Conformation reporter for p53 protein family

Abstract

A reporter for a protein-fragment complementation assay, wherein the reporter is a fused protein comprising a first fragment, a second fragment and a p53 protein family sequence section, wherein the p53 protein family sequence section is derived from a p53 protein family reference sequence, wherein the p53 protein family reference sequence is a wildtype sequence or a patient variation and comprising an oligomerization domain, and the p53 protein family sequence section comprises a sequence modification compared to the p53 protein family reference sequence and said sequence modification is located at the oligomerization domain of the p53 protein family reference sequence as well as a polynucleotide, a cell and methods for conducting such an assay.

Description

CONFORMATION REPORTER FOR P53 PROTEIN FAMILYThe invention relates to a p53 protein family reporter for protein-fragment complementation assays, a polynucleotide and a cell for an intramolecular protein-fragment complementation assay as well as methods of conducting such an assay.BACKGROUND OF THE INVENTIONThe protein p53 plays a crucial role in maintaining genomic stability and preventing the development of cancer. It acts as a tumor suppressor protein by regulating the cell cycle and promoting DNA repair. When DNA damage occurs, p53 can initiate cell cycle arrest to allow for repair or induce apoptosis if the damage is irreparable. On the molecular level, p53 proteins act as DNA-binding transcription factors and are activated upon various stressors such as DNA damage, oncogene activation, hypoxia or nutrient deprivation [Borrero et al., 2021; Humpton et al., 2016; Hafner et al., 2019], The monomeric p53 protein (protomer) has no or reduced effect as transcription factor (TF). Four p53 proteins form a tetrameric complex thus engaging the active protein complex conformation as prerequisite for binding to target-DNA sequences [Joerger et al., 2007], Subsequently, the activated p53 complex is involved in DNA damage repair, cell cycle arrest, senescence, apoptosis and the modulation of metabolism [Fallatah et al., 2023; Kastenhuber et al., 2017], Additionally, p53 is involved in a network of non- canonical functions influenced by different environment conditions [Kastenhuber et al., 2017], Overall, p53 regulates hundreds of target genes including genes encoding for p21 and BCL-2 family proteins [Fallatah et al., 2023; Kastenhuber et al., 2017; Guiley et al., 2023], This happens trough p53 protein stabilization and complex formation kinetics in the nucleus, post- translational modifications, and the presence of p53 binding proteins and cofactors.Structurally, the p53 protein consists of decisive domains. The domain organization and functions are discussed in the following with reference to the residue numbers of the exemplary sequence of human p53, which is identified with the UniProt accession number P04637. N-terminal, two transcription activation domains have been identified (TAD1 and 2; residues 1-62) which allow interactions with cofactors and the p53 regulatory protein and ubiquitin ligase MDM2. This stretch is followed by a proline-rich domain (PRD, residues 63- 97). The PRD is important for the binding of p53 to DNA and for protein stabilization. TheDNA-binding domain (DBD; residues 98-292) is the largest domain in p53 and enables DNA binding to target sequences. The C-terminal domain consists of multiple sub-domains, beginning with a nuclear localization signal (NLS, residues 300-323). The oligomerization domain (OD, residues 324-355) promotes the oligomerization of p53 protomers via proteinprotein interactions and base-pair stacking interactions to form a tetramer structure (dimer of dimers) [Tanaka et al., 2018; Joerger et al., 2007], The function of this domain is regulated by post-translational modifications [Joerger et al., 2007; Borrero et al., 2021; Hafner et al., 2019], Additionally, this domain contains the nuclear export signal (NES); with the NLS it regulates the subcellular localization of p53. Lastly, at the C-terminal end of p53 the basic C-terminal domain (BD; residues 363-393) is located. Diverse post-translational modifications at the C- terminus influence p53 stability, localization and tetramerization [Tanaka et al., 2018; Soussi, 2007],The p53 protein family is comprised of three transcription factors: In addition to p53, it includes p63 and p73. p53 is the best characterized p53 family member. The family members p63 and p73 have been referred also as “tumor protein p63” and “tumor protein p73”, respectively. However, they are identified to have more vital roles in cell regulation and differentiation [Dbtsch et al., 2010],Full-length sequences of human p53 family members p63 and p73 are identified with UniProt accession numbers Q9H3D4 and 015350, respectively. The overall domain organization is conserved amongst the p53 family members [Osterburg et al., 2022], The modular structure of the p53 family members gives rise to various isoforms that originate from a combination of alternative promoter usage and alternative splicing [Osterburg et al., 2022], The full-length proteins share the central domain organization represented by TAD, DBD, and OD. Due to the similarity and functional interactions of p53 with p63 and p73, all family members are discussed to have profound effects on tumorigenesis and anticancer drug responses [Wei et al., 2011; Cai et al., 2022],Mutations in the p53 gene significantly impact protein structure dynamics and thus p53’s function. One common type of mutations are missense mutations, where single nucleotide changes result in the substitution of selective amino acids in the p53 protein. Such alterationsdisrupt the protein’s three-dimensional structure, impairing its ability to form multimers and bind to DNA to regulate gene expression effectively. These structural changes in p53 result in the loss of its tumor-suppressing activities, allowing for the uncontrolled growth of cancer cells. In around 40 to 50 % of all human tumors, mutations of p53 have been identified [Fallatah et al., 2023; Joerger et al., 2007], leading to either abolished p53 tumor suppressor function, altered transactivation spectra or thermodynamic alterations. Pathological p53 mutations are found across different tissues with varying frequencies, mainly in colon, lung, stomach and esophagus, pancreas, ovary and uterus [Fallatah et al., 2023; Borrero et al., 2022], The majority of mutations (~80 %) are missense mutations [Chen et al., 2022], in which a single amino acid is substituted and still allowing the expression of mutated p53 in tumor cells. Most commonly those missense mutations array in the DBD, with six “hotspot” amino acid residues: R175, G245, R248, R249, R273, R282 [Muller et al., 2014], Each of them is assumed to contribute in inducing destabilization of p53 folding by affecting its thermostability and amongst others R175H and Y220C are classified as conformational mutations [Joerger et al., 2007]). The second class of mutations is called contact mutations, which cause DNA interaction loss, like the R248Q and R273H missense mutations [Joerger et al., 2007; Fallatah et al., 2023], Tumors with hotspot mutations appear to be more aggressive when compared to p53 wildtype (wt) tumors [Fallatah et al., 2023; Chen et al., 2022; Muller et al., 2014], indicating that these mutations have a beneficial effect for the tumor (i.e. are tumorigenic). For example, R175H and R273H mutations in the DBD promote tumor cell invasion and migration [Muller et al., 2014], Besides missense mutations, some p53 mutations cause truncations, others are splicing mutations [Chen et al., 2022], Truncation mutations result in a shorting of the p53 protein and to a loss of function, thereby promoting tumorigenesis [Chen et al., 2022; Shirole et al., 2017], Further, mutations outside of the DBD have been identified, which seem to destabilize the tetramerization domain by alterations of secondary protein structure features or weakening the hydrophobic core [Joerger et al., 2007], Mutations of p53 also have been linked to Li-Fraumeni syndrome [Srivastava et al., 1990] and might also be linked to neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease and Huntington’s disease [Jacobs et al., 2006],Given the high frequency of p53 mutations in various cancer types, a variety of drug discovery programs have been unleashed for the development of drug candidates targeting p53. Thegoal is to identify bioactive small molecules which stabilize the wildtype related p53 conformations thereby restoring p53 tumor suppressor function [Fallatah et al., 2023], In parallel, drug discovery programs focus on altering p53 related pathways or e.g. interactions with the p53 degradation machinery involving MDM2 [Zhu et al., 2022], Currently, p53 mutant ‘corrector’ compounds are in clinical trials such as APR-246 (eprenetapopt) [Bykov et al., 2002] and NSC319726 [Yu et al., 2012], The need for drugs aimed at restoring p53 function is significant given the prevalence of p53 mutations. Additionally, as personalized medicine gains traction, targeting specific mutations, such as p53 mutations, with tailored therapies will further drive the development of p53 targeting drug candidates. The goal of drug discovery efforts targeting mutated p53 is to identify and develop bioactive small molecules that can restore the normal function of the mutant protein. By restoring the function of mutated p53, these small molecules aim to inhibit cancer cell growth, induce cell cycle arrest, promote DNA repair, and trigger apoptosis in cancer cells. Successful identification and development of such bioactive small molecules can potentially lead to improved cancer therapies specifically tailored for patients with p53 mutations. To analyze p53 function and to investigate the effect of drug candidate molecules to p53 as well as the other p53 family members, different assays are required.A protein folding molecular imaging biosensor for p53 was described earlier [Paulmurugan et al., 2018] and was shown to monitor the effects of drugs that restore mutant p53 structure. The biosensors were constructed as split luciferase complementation molecular biosensor. The concept of an intramolecular protein-fragment complementation assay (PCA) has been used before for assaying conformational changes, e.g. US 8,178,654 B2 describes an estrogen receptor system. WO 2018 / 060415 discloses several full-length kinase activity conformation reporters. In such intramolecular PCA systems, both PCA fragments are part of one construct. The reporter (biosensor) is a single protein that includes two fragments of a split protein, for example a first and a second fragment of a luciferase protein. In the p53 biosensor according to the prior art [Paulmurugan et al., 2018], the two different fragments are located at the terminal parts of the reporter sequence and the protein of interest is located between them. The construct NRLUC-p53-CRLUC comprises a first split-luciferase fragment from Renilla luciferase (NRLUC), the full length p53 protein, and a second split-luciferase fragment from Renilla luciferase (CRLUC). Besides the wildtype, Paulmurugan et al. also disclosedbiosensors with the three mutations Y220C, G245S and R282W in the DBD and cell lines that specifically expressed the biosensor containing one of the four different p53 proteins. The specific advantages of these constructs were the viability of functional p53 proteins expressed from within the biosensor fusion proteins in cells [Paulmurugan et al., 2018],Due to the increasing importance of p53 targeting compounds, there is and remains a need to provide biosensors that differentiate in their properties in order to study activity profiles of p53 patient mutations and directly acting drug (candidate) compounds.SHORT DESCRIPTION OF THE INVENTIONThe present invention provides a reporter for a protein-fragment complementation assay, wherein the reporter is a fused protein comprising a first fragment, a second fragment and a p53 protein family sequence section, wherein the first fragment and the second fragment are derived from different sections of the same luciferase split protein, and wherein the p53 protein family sequence section intervenes between the first fragment and the second fragment, and the p53 sequence section is derived from a p53 protein family reference sequence, wherein the p53 protein family reference sequence is a wildtype sequence or a patient variation thereof and the p53 protein family reference sequence is comprising an oligomerization domain, wherein the p53 protein family sequence section comprises a sequence modification compared to the p53 protein family reference sequence and said sequence modification is located at an oligomerization domain of the p53 protein family reference sequence.The inventors studied different p53 constructs for a protein-fragment complementation assay based on the human p53 sequence and found that a C-terminal sequence truncation gave access to reports with a remarkably higher readout signal (measured as change in relative luminescence units (RLU) during conditions suitable for detecting the luciferase signal; (compare Example, Fig. 2). The higher readout signal represents an improvement of the reporter over the full-length construct based on the human p53 sequence with full-length and unmodified C-terminal oligomerization section as described by Paulmurugan et al., 2018.Although the structural mechanism behind the increase in the readout signal remains unclear, it is evident that either oligomerization or presence of the oligomerization domain affectsconformational arrangements. The inventors assume that p53 complex formation negatively affects the signals for intramolecular protein-fragment complementation. Thus, the inventors set out to generate another monomeric p53 reporter system.In this line, it was confirmed that the effect is not simply related to the impact of reduction in size, because the single point mutation L344P resulted also in a similarly impressive increase in the readout signal (compare Example, Fig. 3). Said point mutation is located in the OD and was previously described to disrupt the p53 oligomerization [Kamada et al., 2011],Truncation of the C-terminal part and the point mutation in the oligomerization domain share that the p53 protein family sequence section of the resulting reporter comprises a sequence modification compared to the p53 protein family reference sequence and said sequence modification is located at an oligomerization domain of the p53 protein family reference sequence. The high signal leads to an improved sensitivity of the reporter according to the invention and offers several advantages. The reporter can detect subtle changes in protomer 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 p53 protein conformation dynamics, aiding in drug discovery and development efforts.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.In another aspect, the invention relates to a method for measuring an intramolecular interaction within a p53 protein family 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.Finally, the invention provides a method for measuring an effect of a candidate compound on the intramolecular interaction within a p53 protein family reporter, wherein the method for measuring an intramolecular interaction within a p53 protein family 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.DETAILED DESCRIPTION OF THE INVENTIONThe reporter according to the present invention comprises a p53 protein family sequence section derived from a p53 protein family reference sequence including wildtype and patient variation sequences. Preferably, p53 protein family reference sequence is selected from the group consisting of SEQ ID No: 1 including patient variations, SEQ ID No: 2, and SEQ ID No: 3.In a preferred embodiment, the p53 protein family reference sequence is the canonical (wildtype) human p53 sequence SEQ ID No: 1 or a patient variation thereof (SEQ ID No: l including patient variations). Exemplary reporters according to the invention, wherein the p53 protein family reference sequence is the wildtype human p53 sequence are identified in SEQ ID No: 12 and 13.The term “patient variation” is used to cover any sequence variation 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 tumorigenic mutation. Preferred patient variations particularly include function interfering missense mutations in p53. Tumorigenic mutations are typically found in residues N-terminal to the oligomerization domain. Sites of tumorigenic mutations as exemplarily indicated in the sequence listing for SEQ ID No: 1 and the respective missense mutations can be identified by the skilled person based on literature, individual patient data and / or databases (for example, COSMIC: the Catalogue Of Somatic Mutations In Cancer, cancer.sanger.ac.uk). Exemplarily, it is referred to the data in Table 1, confirming that sites of frequent tumorigenic mutationinclude the variation at R273, R248, R175, G245, R248, R282, R249, R179, Y220 etc., as indicated as features in SEQ ID No: 1.Table 1 : Summary on frequent p53 sequence patient variations. The summary is prepared based on a COSMIC data set covering missense mutations in human p53. The amino acid positions with cumulated count of >300 (summarizing frequency in the samples for different mutations at the respective variation site) were extracted and are shown together with the number and nature of the covered mutations.Exemplary reporters according to the invention, wherein the p53 protein family reference sequence is a patient variation of the human p53 protein (SEQ ID No: 1) are identified in SEQ ID No: 14 to 29. In the reporter systems according to the invention, for example, the patient variations at R175 or Y220 did show a reduced bioluminescence measured in relative luminescence units (RLU) in comparison to the reporter based on the wildtype reference sequence (compare Examples, Fig. 4 and 5). The tumorigenic mutations lead to a p53 conformation, representing the protomer with diminished TF function. It can be concludedthat this conformational change observed by the comparative assay reflects the conformation effect of the mutation, which physiologically or pathologically leads to the loss in transcriptional activity towards the target genes. Said findings highlight the value of the reporter according to the invention, wherein the p53 protein family reference sequence is a patient variation of SEQ ID No: 1.For example, the reporter according to the invention is valuable to investigate the specific effect of drug candidates. For example, the specificity of a drug candidate can be monitored as signal response is expected to differ for reporters based on different reference sequences reflecting different patient variations such as tumorigenic mutations. A (time and) concentration dependent change of the luminescence signal should be observed for the reporter with the drug-susceptible p53 protein reference sequences only. Similarly, unspecific interactions with other p53 family members can be studied with the reporters according to the invention (compare Examples, Fig. 6 and 7).The present invention enables to identify means which modify the conformation of the monomeric p53 with missense patient mutations towards the wildtype p53 conformations. The readout with structural implication allows to access new information by a comparative assay using both monomeric p53 reporter and full-length reporter. The effect on reconstitution or disruption of protein fragments may differ between both a monomeric reporter and a full- length reporter depending on the mode of action of the candidate drug. This information allows for the quantification of further differences in the conformational dynamics, enabling another level of characterization of protein and / or candidate behavior.The reporter according to the present invention is characterized by a sequence modification compared to the p53 protein family reference sequence. Said sequence modification is located at the oligomerization domain of the p53 protein family reference sequence. In other words, said modification relates to residues within the oligomerization domain (OD). The carboxyterminal OD, also referred to as tetramerization domain (TD), is highly conserved in the p53 family members. The one of p53 is 38 % identical with that of p63 and p73 [Dbtsch et al., 2010], The OD comprise residues 324 to 355, 394 to 443 and 345 to 386 in human p53 (SEQID No: 1 including patient variations), p63 (SEQ ID No: 2) and p73 reference sequences (SEQ ID No: 3), respectively.Preferably, the sequence modification in the oligomerization domain affects the oligomerization properties of the p53 protein family sequence section, specifically reduces (or completely inhibits) the oligomerization properties of the p53 protein family sequence section in comparison to the oligomerization properties of the p53 protein family reference sequence from which p53 protein family reference sequence the p53 protein family sequence section is derived.The skilled person can readily quantify if a sequence modification affects oligomerization properties and / or rely on modifications studied in the literature [Kamada et al., 2011; Gaglia et al., 2013], The reporter according to the present invention can be referred to as “monomeric” or “protomer” to express distinction to a reporter with full oligomerization properties or to a reporter in oligomer state (dimer or tetramer).In one embodiment, the sequence modification compared to the p53 protein family reference sequence is a truncation. In an embodiment, the truncation implies that the residues of the OD of the p53 protein family reference sequence are not compromised in the p53 protein family sequence section of the reporter. In a preferred embodiment, neither the OD of the p53 protein family reference sequence nor any residues C-terminal thereof are compromised in the p53 protein family sequence section of the reporter. In other words, the p53 protein family sequence section is derived from the p53 protein family reference sequence by way of truncation of the full-length p53 protein family reference sequence. The p53 protein family sequence section consists of or comprises a part of the p53 protein family reference sequence, but not the full-length p53 protein family reference sequence because another part is omitted in the reporter construct, i.e. truncated. These embodiments may be referred to as monomeric reporter by truncation.For example, said truncation implies that the residues 324 to 355 of SEQ ID No: 1 including patient variations are not comprised in the p53 sequence section, i.e. these residues are truncated, or that no residues C-terminal from residue 319 of SEQ ID No: 1 including patient variations are comprised in the p53 sequence section of the reporter.In another embodiment, the sequence modification compared to the p53 protein family reference sequence is a point mutation in one of the residues of the OD. These embodiments may be referred to as monomeric reporter by point mutation.Preferably, the point mutation is a helix breaker mutation in a region of the OD annotated as alpha helix structure. The structural key elements of the OD are interacting alpha helixes. Mutations breaking the typical alpha helical fold are for example proline substitutions. For example, the single point mutation concerns one of the residues 324 to 355 of SEQ ID No: 1 including patient variations. Exemplarily and preferably, said point mutation exchanges a leucine residue at position 344 of SEQ ID No: 1 including patient variations to a proline residue (L344P).The term “fragment” as used according to the invention 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 IFP 1.4, 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 deficientcells. 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.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 receptor according to the invention is a fused protein comprising a first luciferase fragment, a second luciferase fragment and a p53 protein family sequence section, wherein the first and the second luciferase fragments are derived from different sections of the same luciferase, and wherein the p53 protein family sequence section intervenes between the first fragment and the second fragment.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 copepodGaussia princeps. The fragmentation and application of Glue in PCA was described before [Remy et al., 2006],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 due or Glue sequence are sufficient for the fragments.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 p53 protein family sequence section or ii) C-terminally to the p53 protein family 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 p53 protein family 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 p53 protein family 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 p53 protein family 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 p53 protein family sequence section.The reporter according to the invention is constructed to comprise an N-terminal sequence section comprising a fragment, a p53 protein family 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 protein kinase sequence section and / or between the p53 protein family sequence section and the C-terminal fragment. Preferably, two linkers enclose the p53 protein family sequence section; e.g. thereporter comprises two inteijacent 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 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 covered by SEQ ID No: 8 to 10.In a preferred embodiment of the invention with one or two linker(s), 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.The term “polynucleotide” is to be understood synonymous to 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.Moreover, the invention provides a cell comprising a polynucleotide according to the invention, said cell expressing a reporter according to the invention.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 the p53 protein family member and the intramolecular conformation of the p53 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 detailed description.For example, 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.The invention also relates to a method for measuring an intramolecular interaction within the reporter and studying the effect of a candidate molecule.The method includes the essential steps for a PCA assay, which allow to detect the conformation / activities of a reporter with a specific p53 protein family 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.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.In a preferred embodiment, the fragments in the reporter are derived from a luciferase and step b) includes providing a bioluminescence 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.The term “luciferase substrate” refers to so called luciferins, which are compounds that can be oxidized by an active luciferase to form a light emitting molecule. The luciferase substrateprovided 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, CAS70217-82-2), 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.The method is also applicable to study the effect of exogenous factors on the intramolecular interaction of the p53 protein family member. Thus, the method may be for example used to investigate a candidate compound. A “candidate compound” could be any (macro)moleculefor which it is of interest to study the influence on the p53 protein family conformation / activity. 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 p53 conformation, such as specific tumor agents.The method according to the invention indicated the specificity of these agents as the bioluminescence of the reporter derived from the wildtype reference sequence or other p53 protein family members should be unaffected. Thus, the assay provides a method for identifying and characterizing specific compounds for a distinct p53 protein family patient variation.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 1 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.FIGURES AND EXAMPLESThe invention will now be described in more detail by the following figures and non-limiting examples.The figures show:Figure 1 : p53 PCA reporters. The schematical domain structure of the analyzed p53 conformation reporters is shown. N-and C-terminally the fragments of the ue fragments F

[0001] - and -F[2] have been attached. In-between the PCA fragments, the domain organization of p53 sequence section and the dimer breaking L344P mutation is displayed. Interjacent linkers of 10 to 12 amino acids separate the Luciferase PCA fragments from the p53 protein sequence section.Figure 2: Measurements of p53 PCA reporter signals. Intramolecular / due protein complementation was measured using transiently transfected HEK293 cells expressing full length p53 reporter, a truncated p53 reporter and a S6K conformation reporter, / ue PCA signals of the p53 reporters have been normalized to the expression levels of the reporter (n=3 independent experiments, - / +SD are shown). The S6K conformation reporter is used as control.Figure 3: Measurements of p53 PCA reporter signals. p53 (aal-393) and monomeric p53 (aal- 393, L344P) intramolecular / ue protein complementation was measured using transiently transfected HEK293 cells. / ue PCA signals have been normalized to the expression levels of the reporter (n=3 independent experiments, - / +SD are shown). Western blots below the bar chart shows expression levels of the individual reporters with GAPDH as loading control. Protein size in kDa is indicated on the left side of the blots.Figure 4: Impact of p53 patient variation on the p53 PCA reporter signals. Intramolecular / ue protein complementation was measured using transiently transfected HEK293 cells expressing the p53 reporters modified by truncation and displaying indicated patient mutations. / ue PCA signals have been normalized to the expression levels of the reporter (n=3 independent experiments, - / +SD are shown). Western blots below the bar chart shows expression levels of the individual reporters with GAPDH as loading control. Protein size in kDa is indicated on the left side of the blots.Figure 5: Impact of p53 patient variations on the p53 PCA reporter signal. The p53 reporter reporters modified by the point mutation L344P and the indicated tumorigenic mutations have been analyzed using / due PCA measurements, / due PCA signals have been normalized to the expression levels of the reporter (n = 4 to 6 independent experiments, - / +SEM are shown).Figure 6: Application of the p53 PCA reporter signal to study the selective p53-Y220C binding small molecule and reactivator rezatapopt on p53 conformation dynamics using the truncated p53 reporter. The p53 reporter reporters modified by truncation with wildtype (white bars) and the tumorigenic patient mutation Y220C (black bars) were transiently expressed in intactHEK293T cells that were treated with different concentration of rezatapopt or DMSO for Ih before / due PCA measurements (luciferase readout). / due PCA signals have been normalized to the expression levels of the reporter (representative of at least n=3 independent experiments, - / +SEM of technical replicates). Western blots below the bar chart shows expression levels of the individual reporters with GAPDH as loading control. Protein size in kDa is indicated on the left side of the blots. Protein size in kDa is indicated on the left side of the blots.Figure 7 : Application of the p53 PCA reporter signal to study the selective p53-Y220C binding small molecule and reactivator rezatapopt on p53 conformation dynamics using the p53 reporter. The p53 reporter modified by the point mutation L344P only (white bars) and additionally the tumorigenic patient mutation Y220C (black bars) were transiently expressed in intact HEK293T cells that were treated with different concentration of rezatapopt or DMSO for Ih before / ue PCA measurements (luciferase readout). / due PCA signals have been normalized to the expression levels of the reporter (representative of at least n=3 independent experiments, - / +SEM of technical replicates). Western blots below the bar chart shows expression levels of the individual reporters with GAPDH as loading control. Protein size in kDa is indicated on the left side of the blots. Protein size in kDa is indicated on the left side of the blots.EXAMPLES:Material and methodsCell Culture and Antibodies: HEK293T (American Type Culture Collection (ATCC), Manassas, VA, LISA; 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 measurement were performed as described below. Primary antibodies used were rabbit anti-GAPDH (Cell Signaling, Danvers, MA, USA, 2118S) and rabbit recombinant anti-renilla luciferase (abeam, Cambridge, UK, #ab 185926).Expression Constructs for the p53 reporters: Following PCR amplification of the human p53 gene (p53: NM_000546.6; encoding for a protein according to SEQ ID No: 1) and truncated p53 gene (encoding for a protein according to SEQ ID No: 1 truncated after amino acid 318) were fused N-terminally with F[l]- and C-terminally with -F[2] of the Rluc-PCA construct (pcDNA3.1 backbone vector) [Mayrhofer et al., 2020; Rock et al., 2019; Stefan et al., 2007], Interjacent linkers were inserted in the Rluc-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 NEB ridge® Golden Gate Assembly Kit (New England BioLabs, Ipswich, MA, USA, #E1602L). A site-directed mutagenesis approach (New England BioLabs, Ipswich, MA, USA, #E0552S) has been used to generate the sequence modification L344P, which reduces the oligomerization properties of the p53 protein. These reporter sequences are disclosed under SEQ ID No: 11 to 13, wherein SEQ ID No: 11 is a comparative reporter without sequence modification in the oligomerization domain.Further, amino acid substitutions were generated by site-directed mutagenesis to study known patient variations. In particular the tumorigenic mutations R175H and Y220C were studied in the p53 reporter modified by truncation at the oligomerization domain and an extended set of tumorigenic mutations in the p53 reporter modified by the point mutation affecting oligomerization (SEQ ID No: 14 to 29).Luciferase PCA Analyses in Living Cells: HEK293 cells were grown in DMEM supplemented with 10 % FBS. / due 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).Resultsa) Effect of protein sequence modifications on PCA readoutThe different concepts of the intramolecular p53 reporters are exemplarily shown in Figure 1. The first rationally engineered p53 reporter covers the human, full length sequence section and corresponds to the reporter as used in the assays used by Paulmurugan et al. [Paulmurugan et al., 2018], The inventors confirmed that p53 reporters with luciferase-based fragments may be expressed in cells and allow to quantify conformational changes directly in the living cell.Furthermore, the inventors investigated monomeric p53 reporters with diminished dimerization / oligomerization capacity. As shown in Figures 2 and 3, the monomeric p53 reporters lead to surprisingly and unexpected high PCA readout in terms of fold change of RLU in comparison to the p53 reporter with oligomerization capacity and transcription factor function. As control, the analysis of the truncated p53 reporter included the previously published reporter S6K [Mayrhofer et al., 2020] known to display a significant and comparable bioluminescent signal.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 p53 reporter reflects a more open state. A rational could be that the N-terminal and C-terminal domains of the p53 must be more accessible to be engaged in binary interactions such as p53 multimerization and DNA interactions. In the conformation associated with TF functionality, the condition of close contact, which is a prerequisite for a signal associated with the split protein, is not given.Interestingly, both modifications with impact on the oligomerization, based on the truncated p53 and the monomeric point mutation, had the same effect on elevating the RLU signal in the living cell. The helix breaker mutation L344P has been described to disrupt the p53 dimers and tetramers. For the reporter with said mutation L344P, it was shown that this mutation elevated the bioluminescence signal around 15-fold without major effect on the expression level of the reporter hybrid protein (Figure 3).In sum, this data underlines that in the absence of dimerization / oligomerization potential, the p53 reporter engage a more closed biosensor conformation. Thus, a sequence modification compared to the p53 reference sequence located at the oligomerization domain altered the conformation reporter’s readout in a striking way. In particular, the effect was confirmed for sequence modifications known to reduce the oligomerization properties of the p53 protein. b) Studying the effect of patient variationsThe p53 reporters allow integration of disease relevant patient variations, such as tumorigenic mutations, and to study their conformational impact. The tumorigenic p53 mutations are supposed to reduce its ability to tetramerize, a prerequisite for engaging its tumor suppressor function. Accordingly, also conformational impact was expected for p53 hotspot missense mutations.In contrast to the truncated reporter based on the wildtype reference sequence, the truncated p53 reporter displaying the patient mutations R175H and Y220C did show a reduced bioluminescence measured in relative luminescence units (RLU) (Fig. 4). These data underline that the two tested p53-mutants, which represent the inactivated p53 transcription factor, are engaged in a more open p53 conformation.A similar trend was observed when using reference sequences with different patient mutations for the full-length reporter modified by the point mutation L344P (Fig. 5). Herein, for example, patient mutations (compare Table 1) at residues numbers 173-179 and 220 resulted in reduced RLU fold change, in contrast to the monomeric p53 (p53-L344P; black bar) and to the mutations L344P / P151S in the p53 reporter.Taken together, these data in Fig. 4 and 5 underline that indeed the most frequent p53 patient mutations R175H and Y220C alter the monomeric p53’s conformation dramatically with both sequence modifications leading to a lower readout signal. Of note, also Paulmurugan et al. using a tetrameric reporter observed a decrease in the baseline signal for different mutant sensors and in particular also the patient mutation Y220C [Paulmurugan et al., 2018],The sensitivity of the reporter signal to patient mutation indicates their usability to reflect conformational changes with (patho)physiological impact. Hence, it is expected that the reporter with the sequence modification allows to monitor the effects of drugs that restore mutant p53 structure as this has been described for the tetrameric biosensors [Paulmurugan et al., 2018], c) Studying the effect of the mutation specific p53 reactivator rezatapoptThe p53 reporters according to the invention allow analyzing compounds with impact on the p53 conformation and study the sequence specificity of these compounds. The small molecule rezatapopt was specifically designed to bind to a pocket created by the TP53 Y220C mutation. As its binding stabilizes the native p53 structure (as associated with the wild type), rezatapopt should reactivate the tumor suppressor functions of p53.The compound had essentially no effect on the truncated reporter based on the wildtype reference sequence (white bars in Fig. 6). However, the truncated p53 reporter displaying the patient mutations Y220C with a reduced RLU signal under control condition showed a concentration dependent increase of the bioluminescence measured in relative luminescence units (RLU) (black bars in Fig. 6).Similarly, the results presented in Fig. 7 demonstrate a mutation-specific effect of the p53- Y220C specific small molecule rezatapopt on the p53 reporter being monomeric by point mutation L334P and additionally harboring the Y220C mutation (p53-L344P+Y220C).Notably, following 60-minutes treatments with rezatapopt, the selective change in reporter activity of the Y220C reporters at higher concentrations reached a level of RLU activity similar to the monomeric reporters derived from wild type reference sequence (p53(l-318) and p53-L344P reporters). 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Claims

CLAIMS1. A reporter for a protein-fragment complementation assay, wherein the reporter is a fused protein comprising a first fragment, a second fragment and a p53 protein family sequence section, wherein the first fragment and the second fragment are derived from different sections of the same luciferase split protein, and wherein the p53 protein family sequence section intervenes between the first fragment and the second fragment, and the p53 protein family sequence section is derived from a p53 protein family reference sequence, wherein the p53 protein family reference sequence is a wildtype sequence or a patient variation and the p53 protein family reference sequence is comprising an oligomerization domain, characterized in that the p53 protein family sequence section comprises a sequence modification compared to the p53 protein family reference sequence and said sequence modification is located at the oligomerization domain of the p53 protein family reference sequence.

2. The reporter according to claim 1, wherein said sequence modification reduces the oligomerization properties of the p53 protein family sequence section.

3. The reporter according to claim 1, wherein the reporter is a monomeric reporter.

4. The reporter according to any one of claims 1 to 3, wherein the p53 protein family reference sequence is selected from the group consisting of SEQ ID No: 1 including patient variations, SEQ ID No: 2, and SEQ ID No: 3, preferably SEQ ID No: 1 including patient variations.

5. The reporter according to claim 4, wherein the oligomerization domain comprises residues 324 to 355 of SEQ ID No: 1 including patient variations, residues 394 to 443 of SEQ ID No: 2, or residues 345 to 386 of SEQ ID No: 3.

6. The reporter according to one of claims 1 to 5, wherein the sequence modification compared to the human p53 sequence is a truncation.

7. The reporter according to claim 6, wherein the p53 protein family reference sequence is selected from the group consisting of SEQ ID No: 1 including patient variations and said truncation implies that the residues 324 to 355 of SEQ ID No: 1 are not comprised in the p53 sequence section.

8. The reporter according to claim 7, wherein the p53 protein family reference sequence is selected from the group consisting of SEQ ID No: 1 including patient variations and said truncation implies that no residues C-terminal from residue 319 of SEQ ID No: 1 are comprised in the p53 sequence section.

9. The reporter according to one of claims 1 to 5, wherein the sequence modification is a point mutation, preferably in one of the residues 324 to 355 of SEQ ID No: 1 when the p53 protein family reference sequence is selected from the group consisting of SEQ ID No: 1 including patient variations.

10. The reporter according to claim 9, wherein the p53 protein family reference sequence is selected from the group consisting of SEQ ID No: 1 including patient variations and said point mutation exchanges a residue at position 344 of SEQ ID No: 1 to a proline residue.

11. The reporter according to any one of claims 1 to 10, wherein the split protein is a luciferase split protein and preferably the first fragment and the second fragment are derived from a Renilla luciferase protein.

12. A polynucleotide encoding for a reporter according to any one of claims 1 to 11.

13. A cell comprising a polynucleotide according to claim 12 and expressing a reporter according to any one of claims 1 to 11, preferably the cell is established from a cell line selected out of the group consisting of HEK293, SW480 and U2OS.

14. A method for measuring an intramolecular interaction within a p53 reporter in a protein fragment complementation assay comprising the steps of a) providing a reporter according to any one of claims 1 to 11 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.

15. A method for measuring an effect of a candidate compound on the intramolecular interaction within a p53 reporter, wherein the method according to claim 14 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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