Identifying test molecules that mediate targeted degradation

Abstract

The present invention relates to a method for identifying test molecules capable of binding an oligomeric target protein and a RING-type E3 ligase in the presence of a fusion protein, and a cell expressing said fusion protein.

Description

[0001] IDENTIFYING TEST MOLECULES THAT MEDIATE TARGETED DEGRADATION

[0002] Field of the invention

[0003] The present invention relates to a method for identifying test molecules capable of binding an oligomeric target protein and a RING-type E3 ligase in the presence of fusion proteins, in a cell-free (in vitro) system or in a cell expressing said fusion proteins.

[0004] Background to the invention

[0005] Neurodegenerative diseases (NDs) include highly debilitating illnesses, such as Alzheimer’s (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxias, frontotemporal dementia, corticobasal degeneration, progressive supranuclear palsy, chronic traumatic encephalopathy, multiple system atrophy, dementia with Lewy bodies, and prion diseases (PrD).

[0006] Aggregates of intracellular proteins into oligomeric proteins are proposed to drive age-related NDs, such as AD. Progressive accumulation of oligomeric protein aggregates throughout the brain occurs via a prion-like seeded aggregation processes, whereby protein aggregates from one cell are taken up by neighbouring cells where they template the aggregation of the cognate monomer protein. Prevention of seeded protein aggregation and / or targeted degradation of protein aggregates and / or seeds themselves is predicted to have therapeutic benefit.

[0007] RING-type E3 ubiquitin ligases mediate targeted degradation of proteins. TRIM21 is a RING-type E3 ubiquitin ligase that is activated by a target-induced clustering mechanism. TRIM21 ubiquitin ligase activity requires the clustering of multiple TRIM21 molecules around a target to induce intermolecular dimerization of RING domains. TRIM21 recruitment to target proteins that are sufficiently oligomeric (or aggregated) to induce TRIM21 clustering results in ubiquitination and subsequent degradation of the target oligomeric protein, however monomeric proteins targeted in the same manner are not degraded because TRIM21 is not clustered and activated.

[0008] Accordingly, RING-type E3 ubiquitin ligases, such as TRIM21, may be useful therapeutic targets for NDs and other illnesses associated with aggregatory proteins. There is a requirement in the art for assays suitable to identify potential therapeutic agents that can utilise E3 ubiquitin ligases to treat NDs and other illnesses associated with aggregatory proteins.Summary of the invention

[0009] The present invention takes advantage of the fact that protein aggregates present multiple binding sites, in order to provide a screening platform for candidate therapeutics that is particularly efficient, effective and flexible. Conventional methods for detecting the proximity of two proteins, or the ability of an agent or intervention to bring two proteins into proximity, may utilise a split reporter protein that is separated across the two proteins of interest. If the two proteins are brought into proximity, then a signal is generated. In contrast, the present invention provides a method of identifying a test molecule capable of binding an oligomeric target protein and a RING-type E3 ligase that utilises a very different approach, whereby a split reporter protein is separated across two different fusion proteins that both comprise the RING-type E3 ligase or fragment thereof. The oligomeric target protein does not need to be labelled with the split reporter protein, or otherwise modified with any sort of tag. Instead, a signal is generated when multiple RING-type E3 ligase fusions are brought into proximity at the oligomeric target protein. If a test molecule is capable of binding an oligomeric target protein and a RING-type E3 ligase into proximity, then it will be useful for mediating degradation of the oligomeric target protein and may be a candidate therapeutic.

[0010] The methods of the invention provide a powerful platform because it is not necessary to label or otherwise modify the target protein. Therefore, the same reagents can be used to screen for test molecules that may mediate the degradation of any oligomeric target protein of interest. Also, the method of the invention may be used with oligomeric proteins obtained directly from patient samples. Accordingly, for proteopathic seed targets the present invention circumvents the necessity to detect downstream seeded aggregation of the cognate monomer that normally relies on high overexpression, which is problematic for detection of targeted protein degradation activity and relies on engineering different cell lines and assay setups for each different oligomeric target protein to be tested.

[0011] The present invention provides particular advantages for identifying test molecules capable of binding an oligomeric target and TRIM21. Intermolecular TRIM21 clustering precisely correlates with oligomeric target protein degradation. The present invention demonstrates that detection of TRIM21 clustering can be used as a proxy for oligomeric target protein ubiquitination and subsequent degradation.

[0012] The present invention allows for high-throughput screening of test compound libraries for activity that induces clustering of RING-type E3 ligases, in particular TRIM21, only in the presence of oligomeric target proteins. Compounds with such activity are predicted to induce RING-type E3 ligases or TRIM21 recruitment to oligomeric target proteins in a manner that leads to their ubiquitination and degradation. For proteopathic seed targets this activity is predicted to prevent seeded aggregation of the cognatemonomer. Because proteopathic seeds template the identical aggregation of cognate intracellular monomers, such compounds are also predicted to cause RING-type E3 ligase or TRIM21 -mediated degradation of previously seeded intracellular protein aggregates.

[0013] The present invention also allows for the identification of test molecules that can bind a target protein in its oligomeric state, including fibrillar forms. This is important as the development of target-binding molecules, such as antibodies, that selectively bind oligomeric forms of a target protein, is technically challenging. Accordingly, the identification of such test molecules is of substantial value for both diagnostic and therapeutic applications. Thus, the invention allows for high-throughput screening of test compound libraries for compounds with the ability to bind a target protein in an oligomeric state.

[0014] Accordingly, the invention provides a method of identifying a test molecule capable of binding an oligomeric target protein and a RING-type E3 ligase, wherein the method comprises contacting a first fusion protein that comprises the RING-type E3 ligase or fragment thereof and a first portion of a splitreporter protein and a second fusion protein that comprises the RING-type E3 ligase or fragment thereof and a complementary second portion of the split-reporter protein with the test molecule, in the presence of the oligomeric target protein.

[0015] In certain embodiments, the method may be performed in a cell. The first and second fusion proteins may be expressed in the cell. Such methods may allow the effects of a test molecule to be assessed in the cytosol environment. In certain embodiments, the oligomeric target protein may be expressed in the cell. In certain embodiments, the oligomeric target protein may be applied exogenously to the cell, which may allow for the same cell line to be used to screen for any binders to any oligomeric target protein.

[0016] In other embodiments, the method may be performed in a cell-free (in vitro) system. The first and second fusion proteins may be recombinant proteins mixed together with the oligomeric target protein in solution. Such methods do not require the presence of a cell and allow the effects of a test molecule to be assessed in solution.

[0017] In certain embodiments, the oligomeric target protein may be isolated from a sample obtained from a patient. Such methods may allow therapeutically-relevant disease oligomers to be assayed.

[0018] In certain embodiments, the contacting is performed in a cell and the endogenous RING-type E3 ligase is inactivated, for example by gene knockout. Such cells may provide a more sensitive assay because any test molecule that is capable of binding the RING-type E3 ligase will bind the split reporter protein fusions rather than endogenous RING-type E3 ligase.In preferred embodiments, the RING-type E3 ligase or fragment thereof is TRIM21. In preferred embodiments, the fragment thereof is a fragment of TRIM21. TRIM21 ’s enzymatic activity is activated when multiple TRIM21 molecules are brought into close proximity. In some embodiments TRIM21 comprises a mutation that reduces or ablates ubiquitin ligase activity. In some embodiments TRIM21 comprises an amino acid substitution at residues: 118; R55; MIO; and / or M72. Preferably, TRIM21 comprises the following amino acid substitution mutations: I18R; R55A; M10E; and / or M72E. In some embodiments TRIM21 further comprises a mutation, such as I18R; and / or M72E and / or comprises deletion of the RING domain and / or RING-box domain. These mutations prevent degradation of the oligomeric target protein, thereby stabilising activity of the split-reporter protein and the resulting signal.

[0019] In certain embodiments, the method of the invention also comprises isolating a test molecule that is identified as capable of binding the oligomeric target protein and the RING-type E3 ligase. The method may also comprise formulating the test molecule in a pharmaceutically acceptable carrier to provide a pharmaceutical composition comprising the test molecule. The invention also provides a method for producing a molecule capable of binding an oligomeric target protein and the RING-type E3 ligase comprising identifying a molecule capable of binding the oligomeric target protein and the RING-type E3 ligase in accordance with the invention, producing the molecule, and optionally modifying or optimising the molecule.

[0020] Accordingly, the invention also provides a test molecule obtained or isolated from the methods of the invention, as well as the use of that test molecule in the treatment of disease, particularly proteinopathies, such as but not limited to, those diseases listed in Table 1.

[0021] In certain embodiments, the invention provides a cell expressing a first fusion protein that comprises a RING-type E3 ligase or fragment thereof and a first portion of a split-reporter protein and a second fusion protein that comprises a RING-type E3 ligase or fragment thereof and a complementary second portion of the split-reporter protein. Such cells are useful in the methods of the invention and provide a platform that may be used for assaying the ability of any test molecule to mediate degradation of any oligomeric target protein by the RING-type E3 ligase. In preferred embodiments, the RING-type E3 ligase is TRIM21 or a fragment thereof. Optionally, the TRIM21 comprises a mutation that reduces or ablates ubiquitin ligase activity such as an amino acid substitution at residues: 118; R55; MIO; and / or M72, preferably one or more amino acid substitutions selected from: I18R; R55A; M10E; and / or M72E. In certain embodiments, the cell has been engineered to express the oligomeric target protein.

[0022] In some embodiments, the invention provides a method of screening a library of test molecules for a test molecule capable of binding an oligomeric target protein and a RING-type E3 ligase, the methodcomprising contacting a first fusion protein that comprises the RING-type E3 ligase or fragment thereof and a first portion of a split-reporter protein and a second fusion protein that comprises the RING-type E3 ligase or fragment thereof and a complementary second portion of the splitreporter protein with a test molecule in the presence of the oligomeric target protein, detecting a signal from the reporter protein, and selecting the test molecule that generates a signal. Nonlimiting examples of split reporter proteins that can be used in the invention are described in Table 3. In one embodiment, the signal (i.e. readout) from the split reporter is selected from fluorescence, luminescence and biotinylation. In one embodiment the signal is luminescence.

[0023] Brief description of the drawings

[0024] Figure 1. Identification of test molecule clustering of an oligomeric target protein and RING-type E3 ligase or fragment thereof through split-reporter protein detection.

[0025] Figure 2. Identification of therapeutic antibody clustering of an oligomeric target protein and RING-type E3 ligase or fragment thereof through split-reporter protein detection.

[0026] Figure 3. Tau antibody induces clustering of TRIM21 with tau fibrils. LgBiT-TRIM21 and SmBiT-TRIM21 proteins were incubated with titration of tau- 12 antibody in the presence or absence of tau pre-formed fibrils (PFFs). Luminescence signal is caused by proximity of LgBiT-TRIM21 and SmBiT-TRIM21. Baseline is condition containing LgBiT-TRIM21 and SmBiT-TRIM21 only.

[0027] Figure 4. Tau antibody induces TRIM21 clustering with tau fibrils but not tau monomer. LgBiT-TRIM21 and SmBiT-TRIM21 proteins were incubated with a titration of tau- 12 antibody in the presence of either tau PFFs or tau monomer. Luminescence signal is caused by proximity of LgBiT-TRIM21 and SmBiT-TRIM21. Baseline is condition containing LgBiT-TRIM21 and SmBiT-TRIM21 only.

[0028] Figure 5. Tau antibody induces TRIM21 clustering with tau isolated from tauopathy mouse brains. LgBiT-TRIM21 and SmBiT-TRIM21 proteins were incubated with a titration of tau-12 antibody in the presence of either (a) tau PFFs or (b) aged PS 19 mouse brain sarkosyl-insoluble tau (PS19 SI tau). Luminescence signal is caused by proximity of LgBiT-TRIM21 and SmBiT-TRIM21. Baseline is condition containing LgBiT-TRIM21 and SmBiT-TRIM21 only.Figure 6. Small molecule TRIMTAC induces TRIM21 clustering with aggregated tau. LgBiT-TRIM21 and SmBiT-TRIM21 proteins were incubated with FKBPF36V-tagged sarkosyl-insoluble tau (FKBPF36VSI tau) in the presence of a titration of either MRC414 or MRC37. Luminescence signal is caused by proximity of LgBiT-TRIM21 and SmBiT-TRIM21. Baseline is condition containing LgBiT-TRIM21 and SmBiT-TRIM21 only.

[0029] Detailed description of the invention

[0030] The present invention provides a method for identifying a test molecule capable of binding an oligomeric target protein and a RING-type E3 ligase. The present invention comprises contacting a first fusion protein that comprises the RING-type E3 ligase or fragment thereof and a first portion of a splitreporter protein and a second protein that comprises the RING-type E3 ligase or fragment thereof and a complementary second portion of the split-reporter protein with the test molecule in the presence of the oligomeric target protein. The test molecules that induce clustering of the RING-type E3 ligase or fragment thereof and oligomeric target protein enable the first and second portions of the complementary split-reporter protein to come into proximity and generate a signal that can be detected.

[0031] Test molecules identified by the invention have two criteria. First, they allow a ternary complex to form between the test molecule, the RING-type E3 ligase or fragment thereof and the oligomeric target protein. Second, these ternary complexes form part of a higher order structure in which multiple RING-type E3 ligases or fragment thereof are brought into close proximity. Test molecules capable of clustering RING-type E3 ligase or fragment thereof and the oligomeric target protein bring the first portion of a split-reporter protein and complementary second portion of the split-reporter protein within close proximity, leading to a reporter signal being activated. The reporter signal is detected by screening with a reagent and / or high-throughput imaging techniques. This makes it possible to identify test molecules capable of clustering RING-type E3 ligase or fragment thereof and the oligomeric target protein in a cell-free (in vitro) or cell-based system.

[0032] Oligomeric target protein

[0033] The invention relates to identifying a test molecule capable of binding an oligomeric target protein and a RING-type E3 ligase or a fragment thereof.

[0034] In some embodiments, the oligomeric target protein is selected from the list consisting of: tau protein; alpha-synuclein; amyloid P (A ); islet amyloid polypeptide (IAPP); serum amyloid A (SAA); prionproteins; Huntingtin (HTT); misfolded transthyretin protein (TTR); TAR DNA binding protein 43 (TDP-43); N0TCH3 receptor; C9orf72 dipeptide repeat protein; fused in sarcoma (FUS) protein; super oxide dismutase (SOD); rhodopsin (ROD); cGAS; STING; RIG-I, MDA-5; MyD88; IRAK1 / 2 / 4; MAL; ASC; NLRP3; TRAF family; TBK1; RIPK1 / 3; Gasdermin; Apaf-1; PIDD; p53; BRD4; CBX2; PRC 1 / 2; Ras family; Raf; EWS; p-TEFb; YAP; TAZ; TEAD; HSF1; ENL; LAT; Grb2; Sosl; Nek; N-WASP; Arp2 / 3; Axin; APC; GSK3beta; Dishevelled; STAT1 / 2 / 6; SHP2; HSD17B13; PKA; G protein-coupled receptors (GPCRs); Immune checkpoint receptors, such as PD1, PDL1, CTLA-4, LAG-3, TIGIT; Ion channels, such as sodium channel Navi.7; p-glycoprotein; Fc receptors for IgG (FcyRs); Cytokine / chemokine receptors, such as IFNAR; TNFR; Asthma-related receptors, such as IL-33 / IL-1RL1; histamine receptors; TREM2 / CD33; Receptor; tyrosine kinases, such as ALK; and an ALK fusion partner, such as EML4, NPM1, SQSTM1, DCTN1, HIP1, KIF5B, STRN, or RANBP2; Virus receptors, such as ACE2; CCR5; cxcr4; CD4; or LDLR.

[0035] Test molecules that are capable of binding an oligomeric target protein and a RING-type E3 ligase, as identified in accordance with the invention, may be useful for mediating targeted degradation of the oligomeric protein, and treating disease. Examples of oligomeric target proteins for which binding molecules may be identified using of the present invention are set out in Table 1, alongside associated diseases and conditions.

[0036] Table 1 - Oligomeric target proteins and associated diseases

[0037]

[0038]

[0039]

[0040]

[0041]

[0042] In some embodiments, the oligomeric target protein forms / lomo-oligomcrs. In some embodiments, the oligomeric target protein is a homo-oligomer. Homo-oligomers are comprised of multiple copies of the same target protein. Test molecules of the invention capable of binding homo-oligomeric target proteins and a RING-type E3 ligase bring the first and second portions of the split-reporter protein into proximity and result in generation of a signal. Homo-oligomers present multiple identical binding sites that may be bound by multiple test molecules, leading to clustering of the RING-type E3 ligase fusion and activation of the split reporter protein. Thus, test molecules capable of binding homo-oligomeric target proteins and a RING-type E3 ligase are identifiable by the invention.

[0043] The invention may also be used with heterooligomeric proteins. Heterooligomeric target proteins may form disease-relevant biomolecular condensates that cluster multiple binding test molecules, leading to clustering of the RING-type E3 ligase fusion and activation of the split reporter protein.

[0044] In some embodiments, if the target protein is a hetero-oligomeric target, it forms a biomolecular condensate. In some embodiments, the oligomeric target protein is part of a biomolecular condensate. In some embodiments, the hetero-oligomeric target protein may form part of a protein complex, such as: necrosome complex; Apaf-1 apoptosome; PIDD signalling complex; Death-inducing signalling complex (DISC); YAP-TAZ-TEAD complex; LAT-Grb2-Sos 1 complex; Nck-N-WASP-Arp2 / 3 complex; Axin-APC-GSK3beta complex; Axin-dishevelled complex; a polycomb complex; microtubules; microfilaments; intermediate filaments; or NLRP3 inflammasome complex.

[0045] In some embodiments, the oligomeric target protein is composed of any one of: cGAS; STING; RIG-I; MDA-5; MyD88; IRAK1 / 2 / 4; MAL; ASC; NLRP3; TRAF family; TBK1; RIPK1 / 3; Gasdermin; Apaf-1; PIDD; p53; BRD4; CBX2; PRC1 / 2; Ras family; Raf; EWS; p-TEFb; YAP; TAZ; TEAD; HSF1; ENL; LAT; Grb2; Sosl; Nek; N-WASP; Arp2 / 3; Axin; APC; GSK3beta; Dishevelled; STAT1 / 2 / 6; SHP2; PKA; G protein-coupled receptors (GPCRs); HSD17B13; nucleoporin; peripherin; Receptor tyrosine kinases, such as ALK; and an ALK fusion partner, such as EML4, NPM1, SQSTM1, DCTN1, HIP1, KIF5B, STRN, or RANBP2, or an oligomeric domain thereof. In preferred embodiments, the oligomeric target proteins outlined above are expressed in a cell.

[0046] In some embodiments, the oligomeric target proteins are exogenously applied to a cell, or the oligomeric target proteins are in a cell-free (in vitro) system.In some embodiments, the oligomeric target protein is isolated from a sample obtained from a patient diagnosed with: dementia, frontotemporal lobar degeneration (FTLD); Alzheimer’s disease (AD), progressive supranuclear palsy (PSP); corticobasal degeneration (CBD); argyrophylic grain disease (AGD); Huntington's disease (HD); Pick's disease; amyotrophic lateral sclerosis (ALS); chronic traumatic encephalopathy (CTE); multiple system atrophy (MSA); Parkinson’s disease (PD); dementia with Lewy bodies (DLB); or retinitis pigmentosa (RP). In preferred embodiments, the oligomeric target protein is isolated from a sample obtained from a patient and applied exogenously to a cell or tested in a cell-free (in vitro) system.

[0047] In some embodiments, the oligomeric target is composed of any one of: tau protein; alpha-synuclein; amyloid P (A ), islet amyloid polypeptide (IAPP), serum amyloid A (SAA), prion proteins, Huntingtin (HTT); misfolded transthyretin protein (TTR); TAR DNA binding protein 43 (TDP-43); N0TCH3 receptor, C9orf72 dipeptide repeat protein; fused in sarcoma (FUS) protein; super oxide dismutase (SOD); rhodopsin (ROD); or mitochondrial antiviral-signaling protein (MAVS) assemblies. In preferred embodiments, the oligomeric target proteins outlined above are applied exogenously to a cell or tested in a cell-free (in vitro) system. In other embodiments, the oligomeric target proteins outlined above is expressed in a cell.

[0048] In preferred embodiments, the oligomeric target protein is tau protein; TDP-43; alpha-synuclein; Huntingtin (HTT); or RIPK3.

[0049] In some embodiments, the oligomeric target protein is expressed in a cell.

[0050] In some embodiments, the target protein is initially monomeric and oligomerisation is induced during the assay. In some embodiments, addition of a third component in addition to the oligomeric target protein and a RING-type E3 ligase or a fragment thereof results in oligomerisation of the target protein.

[0051] Example oligomeric target proteins are discussed below.

[0052] Tau proteins

[0053] 4R tauopathies are characterised by deposition of filaments with enrichment for 4R tau isoforms. Familial FTLD-tau can be caused by rare mutations in MAPT, demonstrating a critical involvement of tau (Dickson et al., 2011; PMID 21720721). In progressive supranuclear palsy (PSP), tau has a causative role exemplified by instances of familial variants with MAPT mutations (Morris et al 2002 PMID: 11861703). MAPT H1 / H2 haplotype is implicated in disease risk in corticobasal degeneration (CBD)and argyrophylic grain disease (AGD), strongly supporting a causative role of tau in these pathologies (Houlden et al 2001 PMID: 11425937; Conrad et al 2004 PMID: 15030402).

[0054] Rare mutations in MAPT such as G389R cause Pick's disease and FTLD-tau with 3R fdaments, supporting a causative role of 3R tau isoforms in disease etiology (Murrell et al 1999; PMID: 10604746).

[0055] Some tau pathology comprises both 3R and 4R tau isoform, which corresponds closely with disease progression. In AD, pathological tau comprises both 3R and 4R and is closely associated with cognitive decline (Nelson et al 2013 PMID:22487856). A similar but non-identical tau fold comprising all six tau isoforms is observed in chronic traumatic encephalopathy (CTE), (Falcon et al 2019; PMID: 30894745). Rare familial mutations such as V337M and R406W can cause FTLD-tau with both 3R and 4R fdaments (Goedert et al 2017 PMID:28772101). Indicating that oligomeric target protein tau represents an ideal target for target molecule and E3 ligase clustering.

[0056] Huntingtin (HTT)

[0057] It has been reported that expanded CAG repeats encoding poly-glutamine cause Huntington's disease (Genetic Modifiers of Huntington’s Disease (GeM-HD) Consortium, 2019; PMID: 31398342). In one embodiment the RING domain is fused directly or indirectly to a portion of HTT that includes exon 1 or polyglutamine repeats. Indicating that oligomeric target protein HTT represents an ideal target for target molecule and E3 ligase clustering.

[0058] Alpha-synuclein

[0059] Alpha-synuclein is a core component of Lewy bodies (found in PD and DLB) and inclusions found in MSA (Spillantini et al 1997, PMID:9278044; Spillantini et al 1998, PMID:9726379). A causative role in pathogenesis is exemplified by the fact that SNCA copy number variants and point mutations cause inherited forms of PD (Ibanez, 2004 PMID: 15451225; Singleton et al 2003, PMID: 14593171). The filament structure from multiple system atrophy (MSA) has been resolved, and shown to be different to DLB by Schweighauser et al., (2020; PMID: 32461689). Indicating that oligomeric target protein alpha-synuclein represents an ideal target for target molecule and E3 ligase clustering.

[0060] TDP-43

[0061] TAR DNA binding protein 43 (TDP-43) is a 414 amino acid protein found in ALS and FTD-TDP43 inclusions (Arai, 2006; PMID: 17084815). Its role as a cause of neurodegeneration is exemplified bymutations in TARDBP that are causative of ALS and FTLD-U (Sreedharan 2008; PMID: 18309045; Neumann et al 2006; PMID: 17023659). Recent structures of TDP43 filamentous core from patients with Type B pathology reveals a core that extends from amino acid 282-360 (Arseni et al 2022; PMID: 34880495).

[0062] Antibody therapy against TDP43 in vivo appears to work through TRIM21 (Pozzi et al., 2020; PMID: 33021970), indicating that oligomeric target protein TDP-43 represents an ideal target for target molecule and E3 ligase clustering.

[0063] Fused in sarcoma (FUS) protein

[0064] FUS mutations cause ALS (Kwiatkowski Jr et al., 2009; PMID: 19251627) and FTD (Van Langenhove et al., 2010, PMID: 20124201).

[0065] It has been reported that an ASO mediated knockdown of FUS levels in mutant mice (and a human subject) reduces aggregates and improves lifespan (Korobeynikov et al., 2022, PMID: 35075293), indicating that oligomeric target protein FUS represents an ideal target for target molecule and E3 ligase clustering.

[0066] Super oxide dismutase (SOD)

[0067] Mutations in SOD1 can cause Amyotrophic lateral sclerosis (Deng et al., 1993; PMID: 8351519) (Saccon et al., 2013; PMID: 23687121). Mutations have been found along the length of the protein, and all reduce native SOD1 activity. SOD1 is reported to form ThT positive aggregates (Chattopadhyay et al., 2008, PMID: 19022905) which can be propagated in mice (Ayers et al., 2014; PMID: 25262000). Antibody therapy against SOD1 improves lifespan in mice (Maier et al., 2018; PMID: 30518612) and reduces motor neuron 5 toxicity in other models (Benkler et al., 2018; PMID: 30401824), indicating that oligomeric target protein SOD represents an ideal target for target molecule and E3 ligase clustering.

[0068] Apoptosis-associated speck-like protein (ASC) specks

[0069] ASC specks are formed when the protein ASC rapidly assembles into a large complex during inflammasome activation. ASC specks can be detected in the circulation of patients with chronic inflammatory diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). Indicating that oligomeric target protein ASC specks represent an ideal target for target molecule and E3 ligase clustering.RING-type E3 ligase or fragment thereof

[0070] The methods of the invention are useful in identifying a test molecule capable of binding an oligomeric target protein and a RING-type E3 ligase, and such test molecules may be effective to mediate targeted degradation of the oligomeric target protein via the ligase. The methods of the invention utilise a first fusion protein that comprises the RING-type E3 ligase or fragment thereof and a first portion of a splitreporter protein and a second fusion protein that comprises the RING-type E3 ligase or fragment thereof and a complementary second portion of the split-reporter protein with the test molecule.

[0071] The first and second fusion proteins do not necessarily require the entire RING-type E3 ligase, because the method of the invention aims to identify test molecules that bind the RING-type E3 ligase, and the full activity of the ligase and the full sequence are not required. In certain embodiments, the first and second fusion proteins comprise the domain of the RING-type E3 ligase that binds to the Fc portion of antibodies. Test molecules that bind this domain are likely to be useful therapeutics for mediating the RING-type E3 ligase targeted degradation of oligomeric proteins. In one embodiment the RING-type E3 ligase or fragment thereof comprises the “PRYSPRY” (SEQ ID NO: 5) domain of TRIM21. In another embodiment the RING-type E3 ligase or fragment thereof consists of the “PRYSPRY” domain of TRIM21. The “PRYSPRY” domain of TRIM21 is a globular fold that binds to the Fc portion of antibodies. Test molecules that bind this domain are likely to be useful therapeutics for mediating the TRIM21 targeted degradation of oligomeric proteins.

[0072] E3 ligases of the RING-type are characterised by the presence of a RING domain, which is the minimal element required to recruit E2~ubiquitin and stimulate ubiquitin transfer. In the present disclosure, the ligase activity of the RING-type E3 ligase or fragment thereof may be inactivated,

[0073] As described in Balaji V, Hoppe T. Regulation of E3 ubiquitin ligases by homotypic and heterotypic assembly. FlOOORes. 2020 Feb 6;9:F1000 Faculty Rev-88, regulation of these ligases is provided in part by homotypic and heterotypic combination of E3 ligases into oligomeric (i.e. higher order assembly) ubiquitylation complexes. As described in Errington, Wesley I., et al. "Adaptor protein selfassembly drives the control of a cullin-RING ubiquitin ligase." Structure 20.7 (2012): 1141-1153, the RING superfamily consists of many hundreds of E3s that differ greatly in size, structure, subunit stoichiometry, and mode of regulation. Common within the RING superfamily, however, is a remarkable propensity to self-assemble into oligomers to enhance activity.

[0074] Some RING E3s are large multi-subunit complexes. Such E3 ligase complexes of the RING type typically comprise a number of components or subunits which can self-assemble in vivo, which are typically a scaffold component, an adaptor component, a substrate recognition component, as well as acatalytic E3 ligase component. Once assembled these RING-type E3 ubiquitin ligase complexes can facilitate the direct transfer of ubiquitin from E2 -ubiquitin intermediates to the target protein.

[0075] In some embodiments, the RING-type E3 ligase or fragment thereof is selected from the list consisting of: TRIM21; CRBN; VHL; RNF4; XIAP; TRIM5; TRIM7; TRIM25; MDM2; CBL; BRCA1; STUB1 (CHIP); RBX1; BARD1; HTLF; NFX1; and TRIM56. Preferred RING-type E3 ligase for use in the invention are TRIM21, CRBN and VHL, or a fragment thereof. Most preferably, the RING-type E3 ligase is TRIM21 or a fragment thereof.

[0076] In certain embodiments, the RING-type E3 ligase or fragment thereof in the first fusion protein is identical to the RING-type E3 ligase or fragment thereof in the second fusion protein. In other embodiments, the first and second fusion proteins use different fragments of the same RING-type E3 ligase.

[0077] In some embodiments, the RING-type E3 ligase or fragment thereof comprises at least 7, 10, 15, 20, 25, 30 or 35 amino acids. In certain embodiments, the RING-type E3 ligase or fragment thereof is a TRIM21 polypeptide, such as a polypeptide comprising SEQ ID NO: 1 or a variant thereof, for example having 80%, 85%, 90%, 95% or 99% sequence identity thereto. In another embodiment, the RING-type E3 ligase or fragment thereof is a CRBN polypeptide, such as a polypeptide comprising SEQ ID NO: 2 or a variant thereof, for example having 80%, 85%, 90%, 95% or 99% sequence identity thereto. In another embodiment, the RING-type E3 ligase or fragment thereof is a VHL polypeptide, such as a polypeptide comprising SEQ ID NO: 3 or a variant thereof, for example having 80%, 85%, 90%, 95% or 99% sequence identity thereto. In another embodiment, the RING-type E3 ligase or fragment thereof is selected from: a scaffold component, an adaptor component, or a substrate recognition component.

[0078] In some embodiments, mutations are introduced into the RING-type E3 ligase or fragment thereof to reduce, ablate or prevent ubiquitin ligase activity, and thus prevent degradation of the oligomeric target protein and RING-type E3 ligase or fragment thereof, thereby stabilizing the luciferase activity upon recruitment of RING-type E3 ligase or fragment thereof to the oligomeric target protein. Any suitable mutation to ablate ubiquitin ligase activity may be used, including deletions or point mutations. In preferred embodiments, the RING-type E3 ligase or fragment thereof is TRIM21. In preferred embodiments, the fragment thereof is a fragment of TRIM21. In some embodiments TRIM21 comprises a mutation that reduces or ablates ubiquitin ligase activity. In some embodiments TRIM21 comprises an amino acid substitution at residues: 118; R55; M10; and / or M72. Preferably, TRIM21 comprises the following amino acid substitution mutations: I18R; R55A; M10E; and / or M72E. These mutations prevent degradation of the oligomeric target protein, thereby stabilising activity of the splitreporter protein and the resulting signal.E3 ligases comprise a number of components or subunits which can self-assemble in vivo', these include: a substrate receptor (which may in turn comprise an adaptor complex and substrate recognition module such as VHL), a scaffold (e.g. a Cullin2 polypeptide) as well as a catalytic E3 ligase component which is a RING protein (i.e. RING or RING-finger domain containing protein) such as a TRIM or RBX protein. In the present invention mutations may be introduced into the RING-type E3 ligase or fragment thereof to reduce, ablate or prevent ubiquitin ligase activity.

[0079] Those skilled in the art are well aware of examples of scaffold components, adaptor components, substrate recognition components, and catalytic E3 ligase components (see e.g. Buetow and Huang, 2016; PMID: 27485899).

[0080] Scaffold components may include: CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, CUL7, CUL9; adaptor components may include: SKP1, Elongin B, Elongin C, DDB1, BTB; substrate recognition components may include: FBXW1, FBXW11, FBXL1, FBXO1, VHL, LRR1, FEM1, KEAP1, SPOP, KLHL40, BTB6A, DCAF1, DCAF14, DDB2, CRBN; and E3 ligase components may include: RBX1, RBX2.

[0081] A “domain” is a folded protein structure which retains its tertiary structure independently of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and / or of the domain.

[0082] Tripartite motif (TRIM) proteins constitute a protein family based on a conserved domain architecture (known as RBCC) that is characterized by a RING (Really Interesting New Gene) finger domain, one or two B-box domains, a Coiled-coil domain, and a variable C-terminus. The full sequence of the preferred RING type E3 ligase, TRIM21, is shown in SEQ ID NO: 1.

[0083] In another embodiment, the RING type E3 ligase is cereblon (CRBN) or a fragment thereof. Cereblon is an E3 ubiquitin ligase component that recruits substrates for ubiquitination and subsequent proteasomal degradation.

[0084] In another embodiment, the RING type E3 ligase is the VHL (“Von Hippel-Lindau”) domain or protein or a fragment thereof. VHL is an E3 ubiquitin ligase component that recruits substrates for ubiquitination and subsequent proteasomal degradation.These examples of RING-type E3 ligases which can be utilised directly, or modified for use in, the present invention are set out in Table 2.

[0085] Table 2 - RING-type E3 ligases

[0086]

[0087]

[0088] The invention relates to a first fusion protein comprising a RING-type E3 ligase or fragment thereof. In some embodiments, the first fusion protein comprises the full-length E3 ligase fused to a first portion of a split-reporter protein. In other embodiments, the first fusion protein comprises a fragment of the E3 ligase fused to a first portion of a split-reporter protein. In other embodiments, the first fusion protein comprises the “PRYSPRY” domain fused to a first portion of a split-reporter protein. In other embodiments, the first fusion protein consists of the PRYSPRY domain fused to a first portion of a split-reporter protein.

[0089] The invention relates to a second fusion protein comprising a RING-type E3 ligase or fragment thereof. In some embodiments, the second fusion protein comprises the full-length E3 ligase fused to a complementary second portion of a split-reporter protein. In other embodiments, the second fusion protein comprises a fragment of the E3 ligase fused to a complementary second portion of a splitreporter protein. In other embodiments, the second fusion protein comprises the “PRYSPRY” domain fused to a complementary second portion of a split-reporter protein. In other embodiments, the second fusion protein consists of the “PRYSPRY” domain fused to a complementary second portion of a splitreporter protein.The “PRYSPRY” domain is a common C-terminal motif found in the TRIM protein family. In some embodiments, the invention relates to use of a RING-type E3 ligase or fragment thereof comprising of the “PRYSPRY” domain. In other embodiments, the invention relates to use of a RING-type E3 ligase or fragment thereof consisting of the “PRYSPRY” domain.

[0090] Split-reporter protein

[0091] The present invention comprises the step of contacting a first fusion protein that comprises the RING-type E3 ligase or fragment thereof and a first portion of a split-reporter protein and a second fusion protein that comprises the RING-type E3 ligase or fragment thereof and a complementary second portion of the split-reporter protein with the test molecule, in the presence of the oligomeric target protein.

[0092] In some embodiments, the split-reporter protein is a: split-luciferase protein; split-fluorescent protein, split-biotin ligase, split-horseradish peroxidase (HRP); or nano bioluminescence resonance energy transfer (nano-BRET), including a first portion comprising a HaloTag and a second portion comprising a NanoLuc®.

[0093] In some embodiments, the first portion of a split-luciferase protein comprises a large BiT (LgBiT)-E3 ligase and the second portion of a split-luciferase protein comprises a small BiT (SmBiT)-E3 ligase. In some embodiments, LgBiT and SmBiT are fused to the N-terminus of the RING-type E3 ligase or fragment thereof.

[0094] In some embodiments, the first portion of a split-luciferase protein comprises a LgBiT-TRIM21 and the second portion of a split-luciferase protein comprises a SmBiT-TRIM21.

[0095] In some embodiments, the RING-type E3 ligase or fragment thereof is: TRIM21, CBRN, VHL, RNF4, XIAP, TRIM5, TRIM7, MDM2, CBL, BRCA1, STUB1, RBX1, BARD1, HTLF, NFX1, or TRIM56. Preferably, the RING-type E3 ligase or fragment thereof is TRIM21. In some embodiments, the RING-type E3 ligase or fragment thereof comprises or consists of the “PRYSPRY” domain.

[0096] Examples of split reporter-proteins which can be utilised directly, or modified for use in, the present invention are set out in Table 3.Table 3 - Split- reporter proteins

[0097]

[0098] Any appropriate detection method may be used to detect a signal from the split reporter protein that indicates a test molecule is capable of binding the oligomeric target protein and the RING type E3 ligase. Some split reporter proteins may require additional reagents to elicit or detect a signal, and the methods of the invention comprise addition of such reagents as required.Linker

[0099] In some embodiments, the first fusion protein may comprise an amino acid linker connecting the RING-type E3 ligase or fragment thereof and the first portion of the split-reporter protein. In some embodiments, the second fusion protein may comprise an amino acid linker connecting the RING-type E3 ligase or fragment thereof and the second portion of the split-reporter protein.

[0100] In some embodiments, the two portions of the fusion protein may be linked directly by a single peptide bond or through a peptide linker containing one or more amino acid residues. Generally, the two portions and the linker will be in reading frame with each other. In preferred embodiments, the amino acid linker comprises or consists of glycine and serine.

[0101] Typically, fusion proteins will be prepared by DNA recombination techniques standard in the art and may be referred to herein as recombinant fusion proteins.

[0102] The term “recombinant” refers to genetic material (i.e., nucleic acids, the polypeptides they encode, and vectors and cells comprising such polynucleotides) that has been modified to alter its sequence or expression characteristics, such as by mutating the coding sequence to produce an altered polypeptide, fusing the coding sequence to that of another gene, placing a gene under the control of a different promoter, expressing a gene in a heterologous organism, expressing a gene at a decreased or elevated levels, expressing a gene conditionally or constitutively in a manner different from its natural expression profile, and the like. Generally, recombinant nucleic acids, polypeptides, and cells based thereon, have been manipulated by man such that they are not identical to related nucleic acids, polypeptides, and cells found in nature.

[0103] Test molecule

[0104] The invention relates to identification of a test molecule capable of binding an oligomeric target protein and a RING-type E3 ligase or a fragment thereof. Test molecules identified by the invention are capable of binding an oligomeric target protein. Test molecules identified by the invention are also capable of binding and, in the presence of the oligomeric target, clustering the RING-type E3 ligase or fragment thereof.

[0105] The test molecule may be any molecule, either naturally occurring or synthetic that is tested in an assay, such as a screening assay or drug discovery assay. As such, these compounds comprise organic or inorganic compounds. Suitable test molecules include polynucleotides, lipids, hormone analogs, smallpeptides or peptide-like molecules (peptidomimetics) comprising from about 2 to about 40 amino acids and larger polypeptides comprising from about 40 to about 500 amino acids, such as antibodies, antibody fragments or antibody conjugates.

[0106] In certain embodiments, the test molecule is a small molecule (e.g. , having a molecular weight of 1000 Da or less, such as 100 Da to 1000 Da or 100 Da to 600 Da) capable of binding a RING-type E3 ligase or fragment thereof and the oligomeric target protein. The term “small molecule” refers to molecules, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight. Typically, a small molecule is an organic compound (i.e., it contains carbon). The small molecule may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyl, carbonyls, and heterocyclic rings, etc.).In certain embodiments, the text molecule is part of a compound library. The method of the invention is useful for screening compound libraries for candidate therapeutic agents, for example in a high throughput manner. For example, DNA-encoded or peptide-encoded small molecule compound libraries can be used (DELs and PELs, respectively). DELs and PELs allow for the screening of millions to billions of different small molecule compounds. For a DEL-based screen, various DNA-encoded libraries (e.g., oligonucleotide barcodes attached to small molecule compounds) are commercially available. For example, see DELs available from HitGen, X-Chem, WuXi AppTec, and DyNAbind (via Sigma Aldrich).

[0107] Other libraries that may be useful in the practice of the invention include combinatorial or randomized libraries that provide a sufficient range of diversity, such as natural compound libraries, allosteric compound libraries, peptide libraries, antibody fragment libraries, synthetic compound libraries, fragment-based libraries, phage-display libraries, and the like.

[0108] In certain embodiments, the test molecule is a peptide capable of binding a RING-type E3 ligase or fragment thereof and the oligomeric target protein.

[0109] In certain embodiments, the test molecule is a recombinant protein capable of binding a RING-type E3 ligase or fragment thereof and the oligomeric target protein.

[0110] The invention also relates to a method of identifying an antibody or antibody fragment thereof. In some embodiments, the oligomeric target protein of interest is incubated in the presence of the first and second fusions proteins comprising the split-reporter protein and a test antibody or antibody fragment thereof. In some embodiments, the method of identifying an antibody or antibody fragment thereof is in a cell-free (in vitro) or cell-based system. In some embodiments, the split-reporter protein detects clustering of the oligomeric target protein and E3 ligase induced by the test antibody or antibody fragment thereof.Test antibodies or antibody fragments thereof that successfully induce clustering of the oligomeric target protein and E3 ligase are predicted to have therapeutic benefit (Mukadam et al., (2023) Science).

[0111] In preferred embodiments, the test molecule is identified in a cell -based system. In other embodiments, the test molecule is identified in a cell-free (in vitro) system.

[0112] Cell-free (in vitro) system

[0113] The invention also relates to use of purified split-reporter protein tested in a cell-free (in vitro) system. As described above, the split-reporter protein comprises a first protein that comprises the RING-type E3 ligase or fragment thereof and a first portion of a split-reporter protein and a second protein that comprises the RING-type E3 ligase or fragment thereof and a complementary second portion of the split-reporter protein. Preferably the split-reporter protein comprises a first protein, LgBiT-TRIM21, and a second protein, SmBiT-TRIM21. LgBiT and SmBiT are complementary proteins that reconstitute luciferase activity only when in close proximity, i.e. through protein-protein interactions.

[0114] In some embodiments, the first and second fusion proteins are in a cell-free (in vitro) system. In some embodiments the first and second fusion proteins are recombinant proteins mixed together in solution. The cell-free or in vitro system does not require the presence of a cell.

[0115] In some embodiments, the first protein and second protein of the complementary split-reporter protein complex are positioned at the N-terminus of the RING-type E3 ligase, adjacent to the RING domain that normally dimerises upon E3 ligase activation.

[0116] In the cell free system, the purified split-reporter protein and oligomeric target protein are mixed together in suspension. Addition of the split-reporter protein reagent results in a reporter signal if the test compound binds to both the RING-type E3 ligase or fragment thereof and the oligomeric target protein. Preferably, the first fusion protein, which may be LgBiT-TRIM21, and second fusion protein, which may be SmBiT-TRIM21, induce a luminescence signal upon the formation of a complex between the target molecule, oligomeric target protein and RING-type E3 ligase. Luminescence of a splitluciferase reporter is detected using a luminescence reagent kit, preferably the luminescence signal is detected with the Nano-Gio® Luciferase Assay System.Cell-based system

[0117] In some embodiments, the first and second fusion proteins are expressed in a cell.

[0118] In one embodiment, the invention relates to a RING-type E3 ligase knockout cell line stably coexpressing first and second fusion proteins of the invention, such as large BiT (LgBiT)-E3 ligase and a small BiT (SmBiT)-E3 ligase. Preferably, the invention relates to a TRIM21 knockout cell line stably co-expressing LgBiT-TRIM21 and SmBiT-TRIM21. LgBiT and SmBiT are protein fragments that reconstitute luciferase activity only when in close proximity, i.e. through protein-protein interactions (e.g. Promega NanoBiT). Preferably, the LgBiT and SmBiT tags are positioned at the N-terminus of TRIM21 adjacent to the RING domain that normally dimerises when TRIM21 is activated.

[0119] In some embodiments, the cell is contacted with a test molecule and oligomeric target protein of interest. If the test molecule induces TRIM21 clustering around the oligomeric target protein then luciferase activity is measured by luminescence readout using live-cell NanoGio luciferase assay kit.

[0120] Because target proteins are oligomeric they have multiple epitopes for small molecule binders. Thus, compounds that recruit other RING-type E3 ligases to oligomeric target proteins should also result in apparent clustering of multiple copies of that E3 ligase. For example, the same assay setup but with LgBiT-CRBN / VHL and SmBiT-CRBN / VHL.

[0121] In some embodiments, cell-based assays are performed in the presence of ubiquitin-proteasome inhibitors (e.g. TAK-243, MG132) to prevent target protein degradation and stabilize the luciferase signal. In some embodiments, point mutations in CRBN / VHL or CRL complexes are introduced in order to prevent ubiquitin transfer. The platform would screen for compounds that recruit multiple copies of CRBN / VHL to the oligomeric target protein.

[0122] In preferred embodiments, the RING-type E3 ligase of fragment thereof is TRIM21.

[0123] In some embodiments, endogenous TRIM21 or other RING-type E3 ligase is inactivated in the cell by gene knockout using a technique selected from: non-homologous DNA end joining (NHEJ); clustered regularly interspaced short palindromic repeats (CRISPR) editing; transcription activator-like effector nucleases (TALEN) editing; or artificial mutagenesis.

[0124] In some embodiments, RING-type E3 ligase expression is reduced in the cell by gene knockdown of RING-type E3 ligase using a technique selected from: small interfering RNA (siRNA); or short hairpin RNA (shRNA).Preferably, the cell is a human cell. Cells suitable for use in the invention include U2-OS cells, RPE-1 cells, and HEK293 cells.

[0125] In some embodiments, the cell is processed by immunofluorescence imaging. In some embodiments, the cell is fixed prior to immunofluorescence labelling (e.g. using paraformaldehyde (PF A)), or imaged in live culture. In preferred embodiments, the cell is processed in live cells using NanoGio reagent (e.g. Promega N2011) to detect interactions in the cytosol of living cells. In other embodiments, the cell is lysed and NanoGio reagent (e.g. Promega N1110) is used to detect interactions in the cell lysate.

[0126] The invention also relates to a cell expressing a first fusion protein that comprises a RING-type E3 ligase or fragment thereof and a first portion of a split-reporter protein and a second fusion protein that comprises a RING-type E3 ligase or fragment thereof and a complementary second portion of the splitreporter protein.

[0127] In some embodiments, the RING-type E3 ligase or fragment thereof is: TRIM21, CRBN, VHL, RNF4, XIAP, TRIM5, TRIM7, MDM2, CBL, BRCA1, STUB1, RBX1, BARD1, HTLF, NFX1, or TRIM56.

[0128] In some embodiments, TRIM21 comprises a mutation that reduces or ablates ubiquitin ligase activity, such as I18R; R55A; M10E; and / or M72E. In some embodiments TRIM21 further comprises a mutation, such as I18R; and / or M72E and / or deletion of the RING domain and / or RING-box domain.

[0129] In some embodiments, the cell expresses the oligomeric target protein. In other embodiments, the cell has been engineered to express the oligomeric target protein. In other embodiments, the cell is contacted with an exogenously applied oligomeric target protein.

[0130] In one embodiment the RING-type E3 ligase or fragment thereof is TRIM21. TRIM21’s enzymatic activity is activated when multiple TRIM21 molecules are brought into close proximity. In some embodiments TRIM21 comprises a mutation that reduces or ablates ubiquitin ligase activity. In some embodiments TRIM21 comprises an amino acid substitution at residues: 118; R55; MIO; and / or M72. Preferably, TRIM21 comprises the following amino acid substitution mutations: I18R; R55A; M10E; and / or M72E. These mutations prevent degradation of the oligomeric target protein, thereby stabilising activity of the split-reporter protein and the resulting signal.

[0131] TransfectionThe terms “transfect”, “transfection”, “transfected”, and like terms refer to the introduction of a gene into a eukaryotic cell. Transfection is defined as the process of introducing nucleic acids into cells by non-viral methods. Most commonly, transfection techniques rely on the introduction of plasmid DNA into the eukaryotic cell.

[0132] In some embodiments, transfection is used to express the split-reporter protein fusion in a host cell. In other embodiments, transfection is used to express the oligomeric target protein in a host cell.

[0133] A person skilled in the art would be able to construct vectors and design protocols for recombinant gene expression. Suitable vectors can be chosen or constructed, containing, in addition to the elements of the invention described above, appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: A Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press or Current Protocols in Molecular Biology, Second Edition, Ausubel et al., eds., John Wiley & Sons, (1995, and periodic supplements).

[0134] Transduction

[0135] The term "transduction", refers to the introduction of a gene into a eukaryotic cell via, for example, viral-mediated gene transfer, for example by use of recombinant AAV, adenovirus (Ad), retrovirus (e.g., lentivirus), or any other applicable viral-mediated gene transfer platform.

[0136] In some embodiments, transduction is used to express the split-reporter protein in a host cell. In other embodiments, transduction is used to express the oligomeric target protein in a host cell.

[0137] In some embodiments, transduction is used to generate a stable cell line expressing the split-reporter protein of the invention in a stable cell line. In other embodiments, transduction is used to generate a stable cell line expressing the oligomeric target protein in a stable cell line.

[0138] The term “stable” cell line refers to a group of cells that have been genetically modified to express a specific protein or gene. Stable cell lines are generated by transduction techniques as outlined above.

[0139] In certain embodiments of any aspect of the invention, the cells are not RPE-1 cells or the method uses cells that are not RPE-1 cells. In certain embodiments of any aspect of the invention, the oligomeric does not comprise an FK506-binding protein (FKBP) or any part thereof, such as a FKBP tag. In certain embodiments, the oligomeric target protein does not comprise any tag. In certain embodiments, the oligomeric target protein is naturally occurring. In certain embodiments, if the RING-type E3 ligase isTRIM21 and the split-reporter protein is a NanoBiT split luciferase and the contacting is performed in TRIM21 knock out cells, then the first and second fusions proteins do not both comprise the split luciferase portions at their N terminus.

[0140] Sequence identity

[0141] “Substantially identical” in the context of at least two nucleic acids or polypeptides means that a polynucleotide or polypeptide comprises either a sequence that has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a parent or reference sequence, or any other sequence that includes amino acid substitutions, insertions, deletions, or modifications made only to circumvent the present description without adding functionality.

[0142] Calculation of percentage identities between different amino acid / polypeptide / nucleic acid sequences may be carried out as follows. A multiple alignment is first generated by the ClustalX program (pair wise parameters: gap opening 10.0, gap extension 0.1, protein matrix Gonnet 250, DNA matrix IUB; multiple parameters: gap opening 10.0, gap extension 0.2, delay divergent sequences 30%, DNA transition weight 0.5, negative matrix off, protein matrix Gonnet series, DNA weight IUB; Protein gap parameters, residue-specific penalties on, hydrophilic penalties on, hydrophilic residues GPSNDQERK, gap separation distance 4, end gap separation off). The percentage identity is then calculated from the multiple alignment as (N / T)* 100, where N is the number of positions at which the two sequences share an identical residue, and T is the total number of positions compared. Alternatively, percentage identity can be calculated as (N / S)*100 where S is the length of the shorter sequence being compared. The amino acid / polypeptide / nucleic acid sequences may be synthesised de novo, or may be native amino acid / polypeptide / nucleic acid sequence, or a derivative thereof.

[0143] Alternatively, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to any of the nucleic acid sequences referred to herein or their complements under stringent conditions. By stringent conditions, we mean the nucleotide hybridises to filter-bound DNA or RNA in 6x sodium chloride / sodium citrate (SSC) at approximately 45°C followed by at least one wash in 0.2x SSC / 0.1% SDS at approximately 5-65°C. Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the peptide sequences according to the present invention.

[0144] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Degenerative ly equivalent (e.g. codon optimised) nucleotide sequences to any of those described herein may of course be used in their place.Thus, suitable nucleotide variants include those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example, small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0145] Terminology

[0146] The term “derived from” encompasses the terms “originated from”, “obtained from”, “obtainable from”, “isolated from”, and “created from” and generally indicates that one specified material finds its origin in another specified material or has features that can be described with reference to another specified material (which may be termed “reference” or “parent”). The RING-type E3 ligase or a fragment thereof herein may be derived from reference or parent sequences, for example form sequences of a wild-type subunit of a functional multimeric protein assembly and RING-type E3 ligases.

[0147] The terms “polynucleotide” encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single-stranded or double-stranded, and may have chemical modifications. The terms “nucleic acid” and “polynucleotide” are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences which encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in a 5'-to-3' orientation.

[0148] As described herein, a “polypeptide” refers to a molecule comprising a plurality of amino acids linked through peptide bonds. The terms “polypeptide,” “peptide,” and “protein” are used interchangeably. Proteins may optionally be modified (e.g., glycosylated, phosphorylated, acylated, famesylated, prenylated, and sulfonated) to add functionality. The conventional one-letter or three-letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino-to-carboxy terminal orientation (i.e., N-C).As used herein, the terms “wild-type”, “native”, or “reference” refer to polypeptides or polynucleotides that are found in nature. The terms, with respect to a polypeptide, refer to a naturally-occurring polypeptide that does not include a man-made substitution, insertion, or deletion at one or more amino acid positions. The terms with respect to a polynucleotide, refer to a naturally-occurring polynucleotide that does not include a man-made substitution, insertion, or deletion at one or more nucleosides. However, note that a polynucleotide encoding a wild-type or native or reference polypeptide is not limited to a naturally-occurring polynucleotide, and encompasses any polynucleotide encoding that polypeptide.Examples

[0149] Example 1 - Cell-free (in vitro) system

[0150] In order to practice an exemplification of the present invention, recombinant purified fusion proteins may be used, as described in Figure 1.

[0151] To identify test molecules capable of binding an oligomeric target protein and TRIM21, recombinant purified fusion proteins consisting of LgBiT-TRIM21 and SmBiT-TRIM21 may be used. LgBiT and SmBiT are complementary protein fragments of a split-luciferase protein that reconstitute luciferase activity only when in close proximity (Promega NanoBiT), i.e. through protein-protein interactions. LgBiT and SmBiT may be fused to the N-terminus of TRIM21 adjacent to the RING domain that normally dimerises when TRIM21 may be activated.

[0152] Recombinant purified fusion proteins LgBiT-TRIM21 and SmBiT-TRIM21 may be mixed with the oligomeric target protein of interest in vitro. In the absence of a test compound no signal would be expected from the split-luciferase protein, because the LgBiT- and SmBiT-TRIM21 proteins do not interact.

[0153] If the test molecule binds to both TRIM21 and the oligomeric target protein LgBiT-TRIM21 and SmBiT-TRIM21 proteins may come into proximity of one another (due to multiple binding sites for the compound on the oligomeric target protein) resulting in LgBiT: SmBiT NanoLuciferase complementation and thus luminescence reporter signal (NanoGio luciferase assay kit). Presence of the luciferase reporter signal therefore, may be used to indicate identification of a test molecule capable of binding an oligomeric target protein and TRIM21 in a cell-free (in vitro) system.

[0154] Example 2 - Cell-based system

[0155] In order to practice an exemplification of the present invention, a TRIM21 knockout cell line stably co-expressing LgBiT-TRIM21 and SmBiT-TRIM21 may be used.

[0156] To identify test molecules capable of binding an oligomeric target protein and TRIM21, a TRIM21 knockout cell line stably co-expressing LgBiT-TRIM21 and SmBiT-TRIM21 may be used. LgBiT and SmBiT are complementary split-luciferase protein fragments that reconstitute luciferase activity only when in close proximity of one another (Promega NanoBiT), i.e. through protein-protein interactions. LgBiT and SmBiT may be fused to the N-terminus of TRIM21 adjacent to the RING domain that normally dimerises when TRIM21 may be activated.Point mutations may be included in the TRIM21 RING domain to prevent ubiquitin ligase activity, and thus prevent degradation of seeds and TRIM21, thereby stabilizing the luciferase activity upon recruitment of TRIM21 to seeds. For example, point mutations I18R; R55A; M10E; and / or M72E may be used to stabilise luciferase activity.

[0157] The cell line may be contacted with a test molecule and transfected with a oligomeric target protein of interest. Alternatively, the oligomeric target protein of interest may be exogenously applied to the cell line. If the test molecule induces TRIM21 clustering around the oligomeric target protein then luciferase activity may be measured by luminescence readout using live-cell NanoGio luciferase assay kit. Thereby identifying a test molecule capable of binding an oligomeric target protein and TRIM21 in a cell-based system.

[0158] Example 3 - Therapeutic antibodies

[0159] In order to practice an exemplification of the present invention, the cell-based system may be used to screen for therapeutic antibodies that are capable of binding TRIM21 and an oligomeric target protein, as described in Figure 2. For example, an oligomeric target protein of interest may be incubated with a panel of test antibodies and delivered to the cell -free (in vitro) or cell-based assay, as described above. The assays would detect those antibodies that bind oligomeric target protein and TRIM21 in a manner that induces TRIM21 clustering. For proteopathic seed targets this activity may be predicted to prevent seeded aggregation of the cognate monomer and thus may have therapeutic benefit (Mukadam et al., (2023). Science; PMID: 36996217).

[0160] Example 4: The method identifies antibody that induces TRIM21 clustering with tau fibrils but not tau monomer

[0161] To directly measure TRIM21 clustering the NanoBiT split luciferase system was used. In this system luciferase activity is reconstituted only when the two protein fragments, LgBiT and SmBiT are brought into close proximity. Two different recombinant TRIM21 proteins containing either LgBiT tag (LgBiT-TRIM21) or SmBiT tag (SmBiT-TRIM21) were purified from E.coli. The LgBiT and SmBiT tags are encoded at the N-terminus of TRIM21 which contains the RING E3 ligase domain that must cluster to induce target protein ubiquitination (Zheng et al., Nature Structural and Molecular Biology (2021); PMID: 33633400 and Kiss et al., 2023. Nat Commun. PMID: 37061529). Thus, ubiquitination-competent clustered TRIM21 can be inferred by luciferase activity induced by LgBiT: SmBiT proximity.Using the LgBiT-TRIM21 and SmBiT-TRIM21 recombinant proteins an assay was developed to enable identification of molecules that induce TRIM21 clustering in the presence of aggregated target proteins. A candidate antibody was chosen (Tau-12) that binds to an epitope at the N-terminus of tau that is accessible in both tau monomers and fibrils (Ellis et al., (2024)Acta Neuropathologica; PMID: 38761203). Incubation of LgBiT-TRIM21 and SmBiT-TRIM21 with a titration of tau-12 antibody resulted in a tau-12 dose-dependent increase in luminescence only in the presence of tau pre-formed fibrils (PFFs) (Figure 3). This is indicative of the Tau-12 antibody binding to multiple epitopes on the tau fibrils and recruiting multiple TRIM21 proteins in close proximity due to binding to the antibody Fc domain (Figure 2). Luminescence peaked at tau-12 antibody concentrations around 10 nM, consistent with the expected binding affinity of TRIM21 with antibody Fc. Luminescence decreased at tau-12 antibody concentrations above 10 nM consistent with the expected ‘hook effect’ for heterobifiinctional molecules that bind E3 ligase and target protein simultaneously.

[0162] To confirm that the method selectively reports TRIM21 clustering induces by oligomeric target proteins, LgBiT-TRIM21 and SmBiT-TRIM21 were incubated with tau-12 antibody titration in the presence of tau monomer. Whereas tau PFFs induced tau-12 dose-dependent luminescence, tau monomer did not increase luminescence above baseline (Figure 3).

[0163] Example 5: The method is compatible with aggregates isolated from brain samples

[0164] Classical ternary complex assays require the labelling of both E3 ligase and target protein to measure induced proximity and are thus limited to relatively pure recombinant target proteins that can be tagged or labelled in vitro. To test if the present invention is compatible with protein aggregates isolated directly from disease tissue, sarkosyl-insoluble tau was extracted from aged brains of the PS19 P301S tauopathy mouse (Yoshiyama et al., (2007) Neuron. PMID: 17270732). Incubation of PS 19 sarkosyl-insoluble tau (PS19 SI tau) with LgBiT-TRIM21, SmBiT-TRIM21 and a titration of tau-12 antibody resulted in a tau-12 dose-dependent increase in luminescence (Figure 5a), similar to that seen with recombinant tau PFFs (Figure 5b).

[0165] Example 6: The method identifies small molecule that induces TRIM21 clustering with aggregated tau

[0166] To confirm that the present invention enables the identification of small molecules that induce TRIM21 clustering with aggregated target protein, the published small molecule TRIMTAC known as MRC414 (Luptak. (2025), Nature Communications. PMID: 41290659) was tested. MRC414 is a heterobifiinctional molecules consisting of a TRIM21 binder (MRC37), linker and an FKBPF36Vbinder (AP1867). To produce an aggregated target for MRC414 to bind, sarkosyl-insoluble aggregated tautagged with FKBPF36V(FKBPF36V-tau SI) was extracted from cells. Incubation of LgBiT-TRIM21 and SmBiT-TRIM21 with FKBPF36V-tau SI and a titration of MRC414 resulted in a MRC414 dosedependent increase in luminescence (Figure 6). This is indicative of the MRC414 molecule binding to multiple FKBPF36Vsites in the tau aggregates and thus recruiting multiple TRIM21 proteins in close proximity (Figure 1). Luminescence peaked at MRC414 concentrations around 100 nM, consistent with the published binding affinity of MRC414 for TRIM21. Luminescence decreased at MRC414 concentrations above 100 nM consistent with the expected ‘hook effect’ for heterobifunctional molecules that bind E3 ligase and target protein simultaneously. The TRIM21 binding molecule alone (MRC37) did not induce luminescence above baseline, confirming that the method specifically reports small molecules that bind both tau aggregates and TRIM21 concomitantly.

[0167] Methods

[0168] Recombinant TRIM21 protein. Full-length human TRIM21 was expressed with N-terminal 6His-Lipoyl-LgBiT (LgBiT-TRIM21) and 6His-Lipoyl-SmBiT (SmBiT-TRIM21) tags in BL21 E.coli cells and purified by nickel sepharose immobilized metal ion affinity chromatography followed by size exclusion chromatography using a Superdex 20026 / 60 gel filtration column.

[0169] Recombinant tau protein. Tau pre-formed fibrils (SPR-329) and tau monomer (SPR-327) were purchased from Stressmarq.

[0170] PS19 sarkosyl-insoluble tau . Frozen brain hemispheres from 9-month old PS 19 mice (JAX 008169) were homogenised in sarkosyl extraction buffer followed by centrifugation to pellet residual debris. The resulting supernatant was subjected to ultracentrifiigation (>100,000xg) to obtain sarkosyl-insoluble tauP301S aggregates. The concentration of aggregated tau in this sarkosyl-insoluble fraction was quantified via ELISA.

[0171] Sarkosyl-insoluble FKBPF36V-tau. Cells overexpressing FKBPF36V-Tau(P301S) were created via lentiviral transduction and antibiotic selection. To induce FKBPF36V-Tau(P301S) aggregation, cells were treated with sonicated recombinant tau PFFs in the presence of lipofectamine 2000. 72hrs posttreatment cells were lysed in sarkosyl extraction buffer and subjected to ultracentrifiigation (>100,000xg) to obtain sarkosyl-insoluble FKBPF36V-Tau(P301S) aggregates. The concentration of aggregated tau in this sarkosyl-insoluble fraction was quantified via ELISA.

[0172] Test molecules. Tau-12 antibody was purchased from Merck (ZMS1065). Compounds MRC414 and MRC37 are described in Luptak. (2025), Nature Communications. PMID: 41290659TRIM21 clustering assay. LgBiT-TRIM21 and SmBiT-TRIM21 at final concentration 5 nM are incubated with target protein and test molecule (at final concentrations indicated in figures) for Ih at room temperature prior to addition ofNanoGlo substrate (Promega) and luminescence measured on a plate reader. Luminescence values plotted are normalised to conditions containing LgBiT-TRIM21 and SmBiT-TRIM21 only (baseline).Sequence listing

[0173] SEQ ID NO: 1 - TRIM21 full sequence

[0174] MASAARLTMM WEEVTCPICL DPFVEPVSIE CGHSFCQECI SQVGKGGGSV CPVCRQRFLL KNLRPNRQLA NMVNNLKEIS QEAREGTQGE RCAVHGERLH LFCEKDGKAL CWVCAQSRKH RDHAMVPLEE AAQEYQEKLQ VALGELRRKQ ELAEKLEVEI AIKRADWKKT VETQKSRIHA EFVQQKNFLV EEEQRQLQEL EKDEREQLRI LGEKEAKLAQ QSQALQELIS ELDRRCHSSA LELLQEVIIV LERSESWNLK DLDITSPELR SVCHVPGLKK MLRTCAVHIT LDPDTANPWL ILSEDRRQVR LGDTQQSIPG NEERFDSYPM VLGAQHFHSG KHYWEVDVTG KEAWDLGVCR DSVRRKGHFL LSSKSGFWTI WLWNKQKYEA GTYPQTPLHL QVPPCQVGIF LDYEAGMVSF YNITDHGSLI YSFSECAFTG PLRPFFSPGF NDGGKNTAPL TLCPLNIGSQ GSTDY

[0175] SEQ ID NO: 2 - CRBN full sequence

[0176] MAGEGDQQDA AHNMGNHLPL LPAESEEEDE MEVEDQDSKE AKKPNIINFD TSLPTSHTYL GADMEEFHGR TLHDDDSCQV IPVLPQVMMI LIPGQTLPLQ LFHPQEVSMV RNLIQKDRTF AVLAYSNVQE REAQFGTTAE IYAYREEQDF GIEIVKVKAI GRQRFKVLEL RTQSDGIQQA KVQILPECVL PSTMSAVQLE SLNKCQIFPS KPVSREDQCS YKWWQKYQKR KFHCANLTSW PRWLYSLYDA ETLMDRIKKQ LREWDENLKD DSLPSNPIDF SYRVAACLPI DDVLRIQLLK IGSAIQRLRC ELDIMNKCTS LCCKQCQETE ITTKNEIFSL SLCGPMAAYV NPHGYVHETL TVYKACNLNL IGRPSTEHSW FPGYAWTVAQ CKICASHIGW KFTATKKDMS PQKFWGLTRS ALLPTIPDTE DEISPDKVIL CL

[0177] SEQ ID NO: 3 - VHL full sequence

[0178] MASAARLTMM WEEVTCPICL DPFVEPVSIE CGHSFCQECI SQVGKGGGSV CPVCRQRFLL KNLRPNRQLA NMVNNLKEIS QEAREGTQGE RCAVHGERLH LFCEKDGKAL CWVCAQSRKH RDHAMVPLEE AAQEYQEKLQ VALGELRRKQ ELAEKLEVEI AIKRADWKKT VETQKSRIHA EFVQQKNFLV EEEQRQLQEL EKDEREQLRI LGEKEAKLAQ QSQALQELIS ELDRRCHSSA LELLQEVIIV LERSESWNLK DLDITSPELR SVCHVPGLKK MLRTCAVHIT LDPDTANPWL ILSEDRRQVR LGDTQQSIPG NEERFDSYPM VLGAQHFHSG KHYWEVDVTG KEAWDLGVCR DSVRRKGHFL LSSKSGFWTI WLWNKQKYEA GTYPQTPLHL QVPPCQVGIF LDYEAGMVSF YNITDHGSLI YSFSECAFTG PLRPFFSPGF NDGGKNTAPL TLCPLNIGSQ GSTDY

[0179] SEQ ID NO: 4 - PRYSPRY domain of TRIM21VHIT LDPDTANPWL ILSEDRRQVR LGDTQQSIPG NEERFDSYPM VLGAQHFHSG KHYWEVDVTG KEAWDLGVCR DSVRRKGHFL LSSKSGFWTI WLWNKQKYEA GTYPQTPLHL QVPPCQVGIF LDYEAGMVSF YNITDHGSLI YSFSECAFTG PLRPFFSPGF NDGGKNTAPL TLCPLNIGSQ GSTDY

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Claims

CLAIMS1. A method of identifying a test molecule capable of binding an oligomeric target protein and a RING-type E3 ligase, wherein the method comprises contacting a first fusion protein that comprises the RING-type E3 ligase or fragment thereof and a first portion of a split-reporter protein and a second fusion protein that comprises the RING-type E3 ligase or fragment thereof and a complementary second portion of the split-reporter protein with the test molecule, in the presence of the oligomeric target protein.

2. The method of claim 1, wherein the oligomeric target protein is expressed in a cell.

3. The method of claim 1, wherein:(a) the oligomeric target protein is applied exogenously to a cell; or(b) the oligomeric target protein is in a cell-free system.

4. The method of claim 3, wherein the oligomeric target protein is isolated from a sample obtained from a patient.

5. The method of claim 4, wherein the oligomeric target protein is isolated from a sample obtained from a patient diagnosed with: dementia, frontotemporal lobar degeneration (FTLD); Alzheimer’s disease (AD), progressive supranuclear palsy (PSP); corticobasal degeneration (CBD); argyrophylic grain disease (AGD); Huntington's disease (HD); Pick's disease; amyotrophic lateral sclerosis (ALS); chronic traumatic encephalopathy (CTE); multiple system atrophy (MSA); Parkinson’s disease (PD); dementia with Lewy bodies (DLB); or retinitis pigmentosa (RP).

6. The method of any one of the preceding claims, wherein the oligomeric target is composed of any one of: tau protein; synuclein; alpha-synuclein; amyloid P (AP), islet amyloid polypeptide (IAPP), serum amyloid A (SAA), prion proteins, Huntingtin (HTT); misfolded transthyretin protein (TTR); TAR DNA binding protein 43 (TDP-43); NOTCH3 receptor, C9orf72 dipeptide repeat protein; fused in sarcoma (FUS) protein; super oxide dismutase (SOD); rhodopsin (ROD); mitochondrial antiviral-signaling protein (MAVS) assemblies; cGAS; STING; RIG-I; MDA-5; MyD88; IRAK1 / 2 / 4; MAL; ASC; NLRP3;42TRAF family; TBK1; RIPK1 / 3; Gasdermin; Apaf-1; PIDD; p53; BRD4; CBX2; PRC1 / 2; Ras family; Raf; EWS; p-TEFb; YAP; TAZ; TEAD; HSF1; ENL; LAT; Grb2; Sosl; Nek; N-WASP; Arp2 / 3; Axin; APC; GSK3beta; Dishevelled; STAT1 / 2 / 6; SHP2; PKA; G protein-coupled receptors (GPCRs); HSD17B13; nucleoporin; peripherin; Receptor tyrosine kinases, such as ALK; and an ALK fusion partner, such as EML4, NPM1, SQSTM1, DCTN1, HIP1, KIF5B, STRN, or RANBP2, or an oligomeric domain thereof.

7. The method of any one of the preceding claims, wherein:(a) the first fusion protein comprises an amino acid linker connecting the RING- type E3 ligase or fragment thereof and the first portion of the split-reporter protein; and / or(b) the second fusion protein comprises an amino acid linker connecting the RING-type E3 ligase or fragment thereof and the second portion of the splitreporter protein.

8. The method of claim 7, wherein the amino acid linker comprises or consists of glycine and serine.

9. The method of any one of the preceding claims, wherein detection of a signal from the split reporter protein indicates that the test molecule is capable of binding the oligomeric target protein and the RING-type E3 ligase.

10. The method of any one of the preceding claims, wherein:(a) the first and second fusion proteins are expressed in a cell; and / or(b) the first and second fusion proteins are in a cell-free system.

11. The method of any one of the preceding claims, wherein the cell expressing the RING-type E3 ligase or fragment thereof is contacted with a detection reagent to measure fluorescence, luminescence, or biotinylation.

12. The method of claim 11, wherein:(a) the cell is processed by immunofluorescence imaging; and / or(b) the cell is fixed; or live.

13. The method of any one of the preceding claims, wherein the contacting is performed in a cell, wherein the RING-type E3 ligase of fragment thereof is TRIM21, and wherein endogenous TRIM21 is inactivated in the cell.

14. The method of claim 13, wherein:(a) TRIM21 is inactivated in the cell by gene knockout of TRIM21, optionally using a technique selected from: non-homologous DNA end joining (NHEJ); clustered regularly interspaced short palindromic repeats (CRISPR) editing; transcription activator-like effector nucleases (TALEN) editing; or artificial mutagenesis; or(b) TRIM21 expression is reduced in the cell by gene knockdown of TRIM21 optionally using a technique selected from: small interfering RNA (siRNA); or short hairpin RNA (shRNA).

15. The method of any one of the preceding claims, wherein the contacting is performed in a cell, and the cell is exposed to ubiquitin-proteasome inhibitors to prevent protein degradation and stabilise the reporter signal.

16. The method of any one of the preceding claims, wherein:(a) the test molecule is applied exogenously; and / or(b) the test molecule is any one of: a small molecule; peptide; antibody or antibody fragment thereof; or a recombinant protein.

17. The method of any one of the preceding claims, wherein the contacting is performed in a cell and wherein the first and second fusion proteins are expressed in the cell following transient transfection; or stable transduction.

18. The method of any one of the preceding claims, wherein the split-reporter protein is a: split-luciferase protein; split-fluorescent protein, split-biotin ligase, split-horseradish peroxidase (HRP); or nano bioluminescence resonance energy transfer (nano-BRET), including a first portion comprising a HaloTag and a second portion comprising a NanoLuc.

19. The method of claim 18, wherein:44(a) the first fusion protein comprises a large BiT (LgBiT)-E3 ligase fusion and the second fusion protein comprises a small BiT (SmBiT)-E3 ligase fusion, optionally wherein LgBiT and SmBiT are fused to the N-terminus of the RING-type E3 ligase or fragment thereof; and / or(b) the first fusion protein comprises a LgBiT-TRIM21 fusion and the second fusion protein comprises a SmBiT-TRIM21 fusion.

20. The method of any one of the preceding claims, wherein:(a) the RING-type E3 ligase or fragment thereof is: TRIM21, CBRN, VHL, RNF4, XIAP, TRIM5, TRIM7, MDM2, CBL, BRCA1, STUB1, RBX1, BARD1, HTLF, NFX1, or TRIM56; and / or(b) the RING-type E3 ligase or fragment thereof comprises or consists of the PRYSPRY domain; and / or(c) the RING-type E3 ligase or fragment thereof is TRIM21, wherein TRIM21 further comprises a mutation that reduces or ablates ubiquitin ligase activity, such as I18R; R55A; M10E; and / or M72E; and / or(d) the RING-type E3 ligase or fragment thereof is TRIM21, wherein TRIM21 further comprises a mutation, such as I18R; and / or M72E and deletion of the RING domain and / or RING-box domain.

21. The method of any one of the preceding claims, wherein the method further comprises isolating a test molecule that is identified as capable of binding the oligomeric target protein and the RING-type E3 ligase, optionally wherein the method further comprises formulating the test molecule in a pharmaceutically acceptable carrier to provide a pharmaceutical composition comprising the test molecule.

22. A method for producing a molecule capable of binding an oligomeric target protein and the RING-type E3 ligase comprising identifying a molecule capable of binding the oligomeric target protein and the RING-type E3 ligase in accordance with any one of the preceding claims, and optionally modifying or optimising the molecule.

23. A cell expressing a first fusion protein that comprises a RING-type E3 ligase or fragment thereof and a first portion of a split-reporter protein and a second fusion protein thatcomprises a RING-type E3 ligase or fragment thereof and a complementary second portion of the split-reporter protein.

24. The cell of claim 23, wherein the RING-type E3 ligase or fragment thereof is:TRIM21, CRBN, VHL, RNF4, XIAP, TRIM5, TRIM7, MDM2, CBL, BRCA1, STUB1, RBX1, BARD1, HTLF, NFX1, or TRIM56, optionally wherein TRIM21 comprises a mutation that reduces or ablates ubiquitin ligase activity, such as I18R; R55A; M10E; and / or M72E, further optionally wherein TRIM21 comprises a mutation, such as I18R; and / or M72E and / or deletion of the RING domain and / or RING-box domain.

25. The cell of claim 23 or claim 24, wherein:(a) the cell expresses the oligomeric target protein, optionally wherein the cell has been engineered to express the oligomeric target protein; or(b) the cell is contacted with an exogenously applied oligomeric target protein.

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