Method for identifying inhibitors of RAD18

Abstract

The present invention pertains to a method for identifying inhibitors of Rad18 mediated ubiquitination or for determining the inhibitory activity of inhibitors of Rad18 mediated ubiquitination, the method comprising the steps of: a) contacting either (i) a first polypeptide comprising a Rad18 catalytic RING (RING) domain and a Rad18 DNA-binding SAP (SAP) domain, or (ii) a second polypeptide comprising the RING domain but not the SAP domain and a third polypeptide comprising the SAP domain but not the RING domain, wherein at least one first chromophore is bound to an amino acid of or an amino acid in the vicinity of the RING domain and at least one second chromophore is bound to an amino acid of or an amino acid in the vicinity of the SAP domain, and wherein the at least one first chromophore and the at least one second chromophore is a resonance energy transfer (RET) pair, with a test compound; and b) detecting an RET signal in the presence of the test compound and optionally also in the absence of the test compound, wherein (i) a test compound is an inhibitor of Rad18 mediated ubiquitination if no RET signal is detected in step b), or the RET signal is reduced in the presence of the test compound in comparison to a reference RET signal or in comparison to the RET signal detected in the absence of the test compound; or (ii) the inhibitory activity of the test compound is determined by comparing the RET signal detected in step b) with a reference RET signal or with the RET signal detected in the absence of the test compound. The present invention further pertains to a chimeric polypeptide comprising the Rad18 RING domain and the Rad18 SAP domain connected via a heterologous peptide linker.

Description

[0001] EMBLEM Technology Transfer GmbH

[0002] ZSP Ref.: 492-68 PCT

[0003] Method for identifying inhibitors of Radl8

[0004] The present invention pertains to a method for identifying inhibitors of Rad 18 mediated ubiquitination or for determining the inhibitory activity of inhibitors of Rad 18 mediated ubiquitination. The present invention further pertains to a chimeric Rad 18 polypeptide that can be used in the method of the invention.

[0005] Background of the Invention

[0006] Bulky lesions in DNA caused by exposure to UV or other genotoxic agents result in stalling of replication forks with fatal consequences for the cell if left unrepaired. Rad 18 -mediated monoubiquitination of DNA replication sliding clamp, PCNA, triggers the exchange of replicative polymerases with one of the error-prone Y-family polymerases that tolerate various bulky DNA lesions and perform trans-lesion synthesis (TLS) of DNA saving the cells from apoptosis. Due to its fundamental role in DNA damage tolerance, Rad 18 has been identified as a major factor responsible for the resistance of tumors to genotoxic stress inducing chemo / radiotherapy in several cancers including glioma, esophagus squamous cell carcinoma and osteosarcoma (e.g. Lou et al., 2021; Zou et al., 2018). But so far, clear means for targeting Radi 8 activity in tumors do not exist. Although Rad 18 has been the subject of investigation for decades, it is still unclear as to how it specifically recognizes PCNA for ubiquitination to trigger TLS. There is further no established method for identifying inhibitors of Rad 18 ’s ubiquitination activity in tumor settings.

[0007] It is thus an object of the present invention to provide methods for identifying inhibitors of Rad 18 mediated ubiquitination. It is a further object of the present invention to provide methods for determining the inhibitory activity of inhibitors of Radi 8 mediated ubiquitination.

[0008] Summary of the Invention

[0009] According to a first aspect, the present invention provides a method for identifying inhibitors of Radi 8 mediated ubiquitination or for determining the inhibitory activity of inhibitors of Rad 18 mediated ubiquitination, the method comprising the steps of: a) contacting either:

[0010] (i) a first polypeptide comprising a Radl8 catalytic RING (RING) domain and a Radl8 DNA- binding SAP (SAP) domain, or

[0011] (ii) a second polypeptide comprising the RING domain but not the SAP domain and a third polypeptide comprising the SAP domain but not the RING domain, wherein at least one first chromophore is bound to an amino acid of or an amino acid in the vicinity of the RING domain and at least one second chromophore is bound to an amino acid of or an amino acid in the vicinity of the SAP domain, and wherein the at least one first chromophore and the at least one second chromophore is a resonance energy transfer (RET) pair, with a test compound; and b) detecting an RET signal in the presence of the test compound and optionally also in the absence of the test compound, wherein:

[0012] (i) a test compound is an inhibitor of Rad 18 mediated ubiquitination if: no RET signal is detected in step b), or the RET signal is reduced in the presence of the test compound in comparison to a reference RET signal or in comparison to the RET signal detected in the absence of the test compound; or

[0013] (ii) the inhibitory activity of the test compound is determined by comparing the RET signal detected in step b) with a reference RET signal or with the RET signal detected in the absence of the test compound.

[0014] According to one embodiment, the first polypeptide is a chimeric polypeptide comprising the RING and the SAP domain that are optionally connected via a peptide linker. The linker is preferably a glycineserine peptide linker, more preferably a (GSGS)n, (GSGG)n, or (GGGS)npeptide linker with n being an integer of between 1 and 10, most preferably wherein n is an integer of between 4 and 8.

[0015] According to yet another embodiment, the at least one first chromophore and the at least one second chromophore are fluorophores, preferably AF647 and europium (AF647 and terbium; first generation XL665 and terbium, second generation d2 and terbium, first generation XL665 and europium, second generation d2 and europium, and wherein the RET is fluorescence RET (FRET).

[0016] According to a further embodiment, the inhibitor inhibits Rad 18 mediated ubiquitination of proliferating cell nuclear antigen (PCNA).

[0017] According to yet another embodiment, the detecting in step b) is carried out using a fluorescence measuring device, preferably a fluorescence measuring plate reader machine.

[0018] According to a further embodiment, the method further comprises the step of determining the inhibitory activity of the test compound in a PCNA ubiquitination assay.

[0019] According to one embodiment, the ubiquitination assay comprises the steps of i) incubating PCNA or a fragment thereof comprising the ubiquitination domain, the test compound and either the first polypeptide, or the second and third polypeptide in the presence of ubiquitin, ubiquitin- activating enzyme (El) and ATP; and ii) determining ubiquitination of PCNA or fragment thereof.

[0020] According to a further embodiment, the test compound that is determined to be an inhibitor of Radi 8 mediated ubiquitination is formulated with at least one pharmaceutically acceptable excipient or diluent.

[0021] According to a second aspect, the present invention provides a chimeric polypeptide comprising a Radi 8 catalytic RING (RING) domain and a Rad 18 DNA-binding SAP (SAP) domain, wherein the RING domain and the SAP domain are connected via a heterologous peptide linker. According to one embodiment, the chimeric polypeptide does not comprise any other intact Rad 18 domain, preferably wherein the other Rad 18 domain is selected from the group consisting of the ZnF domain, the R6B domain, and the Polrj domain.

[0022] According to yet another embodiment, the heterologous peptide linker is a glycine-serine peptide linker, preferably a (GSGS)n, (GSGG)n, or (GGGS)npeptide linker with n being an integer of between 1 and 15, more preferably wherein n is an integer of between 2 and 10, and most preferably of between 4 and 8.

[0023] According to a particularly preferred embodiment, at least one first chromophore is bound to an amino acid of or an amino acid in the vicinity of the RING domain, and wherein at least one second chromophore is bound to an amino acid of or an amino acid in the vicinity of the SAP domain, wherein the at least one first chromophore and the at least one second chromophore is a resonance energy transfer (RET) pair.

[0024] According to a further preferred embodiment, the chimeric polypeptide comprises a first polypeptide comprising SEQ ID NO: 2 or a sequence at least 80% identical thereto, and a second polypeptide comprising SEQ ID NO: 4 or a sequence at least 80% identical thereto.

[0025] Further aspects and embodiments of the invention will become apparent from the appending claims and the following detailed description. All references cited herein including patent literature and scientific literature are incorporated by reference in their entirety.

[0026] List of Figures

[0027] In the following, the content of the figures comprised in this specification is described. In this context please also refer to the detailed description of the invention above and / or below.

[0028] Fig. 1 shows Rad 18 domain organization and the chimeric Rad 18 construct of the invention. A: Human Rad 18 wt domain organization. Indicated amino acid positions mark start and end of each domain. B: Chimeric Radl8 construct comprising the RING domain and SAP domain interconnected by a linker. The stars indicate chromophores. C: Chimeric Rad 18 construct comprising a first polypeptide comprising the RING domain with attached chromophores (stars), and a second polypeptide comprising the SAP domain with an attached chromophore.

[0029] Fig. 2 shows cross-linked construct for cross-linking mass spectrometry (XL-MS). Expressed and purified Radl8 / Rad6 complex was subjected to cross-linking as detailed in the Examples section. Crosslinked Radl8 / Rad6 was subjected to tryptic digestion and mass spectrometry. Identified inter-molecular cross-links are indicated using solid lines, where the numbers next to the lines refer to the number of crosslinks detected between the specified regions.

[0030] Fig. 3 shows the predicted alignment error (PAE) plot of AlphaFold (AF) predicted full-length Radl8, taken from the AF database (https: / / alphafold.ebi.ac.uk / entry / Q9NS91). The domain organization of Rad 18 as predicted by AF is shown on top of the PAE plot. Fig. 4 shows the interaction interface of human Radl8 RING and SAP domains as predicted by AF. B: Close-up of the Radi 8 RING and SAP interface showing various residues from both domains involved in the interaction.

[0031] Fig. 5 shows the results of in vitro ubiquitination assays performed with wt Radl8 / Rad6 or Radl8 / Rad6 complexes with various mutations interrupting the RING-SAP interaction. PCNA ubiquitination is lost upon disrupting the RING-SAP interaction interface.

[0032] Fig. 6 schematically shows a preferred embodiment of the invention. A: Fluorophores on the RING and SAP domains form a FRET -pair, leading to FRET if both domains interact with each other. B: A test compound such as a potential inhibitor will prevent or weaken the interaction between both domains, leading to a weaker or fully absent FRET signal due to the distance between the fluorophores of the FRET -pair.

[0033] List of Sequences

[0034] SEQ ID NO: 1 (human Rad 18):

[0035] MDSLAESRWPPGLAVMKTIDDLLRCGICFEYFNIAMIIPQCSHNYCSLCIRKFLSYKTQCPTCCVTVTEPD LKNNRILDELVKSLNFARNHLLQFALESPAKSPASSSSKNLAVKVYTPVASRQSLKQGSRLMDNFLIREMS GSTSELLIKENKSKFSPQKEASPAAKTKETRSVEEIAPDPSEAKRPEPPSTSTLKQVTKVDCPVCGVNIPE SHINKHLDSCLSREEKKESLRSSVHKRKPLPKTVYNLLSDRDLKKKLKEHGLSIQGNKQQLIKRHQEFVHM YNAQCDALHPKSAAEIVQEIENIEKTRMRLEASKLNESVMVFTKDQTEKEIDEIHSKYRKKHKSEFQLLVD QARKGYKKIAGMSQKTVTITKEDESTEKLSSVCMGQEDNMTSVTNHFSQSKLDSPEELEPDREEDSSSCID IQEVLSSSESDSCNSSSSDIIRDLLEEEEAWEASHKNDLQDTEISPRQNRRTRAAESAEIEPRNKRNRN

[0036] SEQ ID NO: 2 (Rad 18 RING domain):

[0037] MDSLAESRWPPGLAVMKTIDDLLRCGICFEYFNIAMIIPQCSHNYCSLCIRKFLSYKTQCPTCCVTVTEPD LKNNRILDELVKSLNFARNHLLQFALES

[0038] SEQ ID NO: 3 (Radl8 ZnF domain):

[0039] KVDCPVCGVNIPESHINKHLDSCLSREEKKESL

[0040] SEQ ID NO: 4 (Rad 18 SAP domain):

[0041] SVHKRKPLPKTVYNLLSDRDLKKKLKEHGLSIQGNKQQLIKRHQEFVHMYNAQCDALHPKSAAEIVQEIEN IEKTRMRLEASKLNES

[0042] SEQ ID NO: 5 (R6B domain):

[0043] EKEIDEIHSKYRKKHKSEFQLLVDQARKGYKKIAGMSQKTV

[0044] SEQ ID NO: 6 (PCNA):

[0045] MFEARLVQGSILKKVLEALKDLINEACWDISSSGVNLQSMDSSHVSLVQLTLRSEGFDTY RCDRNLAMGVNLTSMSKILKCAGNEDIITLRAEDNADTLALVFEAPNQEKVSDYEMKLMD LDVEQLGIPEQEYSCVVKMPSGEFARICRDLSHIGDAVVISCAKDGVKFSASGELGNGNI KLSQTSNVDKEEEAVTIEMNEPVQLTFALRYLNFFTKATPLSSTVTLSMSADVPLVVEYK IADMGHLKYYLAPKIEDEEGS

[0046] SEQ ID NO: 7: (Rad 18 Poli) domain):

[0047] HFSQSKLDSPEELEPDREEDSSSCIDIQEVLSSSESDSCNSSSSD

[0048] SEQ ID NO: 8 (Rad6):

[0049] MSTPARRRLMRDFKRLQEDPPVGVSGAPSENNIMQWNAVI FGPEGTPFEDGTFKLVIEFSEEYPNKPPTVR FLSKMFHPNVYADGSICLDILQNRWSPTYDVSSILTSIQSLLDEPNPNSPANSQAAQLYQENKREYEKRVS AIVEQSWNDS

[0050] SEQ ID NO: 9 (Avi-tag):

[0051] GLNDI FEAQKIEWHE

[0052] SEQ ID NO: 10 (peptide linker)

[0053] (GSGS)n, n = 1 to 15

[0054] SEQ ID NO: 11 (peptide linker)

[0055] (GSGG)n, n = 1 to 15

[0056] SEQ ID NO: 12 (peptide linker)

[0057] (GGGS)n, n = 1 to 15

[0058] Detailed Descriptions of the Invention

[0059] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0060] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as being “incorporated by reference”. In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

[0061] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0062] Definitions

[0063] To practice the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, and recombinant DNA techniques are employed which are explained in the literature in the field (cf. , e.g., Molecular Cloning: A Laboratory Manual, 2ndEdition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0064] In the following, some definitions of terms frequently used in this specification are provided. These terms will, in each instance of its use, in the remainder of the specification have the respectively defined meaning and preferred meanings.

[0065] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise.

[0066] The term "specifically binding" refers to the fact that the member of a binding pair interacts with a higher affinity with its binding partner than with other binding partners. The “binding affinity” between two binding partners is determined by the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., CAR) and its binding partner (e.g., LCAR). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). “Specific binding” means that a binding moiety (e.g. an antibody) binds stronger to a target such as an epitope for which it is specific compared to the binding to another target. A binding moiety binds stronger to a first target compared to a second target if it binds to the first target with a dissociation constant (Kd) which is lower than the dissociation constant for the second target. The dissociation constant (Kd) for the target to which the binding moiety binds specifically is more than 10-fold, preferably more than 20-fold, more preferably more than 50-fold, even more preferably more than 100-fold, 200-fold, 500-fold or 1000-fold lower than the dissociation constant (Kd) for the target to which the binding moiety does not bind specifically. The terms "protein" and "polypeptide" are used interchangeably herein and refer to any peptide-bond- linked chain of amino acids, regardless of length or post-translational modification. Proteins usable in the present invention (including protein derivatives, protein variants, protein fragments, protein segments, protein epitopes and protein domains) can be further modified by chemical modification. This means such a chemically modified polypeptide comprises other chemical groups than the 20 naturally occurring amino acids. Examples of such other chemical groups include without limitation glycosylated amino acids and phosphorylated amino acids. Chemical modifications of a polypeptide may provide advantageous properties as compared to the parent polypeptide, e.g. one or more of enhanced stability, increased biological half-life, or increased water solubility.

[0067] The term "amino acid" generally refers to any monomer unit that comprises a substituted or unsubstituted amino group, a substituted or unsubstituted carboxy group, and one or more side chains or groups, or analogs of any of these groups. Exemplary side chains include, e.g., thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynl, ether, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups. Other representative amino acids include, but are not limited to, amino acids comprising photoactivatable cross-linkers, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, photocaged and / or photoisomerizable amino acids, radioactive amino acids, amino acids comprising biotin or a biotin analog, glycosylated amino acids, other carbohydrate modified amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically cleavable and / or photocleavable amino acids, carbon-linked sugar-containing amino acids, redox-active amino acids, amino thioacid containing amino acids, and amino acids comprising one or more toxic moieties. As used herein, the term "amino acid" includes the following twenty natural or genetically encoded alpha-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gin or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V). In cases where "X" residues are undefined, these should be defined as “any amino acid.” The structures of these twenty natural amino acids are shown in, e.g., Stryer et al., Biochemistry, 5th ed., Freeman and Company (2002). Additional amino acids, such as selenocysteine and pyrrolysine, can also be genetically coded for (Stadtman (1996) “Selenocysteine,” Annu Rev Biochem. 65:83-100 and Ibba et al. (2002) “Genetic code: introducing pyrrolysine,” Curr Biol. 12(13):R464-R466). The term "amino acid" also includes unnatural amino acids, modified amino acids (e.g., having modified side chains and / or backbones), and amino acid analogs. See, e.g., Zhang et al. (2004) “Selective incorporation of 5 -hydroxy tryptophan into proteins in mammalian cells,” Proc. Natl. Acad. Sci. U.S.A. 101(24):8882-8887, Anderson et al. (2004) “An expanded genetic code with a functional quadruplet codon” Proc. Natl. Acad. Sci. U.S.A. 101(20):7566-7571, Ikeda etal. (2003) “Synthesis of a novel histidine analogue and its efficient incorporation into a protein in vivo,” Protein Eng. Des. Sei. 16(9):699-706, Chin et al. (2003) “An Expanded Eukaryotic Genetic Code,” Science 301(5635):964-967, James et al. (2001) “Kinetic characterization of ribonuclease S mutants containing photoisomerizable phenylazophenylalanine residues,” Protein Eng. Des. Sei. 14(12):983-991, Kohrer et al. (2001) “Import of amber and ochre suppressor tRNAs into mammalian cells: A general approach to site-specific insertion of amino acid analogues into proteins,” Proc. Natl. Acad. Sci. U.S.A. 98(25): 14310-14315, Bacher etal. (2001) “Selection and Characterization of Escherichia coli Variants Capable of Growth on an Otherwise Toxic Tryptophan Analogue,” J. Bacteriol. 183(18):5414-5425, Hamano-Takaku et al. (2000) “A Mutant Escherichia coli Tyrosyl-tRNA Synthetase Utilizes the Unnatural Amino Acid Azatyrosine More Efficiently than Tyrosine,” J. Biol. Chem. 275(51):40324-40328, and Budisa et al. (2001) “Proteins with {beta}-(thienopyrrolyl) alanines as alternative chromophores and pharmaceutically active amino acids,” Protein Sci. 10(7): 1281- 1292. Amino acids can be merged into peptides, polypeptides, or proteins.

[0068] The terms "variant" and "mutant" are used interchangeably herein and refer to a polypeptide or polynucleotide which differs in comparison to the polypeptide or polynucleotide from which it is derived by one or more changes in its length or sequence. The polypeptide or polynucleotide from which a polypeptide or polynucleotide variant is derived is also known as the parent polypeptide or polynucleotide. The term "variant" comprises "fragments" or "derivatives" of the parent molecule. Typically, "fragments" are smaller in length or size than the parent molecule, whilst "derivatives" exhibit one or more differences in their sequence in comparison to the parent molecule. Also encompassed are modified molecules such as but not limited to post-translationally modified proteins (e.g. glycosylated, biotinylated, phosphorylated, ubiquitinated, palmitoylated, or proteolytically cleaved proteins) and modified nucleic acids such as methylated DNA. Also mixtures of different molecules such as but not limited to RNA-DNA hybrids, are encompassed by the term "variant". Typically, a variant is constructed artificially, preferably by gene- technological means, whilst the parent protein or polynucleotide is a wild-type protein or polynucleotide, or a consensus sequence thereof. However, also naturally occurring variants are to be understood to be encompassed by the term "variant" as used herein. Further, the variants usable in the present invention may also be derived from homologs, orthologs, or paralogs of the parent molecule or from artificially constructed variant, provided that the variant exhibits at least one biological activity of the parent molecule, i.e. is functionally active.

[0069] In particular, the term "peptide variant", "polypeptide variant", "protein variant" is to be understood as a peptide, polypeptide, or protein which differs in comparison to the peptide, polypeptide, or protein from which it is derived by one or more changes in the amino acid sequence. The peptide, polypeptide, or protein, from which a peptide, polypeptide, or protein variant is derived, is also known as the parent peptide, polypeptide, or protein. Further, the variants usable in the present invention may also be derived from homologs, orthologs, or paralogs of the parent peptide, polypeptide, or protein or from artificially constructed variant, provided that the variant exhibits at least one biological activity of the parent peptide, polypeptide, or protein. The changes in the amino acid sequence may be amino acid exchanges, insertions, deletions, N-terminal truncations, or C-terminal truncations, or any combination of these changes, which may occur at one or several sites. A peptide, polypeptide, or protein variant may exhibit a total number of up to 60 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60) changes in the amino acid sequence (i.e. exchanges, insertions, deletions, N-terminal truncations, and / or C-terminal truncations). The amino acid exchanges may be conservative and / or non-conservative. Alternatively or additionally, a "variant" as used herein, can be characterized by a certain degree of sequence identity to the parent peptide, polypeptide, or protein from which it is derived. More precisely, a variant in the context of the present invention exhibits at least 80% sequence identity, more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, and most preferably at least 95% sequence identity to the reference polypeptide. Preferably, the variants of the present invention exhibit the indicated sequence identity, and preferably the sequence identity is over a continuous stretch of 15, 20, 25, 30, 35, 40, 45 or 50 or more amino acids. Most preferably the indicated identity is determined over the entire length of the alignment between the two amino acids, i.e. the reference amino acid and the amino acid that is assessed for its identity. Such amino acid sequence alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Set. USA 90: 5873-5877), with hmmalign (HMMER package, http: / / hmmer.wustl.edu / ) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673- 80) available e.g. on http: / / www.ebi.ac.uk / Tools / clustalw / or on http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html or on http: / / npsa-pbil.ibcp.fr / cgi- bin / npsa_automat.pl?page= / NPSA / npsa_clustalw.html.

[0070] Preferred parameters used are the default parameters as they are set on http: / / www.ebi.ac.uk / Tools / clustalw / or http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html. The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). Preferably, sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1 :154-162) or Markov random fields. When percentages of sequence identity are referred to in the present application, these percentages are calculated in relation to the full length of the longer sequence, if not specifically indicated otherwise.

[0071] The "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window can comprise additions or deletions (i.e. gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. The reference sequence is preferably the sequence having the most amino acids residues or nucleotides, i.e. is the longer sequence of the two sequences to be compared.

[0072] The term "identical" is used herein in the context of two or more nucleic acids or polypeptide sequences, to refer to two or more sequences or subsequences that are the same, i.e. that comprise the same sequence of nucleotides or amino acids. Sequences are "identical" to each other if they have a specified percentage of nucleotides or amino acid residues that are the same. According to the present invention, at least 80% identical includes at least at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity over the specified sequence, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. These definitions also refer to the complement of a test sequence. Accordingly, the term "at least XY% sequence identity" is used throughout the specification with regard to polypeptide and polynucleotide sequence comparisons.

[0073] In the context of the present invention, a protein comprising an amino acid sequence having at least 80% identity to a given SEQ ID NO preferably means that said protein has an amino acid sequence has a sequence identity of at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective to the given SEQ ID NO.

[0074] Likewise, in the context of the present invention, a nucleic acid sequence having at least 80% sequence identity to a given SEQ ID NO or a nucleic acid sequence reverse complementary thereto preferably means that said nucleic acid has a sequence identity of at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the given SEQ ID NO or a nucleic acid sequence reverse complementary to said SEQ ID NO.

[0075] The term "sequence comparison" is used herein to refer to the process wherein one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, if necessary, subsequence coordinates are designated, and sequence algorithm program parameters are designated. Default program parameters are commonly used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. In case where two sequences are compared and the reference sequence is not specified in comparison to which the sequence identity percentage is to be calculated, the sequence identity is to be calculated with reference to the longer of the two sequences to be compared, if not specifically indicated otherwise. If the reference sequence is indicated, the sequence identity is determined on the basis of the full length of the reference sequence indicated by one of the SEQ ID NOs of the present invention, if not specifically indicated otherwise.

[0076] Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), by the homology alignment algorithm of Needleman and Wunsch 1970, by the search for similarity method of Pearson and Lipman 1988, by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)). Algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977), and Altschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negativescoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-87, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, typically less than about 0.01, and more typically less than about 0.001. The term "identical" in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or sub-sequences that are the same, i.e. which comprise the same sequence of nucleotides or amino acids. Sequences are "substantially identical" to each other if they have a specified percentage of nucleotides or amino acid residues that are the same (e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the above sequence comparison algorithms or by manual alignment and visual inspection. These definitions also refer to the complement of a test sequence. Accordingly, the term “at least 80% sequence identity” is used throughout the specification with regard to polypeptide and polynucleotide sequence comparisons. This expression preferably refers to a sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective reference polypeptide or to the respective reference polynucleotide.

[0077] The term "fragment" as used herein refers to naturally occurring fragments (e.g. splice variants) as well as artificially constructed fragments, in particular to those obtained by gene-technological means. Typically, "fragments" are smaller in length or size than the parental molecule. “Fragments” refer to a smaller part of peptides, polypeptides or proteins in size and length than the parental molecule but which is still as functional as the parental protein because it still consists of the essential amino acid sequence or sequences which are responsible for the original features the protein exhibits. In other words, the fragment retains the ability to specifically bind to its target. The term “fragment” can also be understood as that the fragment has a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 amino acids at its N-terminus and / or at its C-terminus and / or internally as compared to the parental polypeptide, peptide or protein preferably at its N-terminus, at its N- and C-terminus, or at its C-terminus.

[0078] The term "nucleic acid" or "polynucleotide" as used in this specification comprises polymeric or oligomeric macromolecules, or large biological molecules, essential for all known forms of life. Nucleic acids, which include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are made from monomers known as nucleotides. Most naturally occurring DNA molecules consist of two complementary biopolymer strands coiled around each other to form a double helix. The DNA strand is also known as polynucleotides consisting of nucleotides. Each nucleotide is composed of a nitrogen-containing nucleobase as well as a monosaccharide sugar called deoxyribose or ribose and a phosphate group. Naturally occurring nucleobases comprise guanine (G), adenine (A), thymine (T), uracil (U) or cytosine (C). The nucleotides are joined to one another in a chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone. If the sugar is desoxyribose, the polymer is DNA. If the sugar is ribose, the polymer is RNA. Typically, a polynucleotide is formed through phosphodiester bonds between the individual nucleotide monomers. In the context of the present invention the term “nucleic acid” includes but is not limited to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof such as e.g. RNA-DNA hybrids (within one strand), as well as cDNA, genomic DNA, recombinant DNA, cRNA and mRNA. A nucleic acid may consist of an entire gene, or a portion thereof, the nucleic acid may also be a miRNA, siRNA, piRNA or shRNA. MiRNAs are short ribonucleic acid (RNA) molecules, which are on average 22 nucleotides long but may be longer and which are found in all eukaryotic cells, i.e. in plants, animals, and some viruses, which functions in transcriptional and post-transcriptional regulation of gene expression. MiRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression and gene silencing. Small interfering RNAs (siRNAs), sometimes known as short interfering RNA or silencing RNA, are short ribonucleic acid (RNA molecules), between 20 - 25 nucleotides in length. They are involved in the RNA interference (RNAi) pathway, where they interfere with the expression of specific genes. A short hairpin RNA (shRNA) or small hairpin RNA (shRNA) is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. PiRNAs are also short RNAs which usually comprise 26 - 31 nucleotides and derive their name from so-called piwi proteins they are binding to. The nucleic acid can also be an artificial nucleic acid. Artificial nucleic acids include polyamide or peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally-occurring DNA or RNA by changes to the backbone of the molecule. The nucleic acids, can e.g. be synthesized chemically, e.g. in accordance with the phosphofriester method (see, for example, Uhhnann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584).

[0079] The term "treat", "treating", "treatment" or "therapy" of a disease or disorder means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in an individual that has previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in individuals that were previously symptomatic for the disorder(s). Accordingly, a moiety having a therapeutic effect treats the symptoms of a disease or disorder by accomplishing one or more of the above named effects (a)-(e).

[0080] As used herein, "prevent", "preventing", "prevention", or "prophylaxis" of a disease or disorder means preventing that such disease or disorder or side effect occur in patients.

[0081] Radi 8 is a 61. 1 kDa, multi-domain protein that is conserved from yeast to humans (Notenboom et al., 2007). The amino acid sequence of human Rad 18 is disclosed under Genebank Access Number AY004333.1, and is shown under present SEQ ID NO: 1. The N-terminal RING domain (residues 1-99; SEQ ID NO: 2) of Radl8 acts as the catalytic domain and is also responsible for Radl8 dimerization (Notenboom et al., 2007, Huang et al., 2011). Binding to proliferating cell nuclear antigen (PCNA) was mapped to the N-terminus of Rad 18, although the exact residues involved and the region on PCNA to which Radi 8 binds remain unknown (Notenboom et al., 2007). The C-terminal Rad6 binding domain (R6BD) (residues 332-372; SEQ ID NO: 5) and the RING domain (RING) (residues 1 to 99; SEQ ID NO: 2) of Radl8 (SEQ ID NO: 1) form non-overlapping interactions with Rad6 (SEQ ID NO: 8) (Notenboom et al., 2007). Conflicting reports propose that the zinc finger (ZnF) domain (residues 201-234; SEQ ID NO: 3) has roles in DNA binding, dimerization and binding to Ub (Miyase et al., 2005; Jones et al., 1988; Tateishi et al., 2000). The SAP domain (residues 236-322; SEQ ID NO: 4) has been shown to be involved in DNA binding (Notenboom et al., 2007). The structures of the ZnF domain, R6BD and RING domain have been reported in (Huang et al., 2011; Hu et al., 2017; Hibbert et al., 2011; Rizzo et al., 2014). A graphic overview on human Radi 8 is shown in Fig. 1A. The following scheme indicates the respective domains in SEQ ID NO: 1:

[0082] MDSLAESRWPPGLAVMKTIDDLLRCGICFEYFNIAMIIPQCSHNYCSLCIRKFLSYKTQCPTC CVTVTEPDLKNNRILDELVKSLNFARNHLLQFALESPAKSPASSSSKNLAVKVYTPVASROSLK QGSRLMDNFLIREMSGSTSELLIKENKSKFSPQKEASPAAKTKETRSVEEIAPDPSEAKRPEPPSTST LKQVT YDCPVCGYNIPESHINKHI^SCI^R EKKESLRSYFHARA Z ATI^AZZYDRDZAAKLA EHGLSIQGNKQQLIKRHQEFVHMYNAQCDALHPKSAAEIVQEIENIEKTRMRLEASKLNESNMN YYYA QYEKEIDEIHSKYRKKHKSEFQLLVDQARKGYKKIAGMSQKTVXYVYYEDYSYYYiYSSNGAGQY DNMTSVTNHFSQSKLDSPEELEPDREEDSSSCIDIQEVLSSSESDSCNSSSSDIIRDLLEEEEAWEAS HKNDLQDTEISPRQNRRTRAAESAEIEPRNKRNRN

[0083] (RING domain bold and underlined; ZnF domain underlined; SAP domain bold italics; R6B domain underlined italics)

[0084] Embodiments

[0085] In the following, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. The present application shows for the first time a novel intramolecular interaction in Radi 8 between the RING and SAP domains which is evolutionarily conserved from yeast to humans. Previous studies using cross-linking mass spectrometry (XL-MS) and pulldown experiments showed that Radi 8 adopts an intricate architecture, where the RING domain and R6BD together interact non-exclusively with Rad6. Without wishing to be bound by any theory, the RING and SAP domains together form an interface that interacts with proliferating cell nuclear antigen (PCNA). Based on this finding, the present application shows that interfering with the interaction between RING and SAP by disrupting the interface between both domains abrogates PCNA monoubiquitination (Figure 5). Radl8 is one of the critical molecules responsible for the resistance to chemo- and radio-therapy in various cancers (Xie et al., 2014; Wu et al., 2019; Li et al., 2022; Du et al., 2022), and compounds that target the RING-SAP interface can thus be potential therapeutics. Inhibiting the interaction of the RING and SAP domains offers a pragmatic approach to silencing Rad 18, without affecting the function of Rad6, which plays an important role in transcription (Kim et al., 2009).

[0086] Radi 8 contains large regions of disorder interspersed with globular domains making it a difficult target for structural studies. The observed intramolecular interactions within Radi 8 were thus used for designing chimeric Rad 18 constructs that bridge domains of importance for PCNA binding, optionally using linkers, which can be used for screening of compounds that interfere with Rad 18 activity and in particular with ubiquitination towards PCNA. A scheme of such constructs is shown in Figs. IB and 1C. The constructs consisting essentially of the RING domain and the SAP domain optionally interconnected by a linker allows easy expression and large scale purification of the otherwise difficult to express protein Radi 8.

[0087] The present invention thus relates to a method for identifying inhibitors of Rad 18 and in particular inhibitors of Rad 18 mediated ubiquitination, or for determining the inhibitory activity of inhibitors of Rad 18 and in particular inhibitors of Rad 18 mediated ubiquitination. The method allows high throughput screening of test compounds. The method comprises the step of contacting either (i) a first polypeptide comprising a Radi 8 catalytic RING (RING) domain and a Radi 8 DNA-binding SAP (SAP) domain, or (ii) a second polypeptide comprising the RING domain but not the SAP domain and a third polypeptide comprising the SAP domain but not the RING domain with a test compound. According to the method of the present invention, at least one first chromophore is bound to an amino acid of or an amino acid in the vicinity of the RING domain and at least one second chromophore is bound to an amino acid of or an amino acid in the vicinity of the SAP domain. The at least one first chromophore and the at least one second chromophore forms or resembles a resonance energy transfer (RET) pair. The method fiirther comprises the step of detecting an RET signal in the presence of the test compound and optionally also in the absence of the test compound. A schematic illustration of respective polypeptides (also termed chimeric constructs) is shown in Figs. IB and 1C. If in an undisturbed state, the RING domain and the SAP domain form interactions as in the natural Rad 18 protein, and the chromophore of one domain will transfer energy to the chromophore of the other domain, thereby exciting the chromophore of the other domain, which excitation can be detected. This concept is also exemplarily shown in Fig. 6A. A test compound interfering with the RINGSAP interaction will enlarge the distance between the two chromophores on the two domains, resulting in a decrease or loss of RET signal, as exemplarily shown in Fig. 6B.

[0088] A test compound is thus considered an inhibitor of Rad 18 or of Rad 18 mediated ubiquitination if either no RET signal is detected, or if the RET signal is reduced in the presence of the test compound in comparison to a reference RET signal or in comparison to the RET signal detected in the absence of the test compound. Alternatively, the inhibitory activity of the test compound is determined by comparing the detected RET signal with a reference RET signal or with the RET signal detected in the absence of the test compound.

[0089] According to a particularly preferred embodiment, the RING domain does not comprise the SAP domain. Likewise, the SAP domain does not comprise the RING domain.

[0090] According to a further embodiment, the RING domain comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 2, or a fragment or variant thereof having at least 80% sequence identity to SEQ ID NO: 2. According to a further embodiment, the SAP domain comprises, consists essentially of or consists of the amino acid sequences of SEQ ID NO: 4, or a fragment or variant thereof having at least 80% sequence identity to SEQ ID NO: 4.

[0091] According to one embodiment of the present invention, the first polypeptide further comprises a linker located between the RING domain and the SAP domain, preferably a heterologous linker. The linker preferably provides flexibility to the first polypeptide. Respective linkers are known to the person of ordinary skill in the art. Examples for such linkers are short polypeptide sequences, e.g. of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues, such as 15, 20, 25, 30, 35, 40 or more amino acid residues. Preferred linkers comprise alanine / glycine / serine linkers, more preferably linkers consisting of glycine and serine residues. Preferred glycine-serine peptide linkers are (GSGS)n(SEQ ID NO: 10), (GSGG)n(SEQ ID NO: 11), or (GGGS)n (SEQ ID NO: 12) peptide linkers, with n being an integer of between 1 and 15, preferably between 2 and 10, more preferably wherein n is an integer of between 4 and 8.

[0092] According to yet another embodiment, the first, the second and / or the third polypeptide do not comprise any other domains of Radl8, such as the ZnF domain, the R6B domain, or the Polij domain.

[0093] The Rad 18 domains described herein with respect to different SEQ ID NOs also include fragments and variants thereof as defined herein. Fragments preferably maintain the biological function of the respective domain, and variants comprise an amino acid sequence at least 80% identical to the indicated SEQ ID NO.

[0094] According to one embodiment, the first, second and / or third polypeptide may additionally comprise a peptide tag. The peptide tag is preferably attached to the C-terminus or to the N-terminus of the respective peptide. According to a preferred embodiment, the peptide tag is selected from the group consisting of a Flag-tag, Myc-tag, HA-tag, His-tag, NE-tag, T7-tag, Avi-tag, Strep-tag, and CaM tag. A preferred tag is the Avi-tag. According to a particularly preferred embodiment, the third polypeptide comprising the SAP domain further comprises an Avi-tag attached to its N- or C-terminus, more preferably wherein the Avi-tag consists of the amino acid sequence as set forth in SEQ ID NO: 9.

[0095] According to the present invention, at least one first chromophore is bound to an amino acid of or an amino acid in the vicinity of the RING domain. This first chromophore can be bound to one or more of the amino acids of the RING domain. Preferably, the at least one first chromophore is bound to a lysine residue of the RING domain, such as but not limited to K17, K57 or K83 of the RING domain according to SEQ ID NO: 2, or to a corresponding amino acid in a RING domain variant. Alternatively, the at least one first chromophore can be bound to an amino acid in the vicinity of the RING domain such as to an amino acid of an N-terminal and / or C-terminal peptide tag or to one or more additional amino acids being present in the polypeptide and which do not belong to the RING domain as defined by SEQ ID NO: 2. According to a preferred embodiment, in the vicinity of the RING domain means up to 20 amino acid positions N- or C-terminal of the RING domain, such as 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position N- or C-terminal of the RING domain as defined herein. According to a particularly preferred embodiment, the at least one first chromophore is bound to K17, K57 or K83 of the RING domain according to SEQ ID NO: 2.

[0096] According to the present invention, at least one second chromophore is bound to an amino acid of or an amino acid in the vicinity of the SAP domain. This second chromophore can be bound to one or more of the amino acids of the SAP domain. Alternatively, the at least one second chromophore can be bound to an amino acid in the vicinity of the SAP domain such as to an amino acid of an N-terminal and / or C-terminal peptide tag or to one or more additional amino acids being present in the polypeptide and which do not belong to the SAP domain as defined by SEQ ID NO: 4. According to a preferred embodiment, in the vicinity of the SAP domain means up to 20 amino acid positions N- or C-terminal of the SAP domain, such as 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position N- or C-terminal of the SAP domain as defined herein. According to a particularly preferred embodiment, the at least second chromophore is bound to a peptide tag at the N- or C-terminus of the SAP domain, more preferably to an Avi-tag.

[0097] According to the present invention, the at least one first chromophore and the at least one second chromophore together form a resonance energy transfer (RET) pair. In RET, electronic energy is transported from one atom or molecule to another, in the present case from the at least one first chromophore to the at least one second chromophore or vice versa. A preferred type of RET is fluorescence resonance energy transfer (FRET), in which a donor chromophore, initially in its electronic excited state, transfers energy to an acceptor chromophore through non-radiative dipole-dipole coupling. The at least one first chromophore and the at least one second chromophore are preferably different from each other. According to a preferred embodiment of the invention, the at least one first and the at least one second chromophore are both fluorophores. More preferably, the at least one first chromophore and the at least one second chromophore are selected from the group consisting of AF647 and europium, AF647 and terbium, first generation XL665 and terbium, second generation d2 and terbium, first generation XL665 and europium, second generation d2 and europium, ULight and europium, Surelight APC and europium, AlexaFluor 647 and europium, Fluorescin / GFP and europium, Fluorescin / GFP and terbium, HiLyte647 and terbium, and Far-red dye and europium. Particularly preferred is the combination AF647 and europium. According to a particularly preferred embodiment, the RET is time-resolved fluorescence resonance energy transfer (TR-FRET). TR- FRET avoids interference from the short-lived fluorescence of the screening compounds by introducing a delay between the excitation and detection of emission signals.

[0098] The RING domain and the SAP domain are located far apart in the amino acid sequence of Radl8 (SEQ ID NO: 1; Fig. 1A) but come together in the natural protein to recognize PCNA and carry out ubiquitination. The RING domain and the SAP domain as used in the polypeptide(s) of the present invention will form the native conformation as they would in the full-length Rad 18 protein. The phenomenon of RET is used to check the interaction status of the RING domain and the SAP domain. In case a test compound interferes with the RING-SAP interaction, it will reduce or prevent transfer of electronic energy from one chromophore to the other and thus reduce or abolish the RET signal that is detected if the RING domain and the SAP domain are in the native or in an interacting conformation (exemplarily shown in Fig. 6B). The inhibitory activity of a test compound can thus be determined by detecting and comparing the RET signal with a reference RET signal, such as the RET signal in the presence of a compound that is known not to interfere with the interaction between the RING domain and the SAP domain, such as a control compound, or with the RET signal obtained when testing a reference Rad 18 polypeptide comprising a mutant polypeptide of the RING and / or the SAP domain, preferably comprising the RING domain having the mutation R51E and shown to result in a low ubiquitination of PCNA. Alternatively, the inhibitory activity of a test compound can be determined by detecting and comparing the RET signal in the presence of the test compound with the RET signal detected in the absence of the test compound.

[0099] Methods for detecting RET signals and in particular FRET signals are not particularly limited and are well known to the person of ordinary skill in the art. According to a preferred embodiment of the present invention, the RET is FRET and the fluorescence is detected using a standard fluorescence detection or measuring device. For a high-throughput method, the incubation of the test compound and the polypeptide(s) comprising the RING and / or the SAP domains is performed in standard laboratory plates and FRET signals are detected using a standard fluorescence measuring plate reader machine such as the Spark® Multimode microplate reader (Tecan). Any standard laboratory plate such a microtiter-plate can be used. Standard microtiter plates include but are not limited to 6-, 12-, 24-, 48-, 96-, and 384-well plates.

[0100] As a control, the RING polypeptide comprising mutation R5 IE or the SAP polypeptide comprising mutation A287D (positions of the mutations with respect to SEQ ID NO: 1) can be used, which were shown to result in essentially no ubiquitination (Fig. 5). Alternatively, a control compound can be used that is known not to interfere with the RING-SAP interaction.

[0101] According to one particularly preferred embodiment, the inhibitor inhibits Rad 18 mediated ubiquitination of proliferating cell nuclear antigen (PCNA).

[0102] The method of the invention may further comprise the step of determining the inhibitory activity of the test compound in a PCNA ubiquitination assay. The ubiquitination assay preferably comprises the steps of incubating PCNA or a fragment thereof comprising the ubiquitination domain, the test compound and either the first polypeptide, or the second and third polypeptide in the presence of ubiquitin, ubiquitin- activating enzyme (El (also UBA1, reviewed e.g. in Lambert-Smith et al., 2020)) and ATP, and determining ubiquitination of PCNA or of the fragment thereof. The ubiquitination assay may include incubating PCNA or a fragment thereof comprising the ubiquitination domain, the test compound and either the first polypeptide, or the second and third polypeptide with ubiquitin, El and ATP in conditions allowing ubiquitination of PCNA or its fragment. Preferred conditions are incubation for between about 15 minutes to about 2 hours, preferably between about 30 to about 60 minutes, at about 30°C. A preferred incubation buffer in which the reaction can be performed comprises 50 mM NaCl, 50 mM Tris, 2.5 mM MgCL and 1 mM DTT.

[0103] The PCNA preferably comprises, consists essentially of or consists of the amino acid sequence as set forth in SEQ ID NO: 6. A fragment of PCNA is any truncated variant of PCNA which can be ubiquitinylated by Radi 8. A preferred PCNA fragment has a C-terminal truncation of between one and six amino acid residues. A particularly preferred PCNA fragment consists of amino acids 1 to 255 of SEQ ID NO: 6. The PCNA or the fragment thereof may also be a variant of PCNA or its fragment. A preferred variant has at least 80% sequence identity to SEQ ID NO: 6 and can be ubiquitinylated by Radi 8. Whether or not the fragment and / or variant of PCNA can be ubiquitinylated by Radi 8 can be determined using the ubiquitination assays as described herein.

[0104] According to one embodiment of the present invention, the test compound that is determined to be an inhibitor of Radi 8 mediated ubiquitination is formulated with at least one pharmaceutically acceptable excipient or diluent. Respective pharmaceutically acceptable excipients and diluents are well-known in the art and the person of ordinary skill in the art may readily select on or more thereof considered suitable in the context of the present invention.

[0105] The method of the invention can thus be used in laboratory scale e.g. using standard microtiter plates such as 6-, 12-, 24-, 48-, 96-, and 384-well plates, or it can be further scaled up to e.g. industrial scale. The method of the invention can thus be used as a high throughput method allowing the simultaneous testing of a vast number test compounds.

[0106] According to a further aspect, the present invention provides chimeric polypeptide comprising a Radi 8 catalytic RING (RING) domain and a Rad 18 DNA-binding SAP (SAP) domain, wherein the RING domain and the SAP domain are connected via a heterologous peptide linker, preferably wherein the linker connects the N-terminus of one domain with the C -terminus of the other domain, more preferably wherein the linker connects the N-terminus of the SAP domain with the C -terminus of the RING domain as exemplarily shown in Fig. IB. According to one embodiment, the RING domain comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 2, or a fragment or variant thereof having at least 80% sequence identity to SEQ ID NO: 2. According to a further embodiment, the SAP domain comprises, consists essentially of or consists of the amino acid sequences of SEQ ID NO: 4, or a fragment or variant thereof having at least 80% sequence identity to SEQ ID NO: 4. According to a particularly preferred embodiment of the present invention, the chimeric construct comprises a polypeptide according to SEQ ID NO: 2 or a sequence at least 80% identical thereto, and a polypeptide according to SEQ ID NO: 4 or a sequence at least 80% identical thereto. According to a more particularly preferred embodiment, the chimeric polypeptide comprises, consists of or consists essentially of a first polypeptide according to SEQ ID NO: 2 and a second polypeptide according to SEQ ID NO:4, wherein both polypeptides are interconnected N- to C-terminally by a linker, preferably wherein the chimeric polypeptide has the order in N- to C-terminal direction of RING domain-linker- SAP domain.

[0107] The linker can be any heterologous peptide linker such as a linker as defined herein. The linker preferably provides flexibility to the first polypeptide. Respective linkers are known to the person of ordinary skill in the art. Examples for such linkers are short polypeptide sequences, e.g. of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues, such as 15, 20, 25, 30, 35, 40 or more amino acid residues. Preferred linkers comprise alanine / glycine / serine linkers, more preferably linkers consisting of glycine and serine residues. Preferred glycine-serine peptide linkers are (GSGS)n, (GSGG)n, or (GGGS)npeptide linkers, with n being an integer of between 1 and 15, preferably between 2 and 10, more preferably wherein n is an integer of between 4 and 8.

[0108] According to one embodiment, the RING domain and / or the SAP domain may additionally comprise a peptide tag. The peptide tag is preferably attached to the C-terminus or to the N-terminus of the respective domain. According to a preferred embodiment, the peptide tag is selected from the group consisting of a Flag-tag, Myc-tag, HA-tag, His-tag, NE-tag, T7-tag, Avi-tag, Strep-tag, and CaM tag. A preferred tag is the Avi-tag. According to a particularly preferred embodiment, the SAP domain further comprises an Avi-tag attached to its N- or C-terminus, more preferably to its C-terminus.

[0109] According to one embodiment of the present invention, the chimeric polypeptide does not comprise any other intact Rad 18 domain except the RING and the SAP domain. Preferably, said other Rad 18 domain is selected from the group consisting of the ZnF domain, the R6B domain, and the Polij domain. According to a preferred embodiment, the chimeric polypeptide does not comprise the ZnF domain. Preferably, the ZnF domain as referred to herein comprises a sequence at least 80% identical to SEQ ID NO: 3. Preferably, the R6B domain comprises a sequence at least 80% identical to SEQ ID NO: 5. Also preferably, the Polrj domain as referred to herein comprises a sequence at least 80% identical to SEQ ID NO: 7.

[0110] According to a further preferred embodiment of the present invention, at least one first chromophore is bound to an amino acid of or to an amino acid in the vicinity of the RING domain. This first chromophore can be bound to one or more of the amino acids of the RING domain. Preferably, the at least one first chromophore is bound to a lysine residue of the RING domain, such as but not limited to K17, K57 or K83 of the RING domain according to SEQ ID NO: 2, or to a corresponding amino acid in a RING domain variant. Alternatively, the at least one first chromophore can be bound to an amino acid in the vicinity of the RING domain such as to an amino acid of an N-terminal and / or C -terminal peptide tag or to one or more additional amino acids being present in the polypeptide and which do not belong to the RING domain as defined by SEQ ID NO: 2. According to a particularly preferred embodiment, the at least one first chromophore is bound to K17, K57 or K83 of the RING domain according to SEQ ID NO: 2. In the vicinity to the respective RING and / or SAP domain means up to 20 amino acid positions N- or C-terminal of the respective domain, such as 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position N- or C- terminal of the domain as defined herein, if the chimeric construct comprises amino acids in addition to those of the RING and the SAP domain and of the linker. According to a preferred embodiment of the invention, in the vicinity of the RING and / or SAP domain does not include the linker.

[0111] According to a preferred embodiment of the present invention, at least one second chromophore is bound to an amino acid of or an amino acid in the vicinity of the SAP domain. The second chromophore can be bound to one or more of the amino acids of the SAP domain. Alternatively, the at least one second chromophore can be bound to an amino acid in the vicinity of the SAP domain such as to an amino acid of an N-terminal and / or C-terminal peptide tag or to one or more additional amino acids being present in the polypeptide and which do not belong to the SAP domain as defined by SEQ ID NO: 4. According to a particularly preferred embodiment, the at least second chromophore is bound to a peptide tag at the N- or C -terminus of the SAP domain, more preferably to an Avi-tag.

[0112] According to the present invention, the at least one first chromophore and the at least one second chromophore together form a resonance energy transfer (RET) pair. A preferred type of RET is fluorescence resonance energy transfer (FRET). The at least one first chromophore and the at least one second chromophore are preferably different from each other. According to a preferred embodiment of the invention, the at least one first and the at least one second chromophore are both fluorophores. More preferably, the at least one first chromophore and the at least one second chromophore are selected from the group consisting of AF647 and europium, AF647 and terbium, first generation XL665 and terbium, second generation d2 and terbium, first generation XL665 and europium, second generation d2 and europium, ULight and europium, Surelight APC and europium, AlexaFluor 647 and europium, Fluorescin / GFP and europium, Fluorescin / GFP and terbium, HiLyte647 and terbium, and Far-red dye and europium. Particularly preferred is the combination AF647 and europium.

[0113] The present invention also provides the use of the chimeric polypeptide of the invention in the method of the invention.

[0114] According to a particularly preferred embodiment of the present invention, the RING domain is as set forth in SEQ ID NO: 2 and the SAP domain is as set forth in SEQ ID NO: 4. One of the fluorophores europium and AF647 is attached to the RING domain, and the other one of europium and AF647 is attached to an Avi-tag of the SAP domain, wherein the Avi-tag is preferably attached to the N- or C-terminus of the SAP domain. The RING and the SAP domain are interconnected by a flexible serine-glycine linker (GSGS)nwith n being an integer of between 4 and 8. RING domain, linker and SAP domain are thus present in a single polypeptide, which lacks any other domains of Radl8. Preferably, the polypeptide consists of the RING domain (comprising the fluorophore), the linker and the SAP domain (comprising the Avi-tag with the fluorophore).

[0115] The present invention also provides a nucleic acid sequence or a plurality of nucleic acid sequences encoding the polypeptide of the present invention, as well as vectors comprising the nucleic acid sequence or a plurality of nucleic acid sequences encoding the polypeptide of the present invention, and a host cell or a culture of host cells comprising the nucleic acid sequence or a plurality of nucleic acid sequences, or the vector of the present invention.

[0116] Any interference of the interaction between the RING domain and the SAP domain detected by the method of the present invention will also reduce or inhibit Radi 8 mediated ubiquitination, in particular Radl8 mediated ubiquitination of PCNA. Inhibitors identified by the present invention can thus be used for reducing or preventing Rad 18 mediated ubiquitination of PCNA, and therefore in cancer treatment.

[0117] Such inhibitors can be formulated into a pharmaceutical composition. Such a pharmaceutical composition preferably comprises one or more of the identified inhibitors and further one or more pharmaceutically acceptable carriers, diluents, excipients, fillers, binders, lubricants, glidants, disintegrants, adsorbents, and / or preservatives. The pharmaceutical composition preferably comprises a therapeutically effective amount of the active ingredient, i.e. the one or more inhibitors as described herein, together with a suitable amount of carrier and / or excipient so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0118] The pharmaceutical compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The pharmaceutical composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.

[0119] In particular embodiments, the pharmaceutical composition is in unit dosage form. In such form the composition may be subdivided into unit doses containing appropriate quantities of the active component. The dosage administered is preferably adapted to achieve a desired reduction of unwanted side effects of CAR therapy. Since the strength of the side effects will vary between patients, the dosage can be individually adapted until the desired reduction of unwanted side effects is achieved.

[0120] The unit dosage form can be a packaged composition, the package containing discrete quantities of the composition, such as packaged tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, an injection vial, a tablet, a cachet, or a lozenge itself, or it can be the appropriate number of any of these in packaged form.

[0121] The pharmaceutical composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0122] Furthermore, such pharmaceutical composition may also comprise other pharmacologically active substance such as but not limited to adjuvants and / or additional active ingredients. Adjuvants in the context of the present invention include but are not limited to inorganic adjuvants, organic adjuvants, oil-based adjuvants, cytokines, particulate adjuvants, virosomes, bacterial adjuvants, synthetic adjuvants, or synthetic polynucleotides adjuvants.

[0123] Examples

[0124] Materials and Methods

[0125] AlphaFold

[0126] To predict the structure of the Radi 8 dimer, the Alphafold multimer (Evans et al. 2022) was used. The prediction resulted in five models of the dimer all containing a similar RING-SAP interface. Interestingly, the RING-SAP intramolecular interface is also observed in Radi 8 monomeric structure, which is available in the Alphafold database under E3 ubiquitin-protein ligase RAD 18 (https: / / alphafold.ebi.ac.uk / entry / Q9NS91; PMID: 34265844 andPMID: 34791371). The AlphaFold model of Rad 18 dimer with the highest-confidence score (ranked 0) was chosen to validate the interface biochemically.

[0127] Protein expression and purification

[0128] Proteins were expressed in E.coli BL21star by growing the bacteria at 37°C in lysogeny broth (LB), supplemented with the appropriate antibiotic, to an OD600 of 0.6 and inducing the cells with 0.2 mM IPTG at 18°C for 16 hours. Radl8 / Rad6, and Rad6 all contained an N-terminal histidine tag (located on Radl8 in the case of Radl8 / Rad6). Following lysis in 200 mM NaCl, 50 mM Tris, 10% Glycerol (lysis buffer), each protein was incubated rolling on TALON® Superflow™ resin for 1 hour at 4°C and eluted with 300 mM imidazole, following numerous washes with lysis buffer and low amounts of imidazole. Proteins were then subjected to size exclusion chromatography (SEC) and eluted in 100 mM NaCl, 20 mM HEPES, 1 mM TCEP (SEC buffer).

[0129] The Radl8 / Rad6 protein complex was produced by cloning Rad 18 and Rad6 cloned into two different expression vectors each with different antibiotic resistance, i.e. ampicillin and kanamycin, respectively. These plasmids were co-transformed into bacteria. Double transformed cells were selected using agar plates with two antibiotics. Selected bacteria were grown and proteins are expressed as described herein.

[0130] Ubiquitination assays

[0131] For examining the impact of the Radl8 R6BD peptide (SEQ ID NO: 5) on PCNA ubiquitination, 1 pM Radl8 / Rad6, 0.3 pM El, 12 pM ubiquitin, 3 pM PCNA (SEQ ID NO: 6), and 2.5 mM ATP were incubated. The assay involving Radi 8 RING and SAP mutants (R5 IE, R51A, A287D, R51A and A287D or R5 IE and A287D, positions with respect to SEQ ID NO: 1) was carried out in the same way but in the absence of the Radi 8 R6BD peptide (SEQ ID NO: 5). All reactions were incubated for 1 hour at 30°C.

[0132] For the autoubiquitination assay for mass spectrometry, 2.5 pM Radl8 / Rad6, 0.75 pM El, 30 pM ubiquitin and 6.25 mM ATP were incubated for 45 minutes at 30°C. The reaction was performed in 50 mM NaCl, 50 mM Tris, 2.5 mM MgC'E and 1 mM DTT. Samples were subsequently loaded onto an SDS PAGE gel and analyzed by western blot. For mass spectrometry analysis, the reaction was performed at 37°C for 1 hour, samples were run on an SDS PAGE gel.

[0133] For in-gel digestion, following SDS-PAGE and coomassie staining, the gel streaks corresponding to autoubiquitinated Radi 8 were excised and subjected to in-gel digestion with trypsin to generate peptides containing the Lys-e-Gly-Gly (diGLY) remnant. Peptides were extracted from the gel by sonication for 15 minutes, followed by centrifugation and supernatant collection. A solution of 50:50 water: acetonitrile, 1% formic acid was added for a second extraction and the samples were again sonicated for 15 minutes, centrifuged and the supernatant was pooled with the first extract. The supernatants were dried down and reconstituted in 10 pL 4% acetonitrile, 1% formic acid in water and analysed by LC-MS / MS.

[0134] Cross-linking

[0135] Full-length Radi 8 / Rad6 was expressed and purified as described herein. Radi 8 / Rad6 was incubated with 0.5 x molar BS3 cross-linker to total lysine residues and the sample was incubated for 10 minutes at room temperature under shaking. Samples were then subjected to Trypsin digest in a 1 :50 protease to protein ratio and incubated at 37°C prior to mass spectrometry analysis.

[0136] Mass spectrometry For cross-linking mass spectrometry, digested peptides were concentrated and desalted using an OASIS® HLB pElution Plate (Waters) according to manufacturer instructions. Crosslinked peptides were enriched using size exclusion chromatography. In brief, desalted peptides were reconstituted with SEC buffer (30% (v / v) ACN in 0.1% (v / v) TFA) and fractionated using a Superdex Peptide PC 3.2 / 30 column (GE) on a 1200 Infinity HPLC system (Agilent) at a flow rate of 0.05 mL / min. Fractions eluting between 50-70 ul were evaporated to dryness and reconstituted in 30 pl 4% (v / v) ACN in 1% (v / v) FA.

[0137] Collected fractions were analyzed by liquid chromatography (LC)-coupled tandem mass spectrometry (MS / MS) using an UltiMate 3000 RSLC nano LC system (Dionex) fitted with a trapping cartridge (p-Precolumn C18 PepMap 100, 5pm, 300 pm i.d. x 5 mm, 100 A) and an analytical column (nanoEase™ M / Z HSS T3 column 75 pm x 250 mm C18, 1.8 pm, 100 A, Waters). Trapping was carried out with a constant flow of trapping solvent (0.05% trifluoroacetic acid in water) at 30 pL / min onto the trapping column for 6 minutes. Subsequently, peptides were eluted and separated on the analytical column using a gradient composed of Solvent A (3% DMSO, 0.1% formic acid in water) and solvent B (3% DMSO, 0.1% formic acid in acetonitrile) with a constant flow of 0.3 pL / min. For the diGLY analysis of ubiquitinated Radi 8, the percentage of solvent B (0. 1% formic acid in acetonitrile, 3% DMSO) was increased from 2% to 8% in 2.2 min, to 23% in 40.9 min, in 5 min from 23% to 38%, followed by an increase of B to 80% in 4 min and a re-equilibration back to 2% B for 6 min. The outlet of the analytical column was coupled directly to an Orbitrap Fusion Lumos (Thermo Scientific, SanJose) mass spectrometer using the nanoFlex source.

[0138] The peptides were introduced into the Orbitrap Fusion Lumos via a Pico-Tip Emitter 360 pm OD x 20 pm ID; 10 pm tip (CoAnn Technologies) and an applied spray voltage of 2.1 kV (2.4 kV for DiGly). The instrument was operated in positive mode. The capillary temperature was set at 275°C. Only charge states of 4-8 were included. The dynamic exclusion was set to 30 sec (60 sec for diGLY analysis), and the intensity threshold was 5e4. Full mass scans were acquired for a mass range 350-1700 m / z in profile mode in the orbitrap with resolution of 120,000. The AGC target was set to Standard and the injection time mode was set to Auto. The instrument was operated in data dependent acquisition (DDA) mode with a cycle time of 3 sec between master scans and MSMS scans were acquired in the Orbitrap with a resolution of 30,000, with a fill time of up to 100 ms and a limitation of 2e5 ions (AGC target). A normalized collision energy of 32 (34 for diGLY) was applied. MS2 data was acquired in profile mode. The DiGly samples were processed in the same way with few exceptions, as noted above.

[0139] For cross-linking mass spectrometry, all data were analyzed using the cross-linking module in Mass Spec Studio v2.4.0.3524. Parameters were set as follows: Trypsin (K / R only), charge states 4-8, peptide length 7-50, percent Evalue threshold = 50, MS mass tolerance = 10 ppm, MS / MS mass tolerance = 10, elution width = 0.5 min. BS3 cross-links residue pairs were constrained to K on one end and one of KSTY on the other. Identifications were manually validated, and cross-links with an E-value corresponding to <0.05% FDR were rejected. The data export from the Studio was filtered to retain only cross-links with a unique pair of peptide sequences and a unique set of potential residue sites.

[0140] For DiGly analysis, raw data was processed with IsobarQuant and as search engine Mascot (v2.2.07) was used. Data was searched against the Uniprot Escherichia coli database (UP000000625, 4518 entries, including common contaminants and reverse hits, May 2016) with the amino acid sequences of interest added (His-tagged Rad 18, His-tagged El, USP46, Rad6, UBB, UBC, UBD, RL40 and RS27A, all from homo sapiens). Carbamidomethyl (C) was set as fixed modification, Acetyl (Protein N-term), Oxidation (M) and GlyGly (K) as variable modifications. The mass error tolerance for the full scan MS spectra was set to 10 ppm, for MS / MS spectra to 0.02 Da. Enzyme was set to Trypsin / P. A maximum of 2 missed cleavages were allowed. A false discovery rate below 0.01 were required on the peptide and protein level.

[0141] Example 1 : Intramolecular interactions between the RING and SAP domains of Radi 8

[0142] A cross-linking mass spectrometry (XL-MS) experiment was performed on the Radl8 / Rad6 complex cross-linked using BS3 (bis(sulfosuccinimidyle)suberate) (Fig. 2) as described in Materials and Methods above, followed by the analysis of the cross-linked peptides using mass spectrometry. This experiment revealed cross-links between Rad6 and the SAP domain of Radl8 as indicated in Fig. 2. To assess if these cross-links reflect direct interactions between Rad6 and the SAP domain of Rad 18 or mere physical proximity in the complex, the AlphaFold (AF) structure of full-length Radl8 (SEQ ID NO: 1) was analyzed. The predicted aligned error (PAE) plot of the Rad 18 model suggested an inter domain interaction between the RING and the SAP domains of Rad 18 (Fig. 3). Consistently, also multiple cross-links between the RING domain and the SAP domain of Radl8 in the XL-MS analysis were detected. In the AF model of Radi 8, the central helix of the SAP domain packs against the sole helix of the core RING domain.

[0143] The predicted interface of RING and SAP includes two salt bridges; one between R51 of RING and D290 of SAP, and another between E69 of RING and K245 of SAP (Fig. 4). A hydrophobic interaction network composed of residues A287, M284 of SAP and L48 of the RING domain further strengthens the RING-SAP interaction. Importantly, multiple sequence alignments of Radi 8 revealed conservation of residues involved in the RING-SAP interface and confirmed that this RING-SAP interaction is evolutionarily conserved from yeast to humans. Several mutants of Rad 18 (R51A, R51E, A287D, R51A_A287D and R51E_A287D) were generated to disrupt the interface between the RING and SAP domains. Note that all amino acid positions mentioned in this example refer to SEQ ID NO: 1. Mutant Radl8 / Rad6 complexes were purified and tested in PCNA monoubiquitination assays (Fig. 5). Strikingly, none of the mutants exhibited any PCNA monoubiquitination activity while they retained Rad 18 autoubiquitination activity to a large extent. Interestingly, PCNA was previously shown to interact with the N-terminus of Rad 18 but binding experiments with individual domains of Rad 18 failed to detect any direct interaction with PCNA (Notenboom et al., 2007). Considering the SAP -RING interaction disclosed here, along with the previous studies that implicated the SAP domain in interactions with DNA (Notenboom et al., 2007), and without wising to be bound by any theory, it was concluded that the RING and the SAP domains cooperate through intramolecular interactions to specifically recognize and engage PCNA loaded onto DNA for ubiquitination.

[0144] Example 2: Chimeric Radl8 constructs and screening assay

[0145] Based on the observations made in Example 1, different chimeric constructs comprising the Rad 18 RING domain and SAP domain are produced. As a preferred fluorophore, europium is attached to the N- terminus or one of K 17, K57 and K83 of the RING polypeptide. An Avi-tag is preferably attached to the C- terminus or N-terminus of the SAP polypeptide. Fluorophore AlexaFluor 647 (AF647) is preferably attached to the Avi-tag. A schematic illustration of respective chimeric constructs is shown in Figs. IB and 1C. Such chimeric Rad 18 construct is used for determining the effect of a test compound, in particular the effect of the test compound on energy transfer between the two fluorophores. In cases of europium and AF647 forming the FRET-pair, if excited with light at a wavelength of 320 nm, europium will transfer energy to excite AF647 to emit light at a wavelength of 665 nm. This signal is detected and serves as a positive control for an undisturbed interaction between the RING domain and the SAP domain (schematically shown in Fig. 6A). A test compound interfering with the RING-SAP interaction will enlarge the distance between the two fluorophores resulting in a decrease or loss of FRET signal (schematically shown in Fig. 6B). For a high- throughput method, the chimeric Rad 18 polypeptide and the test compounds are mixed in standard wellplates and FRET signals are detected using a fluorescence measuring plate reader machine such as the Spark® Multimode microplate reader (Tecan).

[0146] The test compounds identified in the FRET analysis to result in a loss of signal or a diminished FRET signal are optionally further tested in a ubiquitination activity test, which is performed analogous to the ubiquitination assay described above. In brief, PCNA or a fragment thereof comprising the ubiquitination domain, the identified test compound, and the full-length Radl8 / rad6 complex is incubated in the presence of ubiquitin, ubiquitin-activating enzyme (El) and ATP. A preferred incubation solution comprises 50 mM NaCl, 50 mM Tris, 2.5 mM MgC’F and 1 mM DTT for 30 to 60 minutes at 30°C. After incubation, ubiquitination of PCNA or its fragment is determined. As a control, the RING polypeptide comprising mutation R5 IE can be used which was shown to result in essentially no ubiquitination of PCNA (cf. Fig. 5). Samples are subsequently loaded onto an SDS PAGE gel and analyzed by western blot. For mass spectrometry analysis, the reaction is performed at 37°C for 1 hour, and the samples are run on an SDS PAGE gel. List of non-patent literature

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[0148] Du M, Gu J, Liu C, Liu N, Yu Z, Zhou C, et al. Genome-wide CRISPR screen identified Rad 18 as a determinant of doxorubicin sensitivity in osteosarcoma. J Exp Clin Cancer Res. 41(1) (2022).

[0149] Evans R, O’Neill M, Pritzel A. et al. Protein complex prediction with AlphaFold-Multimer. bioRxiv, 2022, doi: https: / / doi.Org / 10.1101 / 2021.10.04.463034.

[0150] Hibbert RG, Huang A, Boelens R, Sixma TK. E3 ligase Rad 18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6. Proc. Natl. Acad. Sci. USA. 108(14): 5590-5 (2011).

[0151] Hu Q, Botuyan MV, Cui G, Zhao D, Mer G. Mechanisms of Ubiquitin-Nucleosome Recognition and Regulation of 53BP1 Chromatin Recruitment by RNF168 / 169 and Radl8. Mol. Cell 66(4):473-87 (2017

[0152] Huang A, Hibbert RG, de Jong RN, Das D, Sixma TK, Boelens R. Symmetry and Asymmetry of the RINGRING Dimer of Rad 18. J. Mol. Biol. 410(3): 424-35 (2011).

[0153] Jones JS, Weber S, Prakash L. The Saccharomyces cerevisiae Radl8 gene encodes a protein that contains potential zinc finger domains for nucleic acid binding and a putative nucleotide binding sequence. Nucleic Acids Res. 16(14): 7119-31 (1988).

[0154] Kim J, Guermah M, McGinty RK, Lee JS, Tang Z, Milne TA, et al. RAD6-Mediated transcription-coupled H2B ubiquitylation directly stimulates H3K4 methylation in human cells. Cell. 137(3):459— 71 (2009).

[0155] Lambert-Smith IA, Saunders DN and Yerbury JJ. The pivotal role of ubiquitin-activating enzyme El (UBA1) in neuronal health and neurodegeneration. Int J Biochem Cell Biol. 2020 Jun: 123: 105746.

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[0159] Miyase S, Tateishi S, Watanabe K, Tomita K, Suzuki K, Inoue H, Yamaizumi M. Differential regulation of Radl8 through Rad6-dependent mono- and polyubiquitination. J. Biol. Chem. 280(1): 515-24 (2005). Notenboom V, Hibbert RG, van Rossum-Fikkert SE, Olsen JV, Mann M, Sixma TK. Functional characterization of Rad 18 domains for Rad6, ubiquitin, DNA binding and PCNA modification. Nucleic Acids Res. 35(17): 5819-30 (2007).

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Claims

Claims1. A method for identifying inhibitors of Rad 18 mediated ubiquitination or for determining the inhibitory activity of inhibitors of Rad 18 mediated ubiquitination, the method comprising the steps of: a) contacting either:(i) a first polypeptide comprising a Radi 8 catalytic RING (RING) domain and a Rad 18 DNA-binding SAP (SAP) domain, or(ii) a second polypeptide comprising the RING domain but not the SAP domain and a third polypeptide comprising the SAP domain but not the RING domain, wherein at least one first chromophore is bound to an amino acid of or an amino acid in the vicinity of the RING domain and at least one second chromophore is bound to an amino acid of or an amino acid in the vicinity of the SAP domain, and wherein the at least one first chromophore and the at least one second chromophore is a resonance energy transfer (RET) pair, with a test compound; and b) detecting an RET signal in the presence of the test compound and optionally also in the absence of the test compound, wherein:(i) a test compound is an inhibitor of Rad 18 mediated ubiquitination if: no RET signal is detected in step b), or the RET signal is reduced in the presence of the test compound in comparison to a reference RET signal or in comparison to the RET signal detected in the absence of the test compound; or(ii) the inhibitory activity of the test compound is determined by comparing the RET signal detected in step b) with a reference RET signal or with the RET signal detected in the absence of the test compound.

2. The method according to claim 1, wherein the first polypeptide is a chimeric polypeptide comprising the RING and the SAP domain that are optionally connected via a peptide linker.

3. The method according to claim 2, wherein the linker is a glycine-serine peptide linker, preferably a (GSGS)n, (GSGG)n, or (GGGS)n peptide linker with n being an integer of between 1 and 15, more preferably wherein n is an integer of between 2 and 10, and most preferably an integer of between 4 and 8.

4. The method according to any one of claims 1 to 3, wherein the at least one first chromophore and the at least one second chromophore are fluorophores, preferably AF647 and europium, and wherein the RET is fluorescence RET (FRET).

5. The method according to any one of claims 1 to 4, wherein the inhibitor inhibits Radi 8 mediated ubiquitination of proliferating cell nuclear antigen (PCNA).

6. The method according to any one of claims 1 to 5, wherein the detecting in step b) is carried out using a fluorescence measuring device, preferably a fluorescence measuring plate reader machine.

7. The method according to any one of claims 1 to 6, further comprising the step of determining the inhibitory activity of the test compound in a PCNA ubiquitination assay.

8. The method according to claim 7, wherein the ubiquitination assay comprises the steps of i) incubating PCNA or a fragment thereof comprising the ubiquitination domain, the test compound and either the first polypeptide, or the second and third polypeptide in the presence of ubiquitin and ATP; and ii) determining ubiquitination of PCNA or fragment thereof.

9. The method according to claims 1 to 8 , wherein the test compound that is determined to be an inhibitor of Radi 8 mediated ubiquitination is formulated with at least one pharmaceutically acceptable excipient or diluent.

10. A chimeric polypeptide comprising a Radl8 catalytic RING (RING) domain and a Radl8 DNA- binding SAP (SAP) domain, wherein the RING domain and the SAP domain are connected via a heterologous peptide linker.

11. The chimeric polypeptide according to claim 10, wherein the chimeric polypeptide does not comprise any other intact Radi 8 domain, preferably wherein the other Rad 18 domain is selected from the group consisting of the ZnF domain, the R6B domain, and the Pol ij domain.

12. The chimeric polypeptide according to claim 10 or 11, wherein the heterologous peptide linker is a glycine-serine peptide linker, preferably a (GSGS)n, (GSGG)n, or (GGGS)npeptide linker with n being an integer of between 1 and 10, more preferably wherein n is an integer of between 4 and 8.

13. The chimeric polypeptide according to any one of claims 10 to 12, wherein at least one first chromophore is bound to an amino acid of or an amino acid in the vicinity of the RING domain, and wherein at least one second chromophore is bound to an amino acid of or an amino acid in the vicinity of the SAP domain, wherein the at least one first chromophore and the at least one second chromophore is a resonance energy transfer (RET) pair.

14. The chimeric polypeptide according to any one of claims 10 to 13, wherein the chimeric polypeptide comprises a first polypeptide comprising SEQ ID NO: 2 or a sequence at least 80% identical thereto, and a second polypeptide comprising SEQ ID NO: 4 or a sequence at least 80% identical thereto.

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