptor associated factors (TRAF), or RING finger (RNF). Others are categorized as miscellaneous (Misc).

[0070] Figure 2: Intramolecular PCA reporters for the human E3 ubiquitin-protein ligase parkin (SEQ ID NO: 1). A) Domain organization of the human E3 ubiquitin-protein ligase parkin (PRKN) annotated with respective amino acid positions. B) Basal bioluminescent readout of full-length (f.l.) and truncated parkin reporter. C) Modulation of the truncated parkin reporter readout via co-expression of two PINK1 variants. D) Western blot analysis showing protein levels of the over-expressed parkin reporter.

[0071] Figure 3: Intramolecular PCA reporters for human E3 ubiquitin-protein ligase MDM2. A) Domain organization of the human E3 ubiquitin-protein ligase MDM2 (mdm2, SEQ ID NO: 2) annotated with respective amino acid positions. B) Basal bioluminescent readout of full- length (f.l.) and truncated MDM2 reporters. C) Modulation of the MDM2 reporter readout via treatment with various doses of the bioactive small molecule Nutlin3a. Applied Nutlin3a doses: 0.2pM, 2pM, or 20pM. A BRAF reporter was used as specificity control (gray bars).

[0072] Figure 4: Intramolecular PCA reporters for cereblon (CRBN), a human substrate receptor forming part of a multi-subunit ubiquitin E3 ligase complex. A) Domain organization of the human cereblon protein (CRBN, SEQ ID NO: 3) annotated with respective amino acid positions. B) Basal bioluminescent readout of full-length (f.l.) and truncated CRBN reporters. C) Bioluminescent readout for a full-length RLuc control and various CRBN reporters as modulated by treatment with IpM or 5pM of the Thalidomide-derivate Lenalidomide. D) Bioluminescence readout for the CRBN reporter variants comprising a truncated CRBN sequence and the indicated point mutation without and with pretreatment of reporter expressing HEK293 cells with Lenalidomide.

[0073] Figure 5: Intramolecular PCA reporters for cIAP A) Domain organization of the human cIAP protein (cIAP, SEQ ID NO: 27) annotated with respective amino acid positions. B) Basal bioluminescent readout of full-length (f.l.) and one truncated cIAP reporter upon drug candidate exposure (IpM, HEK293, Ih). C) Time-dependent readout for the two candidates.

[0074] EXAMPLES:

[0075] Material and methods

[0076] Cell Culture and Antibodies: HEK293T (American Type Culture Collection (ATCC), Manassas, VA, USA; CRL11268) cells were grown in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS). For transient transfections of HEK293T 80,000 cells per well were seeded in a 24-well plate and cultivated overnight. Transfection was performed with Transfectin reagent (Biorad, Hercules, California, USA, #1703352) according to the manufacturer’s instructions. At 48 h post transfection bioluminescence measurements were performed as described below. Primary antibodies used were rabbit recombinant n -Renilla luciferase (abeam, Cambridge, UK, #ab 185926), and a flag-antibody for the detection of co-expressed PINK1 variants.

[0077] Expression Constructs for the reporters: Following PCR amplification of the genes encoding the E3 ligase sequence sections were fused N-terminally with F

[0001] - and C-terminally with -F[2] of the / duc-PCA construct (pcDNA3.1 backbone vector) [Mayrhofer et al., 2020; Rock et al., 2019; Stefan et al., 2007], Interjacent linkers were inserted in the 7?luc-PCA construct comprising a 12-amino acid section of glycine and serine residues and potential flanking residues. Fusion of the insert and vector was performed according to the manufacturer’s instructions of NEBridge® Golden Gate Assembly Kit (New England BioLabs, Ipswich, MA, USA, #E1602L). Amino acid substitutions were generated by site- directed mutagenesis to study point mutations of interest.

[0078] Luciferase PCA Analyses in Living Cells: HEK293 cells were grown in DMEM supplemented with 10 % FBS. / ue PCA-based reporters were transiently overexpressed in 24-well plate formats (48 h expression, 80,000 cells seeded). For the luciferase PCA measurements, the growth medium was carefully removed, and the cells were washed with PBS. Cell suspensions were transferred to 96-well plates and subjected to luminescence analysis using the PHERAstar Biomolecules 2021, 11, 518 4 of 13 FSX (BMG labtech, Ortenberg, Germany). Luciferase luminescence signals were integrated for 10 s following addition of the / due substrate benzyl -coelenterazine (Nanolight, #301).

[0079] Example 1: Intramolecular PCA reporters for the human E3 ubiquitin-protein ligase parkin.

[0080] Reporter construction:

[0081] Domain organization of the full-length human E3 ubiquitin-protein ligase parkin (PRKN, NCBI Reference Sequence: NP_004553.2; Uniprot: 060260, SEQ ID NO: 1) is shown in Fig. 2A. Different domains with respective amino acid positions are annotated with the abbreviations as follows UBL: ubiquitin-like domain, UPD: unique parkin domain, RING: really interesting new gene, IBR: in-between-RING, REP: repressor element.

[0082] Besides a full-length parkin reporter with SEQ ID NO: 11, wherein the E3 ligase sequence section consists of the full-length human parkin sequence (residues 1-465), a truncated parkin reporter with SEQ ID NO: 12 was derived from the parkin reference sequence by omitting the REP and RING2 domain. In Fig. 2A, the dashed line indicates the C-terminus of the truncated parkin variant (1-377).

[0083] Effect of sequence truncation on PCA readout:

[0084] Basal bioluminescent readout of full-length (f.l.) and truncated parkin reporters (residues 1- 377) are shown in Fig. 2B. Bars represent the mean fold change of relative light units (RLU), relative to the full-length variant with error bars for standard deviation. Nodes represent the individual results of n=3 independent experiments.

[0085] A substantially higher signal (RLU fold change) was observed for the reporter with a C- terminally truncated Parkin section (SEQ ID NO: 12, comprising only residues 1-377 of SEQ ID NO: 1) in comparison to the reporter, wherein the Parkin section comprised the full-length sequence of the reference protein Parkin (SEQ ID NO: 11, comprising full-length SEQ ID NO: 1). The high signal is the consequence of a more closed conformation of the luciferase PCA fragments. In this conformation, the two terminal fragments F[l]- and -F[2] of the reporter arrange to form a functional unit. In contrast, the full-length reporter results in a more open state.

[0086] Effect of PINK1 co-expression on PCA readout:

[0087] The truncated parkin reporter was co-expressed with two variants of the kinase PINK1, which is known as modulator of parkin activity.

[0088] Results are shown in Fig. 2C and Fig. 2D. In Fig. 2C, bioluminescence readout of the reporters is shown with error bars indicating standard deviation. Additionally, to the C-terminally truncated Parkin reporter, two unrelated PCA reporters were used as specificity controls (gray bars). Asterisks indicate statistical significance of the observed signal changes: * p<0.05; ** p<0.01; via one-sample t-test. The expression of the reporter as well as the co-expression of the PINK1 variant were verified by Western Blot analysis as shown in Fig. 2D. Protein level demonstrate the over-expression of the parkin reporter, using a Renilla luciferase antibody, and the co-expressed PINK1 variants, using a flag antibody. Apparent molecular weight of the detected bands is indicated.

[0089] The readout signal was significantly increased in presence of the co-expression of the indicated amounts of the two PINK1 variants.

[0090] The modulation of the reporter signal by the physiologically relevant upstream factor (PINK1) confirms the sensitivity of the reporter towards endogenous factors.

[0091] Example 2: Intramolecular PCA reporters for the human E3 ubiquitin-protein ligase MDM2.

[0092] Reporter construction:

[0093] Domain organization of the full-length human E3 ubiquitin-protein ligase mdm2 (MDM2, NCBI Reference Sequence: NP_002383; Uniprot: Q00987, SEQ ID NO: 2) is shown in Fig. 3A. Different domains with respective amino acid positions are annotated with the abbreviations as follows p53 BD: p53-binding domain, ZnF: zinc finger, RING: really interesting new gene.

[0094] First, the inventors studied a full-length MDM2 reporter with SEQ ID NO: 13, wherein the E3 ligase sequence section consists of the full-length MDM2 sequence (residues 1-497). Second, two truncated MDM2 reporters with SEQ ID NO: 14 and 15 were derived from the MDM2 reference sequence by omitting the C-terminal RING domain and further C-terminal residues without annotation. In Fig. 3A, the dashed line indicates the C-terminus of the truncated MDM2 sequence sections as incorporated in the reporters according to SEQ ID NO: 14 and 15, respectively.

[0095] Effect of sequence truncation on PCA readout:

[0096] Basal bioluminescent readout of full-length (f.l.) and truncated MDM2 reporters (residues 1- 433 and 1-386) are shown in Fig. 3B. Bars indicate mean fold-change of relative light units (RLU), relative to the full-length variant. Error bars represent standard deviation. Shown is one representative experiment of at least n=3, performed in technical triplicates.

[0097] A substantially higher signal (RLU) was observed for the two reporters with a C-terminally truncated MDM2 section (comprising only residues 1-433 or 1-386 of SEQ ID NO: 2) in comparison to the reporter, wherein the MDM2 section comprised the full-length sequence of the reference protein MDM2 (comprising full-length SEQ ID NO: 2).

[0098] The high signal is the consequence of a more closed conformation of the luciferase PCA fragments. In this conformation, the two terminal fragments F[l]- and -F[2] of the reporter arrange to form a functional unit. In contrast, the full-length reporter displays much weaker bioluminescence signals underlining structural hinderance for reporter complementation.

[0099] Effect of the small molecule Nutlin3a on PCA readout:

[0100] The MDM2 reporters were investigated in presence of the various doses of the small molecule Nutlin3a. Nutlin3a is known as an inhibitor of MDM2-p53 protein-protein interaction. Nutlin3a doses of 0.2 pM, 2pM, or 20pM were applied for Ih before determination of the luciferase activity. Fig. 3C shows the RLU readout, wherein bars represent mean fold change of RLU, relative to untreated (-) condition, with error bars representing SD. The BRAF reporter (KinCon Biolabs GmbH, WO 2018 / 060415 Al) was used as specificity control (gray bars). Shown is one representative experiment of at least n=3, performed in technical triplicates. Asterisks indicate statistical significance of the observed signal changes: * p<0.05; ** p<0.01; *** p<0.001 via two-way ANOVA.

[0101] The full-length MDM2 reporter and the negative control (a PCA reporter comprising a BRAF kinase sequence) showed essentially no changes of bioluminescence signal upon small molecule exposure. However, the RLU fold change significantly increased in the presence of Nutlin3a inhibitor for the reporters with the MDM2 sequence section truncated at residue 433 or 386 of the MDM2 reference sequence. The effect of Nutlin3a increased with the increase in concentration from 0.2 pM to 2 pM, whereas the even higher concentration of 20pM had no additional effect.

[0102] The increase of the RLU signal results from complementation of the fragments of the luciferase split protein and does represents a conformational change, which obviously results from the engagement of the inhibitor with the MDM2 sequence section, which was truncated at the C terminus. The E3 ubiquitin ligase activity of MDM2 is harbored in evolutionarily conserved C-terminal RING finger domain. However, the N-terminal part is responsible for the engagement with the target protein p53 and the Nutlin3a binding.

[0103] Evidently, the C-terminal truncation allows deciphering conformation alterations reported through fragment complementation of the split protein, while the reporter signal is sensitive towards the engagement with an established inhibitor of MDM2. Hence, the reporter is suitable for measuring the intramolecular movements of an E3 ligase to be used as target engagement reporter for investigating small molecule interactions with the respective E3 ligases, to determine their specificity and efficacy through altering conformation states in a cellular system. Example 3: Intramolecular PCA reporters for the human E3 ubiquitin-protein ligase CRBN.

[0104] Reporter construction:

[0105] Domain organization of the full-length human cereblon protein (CRBN, NCBI Reference Sequence: NP_057386; Uniprot: Q96SW2, SEQ ID NO: 3) is shown in Fig. 4A. Different domains with respective amino acid positions are annotated with the abbreviations as follows: disord.: disordered region, CULT: Cereblon domain of Unknown activity, binding cellular Ligands and Thalidomide.

[0106] First, the inventors studied a full-length CRBN reporter with SEQ ID NO: 16, wherein the E3 ligase sequence section consists of the full-length CRBN sequence (residues 1-442 of the CRBN reference sequence with SEQ ID NO: 3).

[0107] Second, various truncated CRBN reporters with SEQ ID NOs: 17 to 21 were derived from the CRBN reference sequence by omitting the N-terminal residues 1-40 and 1-48 as the residues 1-45 are annotated as disordered region, by omitting the C-terminal residues 426 to 442 without annotation or by omitting N- and C-terminal sections. In Fig. 4A, the dashed lines indicate the different N- and C-termini of the truncated CRBN sequence sections as incorporated in the reporters according to SEQ ID NO: 17 to 21.

[0108] Finally, the truncated CRBN reporter with SEQ ID NO: 21 (comprising only residues 1-425 of the CRBN reference sequence with SEQ ID NO: 3) was further modified by introducing individual point mutations. Of the investigated mutations, two were previously identified as patient mutations and associated with resistance [Kortum et al., 2016] and other three point mutations were located at sites previously identified as hotspots [Hanzl et al., 2023] as shown in Table 1 below.

[0109] Table 1: CRBN mutations as investigated by incorporation in a truncated CRBN reporter

[0110] Effect of sequence truncation on PCA readout:

[0111] Basal bioluminescent readout of full-length (f.1.) and various truncated CRBN reporters are shown in Fig. 4B. Bars indicate fold change of relative light units (RLU), relative to the full- length CRBN reporter. Error bars indicate standard deviation, and nodes indicate individual results of n=4 independent experiments. A BRAF kinase reporter (KinCon Biolabs GmbH, WO 2018 / 060415 Al) was used as control (gray bar).

[0112] A lower basal signal (RLU fold change) was observed for the reporters with an N -terminally truncated CBRN section (wherein the first 40 or 18 residues of SEQ ID NO: 3 were omitted) in comparison to the reporter, wherein the CRBN section comprised the full-length sequence of the reference protein (SEQ ID NO: 3 in full length). However, the highest signals were observed for the C-terminally truncated reporter with SEQ ID NO: 21 comprising only residues 1-425 of SEQ ID NO: 3.

[0113] Effect of the PROTAC component lenalidomide on the respective PCA readout:

[0114] The CRBN reporters were investigated in presence of two doses of the small molecule Lenalidomide. Lenalidomide is a derivative of Thalidomide and used as PROTAC-warhead for CRBN targeting and known to interact with the CRBN protein.

[0115] Lenalidomide doses of 1 pM or 5pM were applied for Ih before determination of the luciferase activity. Fig. 4C shows the RLU readout, wherein bars represent fold change of RLU, relative to the untreated condition. A modified full-length Renilla luciferase was used as specificity control (gray bars). Error bars indicate standard deviation, and nodes indicate individual results of n=4 independent experiments. Asterisks indicate statistical significance of the observed signal changes: * p<0.05; ** p<0.01; *** p<0.001 via one-sample t-test.

[0116] The full length CRBN reporter showed a significant but weak change of the readout signal in presence of 1pm or 5pM Lenalidomide. A more pronounced effect in the RLU fold change with the presence of Lenalidomide was seen for the reporters with the CRBN sequence section truncated at residue 425 of the MDM2 reference sequence. The effect of the N-terminal truncation alone was less pronounced.

[0117] Effect of the CRBN mutations:

[0118] The immunomodulatory imide drugs (IMiDs) Thalidomide and Lenalidomide are successfully used in treatment of multiple myeloma. However, drug resistances were previously reported, and the resistance mechanisms include point mutations of the CRBN protein.

[0119] Different mutations described in literature were investigated in the reporter truncated at residue 425 (see Table 1). The impact of Lenalidomide on the bioluminescence readout for the CRBN reporter variants is shown in Fig. 4D, wherein the bar chart shows changes upon treatment with IpM of Lenalidomide for 30min in relative light units (RLU). Bars indicate mean RLU fold change of n=3 independent experiments, error bars indicate standard deviation. The bioluminescence signal of lenalidomide-treated cells (black bars) was normalized to that of DMSO-treated cells (white bars). Asterisks indicate mutations, which were previously identified as patient variations [Kortum et al., 2016],

[0120] The results demonstrate that the investigated mutations suppress the Lenalidomide mediated conformation changes as seen for the truncated reference sequence. Hence, the data demonstrate a sequence specific effect on the reporter signal and indicate that the reporter concept allows to study the small molecule engagement of E3 ligase variations including patient mutations.

[0121] Example 4: Intramolecular PCA reporters for cIAPl.

[0122] Reporter construction:

[0123] Domain organization of the full-length Cellular Inhibitor of Apoptosis Protein 1 (cIAPl, also named BIRC2, NCBI Reference Sequence: NP 001157; Uniprot: Q13490; SEQ ID NO: 27) is shown in Fig. 5A. Different domains with respective amino acid positions are annotated with the abbreviations as follows: BIR: inhibitor of apoptosis domain, also known as IAP repeat, Baculovirus Inhibitor of apoptosis protein Repeat, or BIR, UBA: Ubiquitin-Associated Domain, CARD: card domain, RING: RING: really interesting new gene domain.

[0124] The inventors studied a full-length cIAPl reporter with SEQ ID NO: 28, wherein the E3 ligase sequence section consists of the full-length cIAPl sequence (residues 1-618). Second, a truncated reporter with SEQ ID NO: 29 was derived from the cIAPl reference sequence by omitting the N-terminal BIR1 domain. In Fig. 5 A, the dashed line indicates the N-terminus of the truncated cIAPl sequence sections as incorporated in the reporters according to SEQ ID NO: 29 (residues 173-618 of SEQ ID NO: 27).

[0125] Effect of sequence truncation and various drug candidate molecules on PCA readout:

[0126] Basal bioluminescent readout of full-length (f.l.) and the truncated cIAPl reporter are shown in Fig. 5B. Bars indicate fold change of relative light units (RLU) normalized to the untreated DMSO reporter. Error bars indicate standard deviation, and nodes indicate individual results of independent experiments.

[0127] The full-length cIAPl reporter showed a higher standard variation in all investigated conditions in comparison to the reporter with the truncated cIAPl reporters. This effect is particularly evident when the reports were investigated in presence of the compounds LCL61, CDC-0152, Xevinapant, and Tolinapant. These molecules are investigational new drugs or candidates that are being evaluated to drug therapy (for example of cancer) because they function as inhibitors of the IAP protein family. The reporter expressing HEK293 cells were incubated with the candidate compounds at a concentration of IpM for Ih. The presence of these molecules resulted in a decrease of the RLU fold change signal compared to the untreated control. The decrease was more significant in the truncated version (right panel of Fig. 5B). The sensitivity of the truncated reporter allowed for studying time dependent effects of the two candidate compounds LCL161 and GDC-0152 as shown in Fig. 5C. The slight increase of the luminescence signal over time might indicate a reversible effect of the drug candidate on the intramolecular conformation. Accordingly, the cIAPl reporters represent a further dynamic E3 ligase reporter example, which showed increased conformation dynamics with the truncated version missing one regulatory N-terminal domain (BIR1).

[0128] Although, the data are of preliminary nature, it is evident from the Examples 1 to 4 as shown above that the reporters according to the invention will be of valuable impact for drug development as they allow to monitor and evaluate various factors with influence on the E3 ligase conformation in particular engagement with small molecules in a dose-dependent and sequence specific manner.

[0129] The sequences as identified through the description (SEQ ID No. 1 to SEQ ID No. 29) are disclosed in the sequence listing (generated with the Software WIPO Sequence version 2.3.0 compliant with WIPO Standard ST.26) and form part of the original disclosure.

[0130] REFERENCES

[0131] Bekes, M., Langley, D. R., & Crews, C. M. (2022). PROTAC targeted protein degraders: The past is prologue. Nature Reviews Drug Discovery, 27(3), 181-200. DOI: 10.1038 / s41573-021- 00371-6

[0132] Dale, B., Cheng, M., Park, K.-S., Kaniskan, H. U., Xiong, Y., & Jin, J. (2021). Advancing targeted protein degradation for cancer therapy. Nature Reviews Cancer, 21( 0), 638-654. DOI: 10.1038 / s41568-021-00365-x

[0133] George, A. J., Hoffiz, Y. C., Charles, A. J., Zhu, Y., & Mabb, A. M. (2018). A comprehensive atlas of E3 ubiquitin ligase mutations in neurological disorders. Frontiers in Genetics, 9, 29. DOI: 10.3389 / fgene.2018.00029

[0134] Gooding, S., Ansari-Pour, N., Towfic, F., Ortiz Estevez, M., Chamberlain, P. P., Tsai, K. T., Flynt, E., Hirst, M., Rozelle, D., Dhiman, P., Neri, P., Ramasamy, K., Bahlis, N., Vyas, P., & Thakurta, A. (2021). Multiple cereblon genetic changes are associated with acquired resistance to lenalidomide or pomalidomide in multiple myeloma. Blood, 137(2), 232-237. DOI: 10.1182 / blood.202000708

[0135] Hanzl, A., Casement, R., Imrichova, H., Hughes, S. J., Barone, E., Testa, A., Bauer, S., Wright, J., Brand, M., Ciulli, A., & Winter, G. E. (2023). Functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders. Nature Chemical Biology, 19(3), 323-333. DOI: 10.1038 / s41589-022-01177-2

[0136] Kortum, K. M., Mai, E. K., Hanafiah, N. H., Shi, C. X., Zhu, Y. X., Bruins, L., Barrio, S., Jedlowski, P., Merz, M., Xu, J., Stewart, R. A., Andrulis, M., Jauch, A., Hillengass, J., Goldschmidt, H., Bergsagel, P. L., Braggio, E., Stewart, A. K., & Raab, M. S. (2016). Targeted sequencing of refractory myeloma reveals a high incidence of mutations in CRBN and Ras pathway genes. Blood, 128(9), 1226-1233. DOI: 10.1182 / blood-2016-02-698092

[0137] Mayrhofer, J. E., Enzler, F., Feichtner, A., Rock, R., Fleischmann, J., Raffeiner, A., et al. (2020). Mutation-oriented profiling of autoinhibitory kinase conformations predicts RAF inhibitor efficacies. Proceedings of the National Academy of Sciences of the United States of America, 777(49), 31105-31113. DOI: 10.1073 / pnas.2012150117

[0138] Michnick, S. W ., Ear, P. H., Manderson, E. N., Remy, I., & Stefan, E. (2007). Universal strategies in research and drug discovery based on protein-fragment complementation assays. Nature Reviews Drug Discovery, 7, 569-582. DOI: 10.1038 / nrd2311

[0139] Remy, I., & Michnick, S. W. (2006). A highly sensitive protein-protein interaction assay based on Gaussia luciferase. Nature Methods, 12, 977-979. DOI: 10.1038 / nmeth979

[0140] Rock, R., Mayrhofer, J. E., Torres-Quesada, O., Enzler, F., Raffeiner, A., Raffeiner, P., et al. (2019). BRAF inhibitors promote intermediate BRAF(V600E) conformations and binary interactions with activated RAS. Science Advances, 5(8), eaav8463. DOI:

[0141] 10.1126 / sciadv.aav8463

[0142] Samarasinghe, K. T. G., & Crews, C. M. (2021). Targeted protein degradation: A promise for undruggable proteins. Cell Chemical Biology, 28(7), 934-951. DOI:

[0143] 10.1016 / j.chembiol.2021.04.011

[0144] Sluimer, J., & Distel, B. (2018). Regulating the human ELECT E3 ligases. Cellular and Molecular Life Sciences, 75, 3121-3141. DOI: 10.1007 / s00018-018-2848-2

[0145] Stefan, E., Aquin, S., Berger, N., Landry, C. R., Nyfeler, B., Bouvier, M., & Michnick, S. W. (2007). Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo. Proceedings of the National Academy of Sciences of the United States of America, 104(43), 16916-16921. DOI: 10.1073 / pnas.0704257104 Spratt, D. E., Walden, H., & Shaw, G. S. (2014). RBR E3 ubiquitin ligases: new structures, new insights, new questions. The Biochemical Journal, 458(3), 421-437. DOI: 10.1042 / BJ20140006

[0146] Zhao, L., et al. (2022). Targeted protein degradation: mechanisms, strategies and application. Signal Transduction and Targeted Therapy, 7(1), 113. DOI: 10.1038 / s41392-022-00966-4

[0147] Zheng, N., & Shabek, N. (2017). Ubiquitin Ligases: Structure, Function, and Regulation. Annual Review of Biochemistry, 86, 129-157. DOI: 10.1146 / annurev-biochem-060815- 014922

Claims

CLAIMS1. A reporter for an intramolecular protein-fragment complementation assay, wherein the reporter is a fused protein comprising a first fragment, a second fragment and an E3 ligase sequence section, wherein the first fragment and the second fragment are derived from different sections of the same split protein, and fragments of the split protein are configured for providing a signal indicating an assembly of the first fragment and the second fragment, and wherein the E3 ligase sequence section intervenes between the first fragment and the second fragment, and the E3 ligase sequence section is derived from an E3 ligase reference sequence2. The reporter according to claim 1, wherein the E3 ligase sequence section comprises at least a substrate recognition site of the E3 ligase reference sequence.

3. The reporter according to claim 1 or 2, wherein the E3 ligase reference sequence is an amino acid sequence of a full-length, human protein with E3 ligase activity, preferably a protein identified as autonomous ubiquitin E3 ligase or substrate receptor of multisubunit ubiquitin E3 ligase.

4. The reporter according to any one of claims 1 to 3, wherein the E3 ligase sequence section comprises or consists of an amino acid sequence with a sequence identity of at least 95 %, preferably 100 %, to the full-length of the E3 ligase reference sequence or to a part of the E3 ligase reference sequence.

5. The reporter according to any one of claims 1 to 4, wherein the E3 ligase sequence section comprises or consists of an amino acid sequence with a sequence identity of at least 95 %, preferably 100 %, to a part of the E3 ligase reference sequence, wherein said part comprises a substrate recognition domain of the E3 ligase reference sequence.

6. The reporter according to any one of claims 1 to 5, wherein the E3 ligase sequence section comprises or consists of an amino acid sequence with a sequence identity of atleast 95 % to a consecutive part of at least 50, 60, 70, 75, 80, or 85%, preferably 75%, in length of the full-length E3 ligase reference sequence.

7. The reporter according to any one of claims 1 to 6, wherein the E3 ligase sequence section is derived from the E3 ligase reference sequence by truncation of the full-length E3 ligase reference sequence.

8. The reporter according to claim 7, wherein the E3 ligase reference sequence is truncated N-terminally or C-terminally, preferably C-terminally.

9. The reporter according to claim 7 or 8, wherein the sequence of the E3 ligase sequence section comprises or consists of an amino acid sequence with a sequence identity of at least 95 % to a consecutive part of at least 70% of the E3 ligase reference sequence and is truncated to omit a section with a length of at most 30 % of the E3 ligase reference sequence.

10. The reporter according to any one of claims 1 to 9, wherein the E3 ligase reference sequence is selected from sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 27.

11. The reporter according to any one of claims 1 to 10, wherein the split protein is a luciferase split protein.

12. The reporter according to claim 11, wherein the first fragment and the second fragment are derived from a Renilla luciferase protein.

13. The reporter according to any one of claims 1 to 12, wherein the reporter essentially consists of the first fragment, the second fragment, the E3 ligase sequence section, and intervening linker sequences.

14. A polynucleotide encoding for a reporter according to any one of claims 1 to 13.

15. A cell comprising a polynucleotide according to claim 14 and expressing a reporter according to any one of claims 1 to 13, wherein the cell is established from a cell line preferably selected from the group consisting of HEK293, SW480 and U2OS.

16. A method for measuring an intramolecular interaction within an E3 ligase in a protein fragment complementation assay comprising the steps of a) providing a reporter according to any one of claims 1 to 13 and b) providing conditions suitable for detecting a signal from the split protein, wherein said signal indicates assembling of the first fragment and the second fragment upon an intramolecular interaction within the reporter.

17. A method for measuring an effect of a candidate compound on the intramolecular interaction within an E3 ligase reporter, wherein the method for measuring an intramolecular interaction within an E3 ligase reporter according to claim 16 is conducted in presence of the candidate compound and the effect of the candidate compound on the interaction is determined by comparing the signal as detected in presence of the candidate compound versus the signal in absence of the candidate compound.

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